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https://openalex.org/W2964001028
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https://europepmc.org/articles/pmc6731747?pdf=render
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English
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Liquid chromatography–tandem mass spectrometry metabolic profiling of nazartinib reveals the formation of unexpected reactive metabolites
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Royal Society open science
| 2,019
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| 10,001
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royalsocietypublishing.org/journal/rsos Research
Cite this article: Abdelhameed AS, Attwa MW,
Kadi AA. 2019 Liquid chromatography–tandem
mass spectrometry metabolic profiling of
nazartinib reveals the formation of unexpected
reactive metabolites. R. Soc. open sci. 6: 190852. http://dx.doi.org/10.1098/rsos.190852 Keywords: Keywords:
bioactivation, LC–MS/MS, epidermal growth
factor receptor, human liver microsomes,
iminium ion intermediates, lung cancer MWA, 0000-0002-1147-4960 Nazartinib (EGF816, NZB) is a promising third-generation
human epidermal growth factor receptor (EGFR) tyrosine
kinase inhibitor. This novel irreversible mutant-selective EGFR
inhibitor targets EGFR containing both the resistance mutation
(T790M) and the activating mutations (L858R and Del19), while
it does not affect wild-type EGFR. However, the metabolic
pathway and bioactivation mechanisms of NZB are still
unexplored. Thus, using liquid chromatography–tandem mass
spectrometry, we screened for products of NZB metabolism
formed in vitro by human liver microsomal preparations and
investigated the formation of reactive intermediates using
potassium cyanide as a nucleophile trap. Unexpectedly, the
azepane ring was not bioactivated. Instead, the carbon atom
between the aliphatic linear tertiary amine and electron-
withdrawing system (butenoyl amide group) was bioactivated,
generating iminium intermediates as reactive species. Six NZB
phase I metabolites, formed by hydroxylation, oxidation and
N-demethylation, were characterized. Moreover, two reactive
iminium ions were characterized and their corresponding
bioactivation mechanisms were proposed. Based on our results,
we speculate that bioactivation of NZB can be blocked by small
sterically hindering groups, isosteric replacement or a spacer. This approach might reduce the toxicity of NZB by avoiding
the generation of reactive species. Research Cite this article: Abdelhameed AS, Attwa MW,
Kadi AA. 2019 Liquid chromatography–tandem
mass spectrometry metabolic profiling of
nazartinib reveals the formation of unexpected
reactive metabolites. R. Soc. open sci. 6: 190852. http://dx.doi.org/10.1098/rsos.190852 Ali S. Abdelhameed1, Mohamed W. Attwa1,2
and Adnan A. Kadi1 Ali S. Abdelhameed1, Mohamed W. Attwa1,2 Received: 4 June 2019
Accepted: 22 July 2019
Subject Category:
Chemistry
Subject Areas:
analytical chemistry
Keywords:
bioactivation, LC–MS/MS, epidermal growth
factor receptor, human liver microsomes,
iminium ion intermediates, lung cancer
Author for correspondence:
Mohamed W. Attwa
e-mail: mzeidan@ksu.edu.sa Received: 4 June 2019
Accepted: 22 July 2019 1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
PO Box 2457, Riyadh 11451, Kingdom of Saudi Arabia
2Students’ University Hospital, Mansoura University, Mansoura 35516, Egypt royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 Non-small-cell lung cancer (NSCLC) encompasses a heterogeneous group of lung cancer subtypes [1–5],
which affects 90% of patients with lung cancer [6]. This class of lung cancer is associated with several
mutations, such as those in human epidermal growth factor receptor (EGFR). Tyrosine kinase
inhibitors (TKIs) regulate the activity of human EGFR and have become the standard treatment for
patients suffering from advanced EGFR-mutant NSCLC. The first-generation EGFR TKIs (e.g. gefitinib
and erlotinib) bind reversibly and competitively to the ATP-binding site of the EGFR tyrosine kinase
(TK) domain, which improves the outcome of NSCLC patients bearing EGFR-activating mutations
(L858R and Del19) [7,8]. However, after satisfactory responses for a period, patients’ tumours acquired
resistance to first-generation TKIs because of the development of a T790M mutation, which affects the
ATP-binding site of the human EGFR [9–12]. Thus, second-generation EGFR TKIs (e.g. avitinib and dacomitinib) were designed to target
tumours with T790M mutation and EGFR-activating mutations. These compounds showed
promising anti-T790M activity in laboratory experiments. However, their clinical activity towards
T790M-associated NSCLC was limited because of their inhibitory effects on wild-type EGFR,
which resulted in toxicity and a narrow therapeutic index [13–15]. More recently, third-generation
EGFR TKIs (e.g. osimertinib and nazartinib (NZB)) were developed. They irreversibly and
selectively target EGFR with T790M and other mutations, whereas they have little effect on wild-
type EGFR activity [13,14]. Third-generation EGFR TKIs were developed to overcome EGFR
T790M-mediated resistance to first- and second-generation EGFR TKIs with minor toxicity. Third-
generation EGFR TKIs combine effectiveness against NSCLC that is resistant to both first- and
second-generation EGFR TKIs [16,17]. Osimertinib, for example, is approved by both the
American and European regulatory agencies for the management of patients with metastatic
EGFR T790M NSCLC [18]. Pre-clinical data show that NZB, another third-generation EGFR TKI
[19], does not affect wild-type EGFR activity and presents selectivity against mutated EGFR,
similar to other third-generation EGFR TKIs. Nevertheless, it presents some side effects, such as
diarrhoea, pruritus and rash [20]. In addition to the drug itself, by-products of detoxification pathways may be responsible for such
adverse effects in patients. Detoxification involves metabolic reactions that transform endogenous
compounds and xenobiotics, increasing their polarity to be excreted from the human body. Although
metabolites usually exhibit less toxicity than their parents, in some cases, bioactivation may generate
reactive intermediates that are more toxic than the unmetabolized molecules [21–23]. Author for correspondence: Author for correspondence:
Mohamed W. Attwa
e-mail: mzeidan@ksu.edu.sa Mohamed W. Attwa
e-mail: mzeidan@ksu.edu.sa This article has been edited by the Royal Society
of Chemistry, including the commissioning, peer
review process and editorial aspects up to the
point of acceptance. © 2019 The Authors. Published by the Royal Society under the terms of the Creative
Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits
unrestricted use, provided the original author and source are credited. 2 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 Soc. open sci. 6: 190852
3 benzimidazole
isonicotinamide
butenone
azepane
molecular weight: 495.02
nazartinib
tertiary
nitrogen
Figure 1. Chemical structure of NZB showing its building blocks. royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
3 3 isonicotinamide benzimidazole azepane nazartinib Figure 1. Chemical structure of NZB showing its building blocks. Figure 1. Chemical structure of NZB showing its building blocks. 2.2. Chromatographic conditions Resolution and identification of in vitro NZB metabolites and its related cyano adducts from the HLM
incubation mixtures was performed on an Agilent Triple Quadrupole system comprising an Agilent
rapid resolution liquid chromatography (RRLC) 1200 as an HPLC system and an Agilent 6410 triple
quadrupole (QqQ) as a mass detector (Agilent Technologies, Palo Alto, CA, USA) with an electrospray
ionization (ESI) source. Chromatographic resolution of the metabolic mixtures components was done on
a C18 column (length, 150 mm; internal diameter, 2.1 mm; and particle size, 3.5 µm). The column
temperature was fixed at 22 ± 1°C, and we used a gradient mobile phase at a flow rate of 0.2 ml min−1
and consisting of 10 mM ammonium formate (solvent A; pH 4.2) and acetonitrile (solvent B). The
gradients steps involved solvent B (5%; 0–5 min), solvent B (5–50%; 5–35 min), solvent B (50–90%;
35–50 min) and solvent B (90–5%; 50–60 min), with a post time of 15 min. The sample injection volume
was 10 µl. The run time was 60 min, with the chromatographic and mass parameters preoptimized for
NZB. The generation of daughter ions (DIs) of NZB metabolites and cyano adducts was done in the
collision cell by collision-induced dissociation (CID). Mass analysis was performed on a mass detector
using positive ESI source. Nitrogen (N2) was used as drying gas at a flow rate of 11 l min−1, and as
collision gas at a pressure of 55 psi. Capillary voltage, source temperature, fragmentor voltage and
collision energy were set to 4000 V, 350°C, 140 V and 18 eV, respectively. Agilent Mass Hunter software
was used for controlling instrument and data acquisition. 2.1. Chemicals NZB was obtained from MedChem Express (Monmouth Junction, NJ, USA). Formic acid, ammonium
formate, potassium cyanide, pooled human liver microsomes (HLMs, M0567) and acetonitrile were
procured from Sigma-Aldrich (St Louis, MO, USA). High-performance liquid chromatography
(HPLC)-grade water (H2O) was generated by an in-house Milli-Q Plus purification system (Burlington,
MA, USA). All other solvents and chemicals were of analytical grade. royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 Reactive
intermediates are unstable and can modify DNA and proteins by the formation of covalent bonds,
which is considered the initial step in drug-induced organ toxicity [24,25]. Thus, the identification of
generated reactive metabolites is crucial for understanding drug-induced toxicity. However, reactive
metabolites are usually generated by phase I metabolic pathways and their identification is hindered
by their transient nature. To overcome this limitation, a nucleophile can be used to capture reactive
intermediates, and the resulting adducts can be characterized and identified by mass spectrometry
technique [26,27]. The
chemical
structure
of
NZB
(N-(7-chloro-1-{(3R)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-3-
azepanyl}-1H-benzimidazole-2-yl)-2-methyl isonicotinamide; figure 1) contains two tertiary nitrogen
atoms (an azepane ring and a terminal dimethylamino group) that can be bioactivated, generating
iminium ion intermediates [28–31]. The formation of unstable intermediates reveals side effects of
NZB as was approved with similar drugs. Cyclic tertiary amine rings can perform bioactivation by
iminium ion generation [28–31]. These intermediates react poorly with glutathione; however, they can
be trapped using potassium cyanide [21,28,29]. The obtained reactive iminium intermediates trapped
efficiently using cyanide to form cyano conjugates can be characterized by mass spectrometry
[26–28,32,33]. Moreover, although the azepane ring was expected to undergo bioactivation during
NZB metabolism, this does not occur. Instead, the carbon between the aliphatic linear tertiary amine
and the unsaturated conjugated system are bioactivated. It is hypothesized that these reactive metabolites might be responsible for the side effects of NZB. However, there are no reports on specific metabolic pathways associated with the bioactivation
mechanism of NZB. Thus, the aim of this work was to use in vitro experiments to characterize the
bioactivation pathways of NZB that form reactive intermediates. To do so, we used a scavenging
molecule (potassium cyanide) to trap reactive intermediates of NZB metabolism. This approach was
used because when reactive metabolites form in vivo, they bind to DNA and proteins via covalent
bonds and hence cannot be detected [24,27,32]. 2. Material and methods
2.1. Chemicals
NZB was obtained from MedChem Express (Monmouth Junction, NJ, USA). Formic acid, ammonium
formate, potassium cyanide, pooled human liver microsomes (HLMs, M0567) and acetonitrile were
procured from Sigma-Aldrich (St Louis, MO, USA). High-performance liquid chromatography
(HPLC)-grade water (H2O) was generated by an in-house Milli-Q Plus purification system (Burlington,
MA, USA). All other solvents and chemicals were of analytical grade. benzimidazole
isonicotinamide
butenone
azepane
molecular weight: 495.02
nazartinib
tertiary
nitrogen
Figure 1. Chemical structure of NZB showing its building blocks. royalsocietypublishing.org/journal/rsos
R. 2.3. Human liver microsomes incubation 6: 190852
4 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min) + product ion (37.210 min) (495.0 - >**) NZB HLMs 495.d
×103
0.5
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
58.1
84.0
97.9
164.1
209.3
112.1
287.0
495.2
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
q
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
(b)
Figure 2. PI chromatogram of NZB (a) and DIs mass spectrum at 37.21 min (b). (b) Figure 2. PI chromatogram of NZB (a) and DIs mass spectrum at 37.21 min (b). results. The purification of the incubated solutions was performed by a protein precipitation method
involving: (i) centrifugation at 9000g for 15 min at 4°C, (ii) the transfer of the supernatant into clean
vials, and (iii) concentration of the extracts by evaporation under nitrogen stream, followed by
reconstitution in 0.5 ml of mobile phase. To analyse the composition of each sample, 10 µl was
injected into a liquid chromatography tandem mass spectrometry (LC–MS/MS) [36–38]. Controls were
prepared following the same steps except the addition of the drug or NADPH. results. The purification of the incubated solutions was performed by a protein precipitation method
involving: (i) centrifugation at 9000g for 15 min at 4°C, (ii) the transfer of the supernatant into clean
vials, and (iii) concentration of the extracts by evaporation under nitrogen stream, followed by
reconstitution in 0.5 ml of mobile phase. To analyse the composition of each sample, 10 µl was
injected into a liquid chromatography tandem mass spectrometry (LC–MS/MS) [36–38]. Controls were
prepared following the same steps except the addition of the drug or NADPH. 2.4. Identification of NZB reactive intermediates Full mass spectrometry scans and extracted ion chromatograms of the detected mass to charge ratio (m/z)
peaks were used to identify the in vitro metabolites in the incubation mixtures. Molecular ions were used
as parent ions (PIs) for fragmentation into daughter ions (DIs). The fragmentation behaviour was used to
characterize the reactive metabolites formed during NZB metabolism by HLMs in vitro. 2.3. Human liver microsomes incubation We first exposed HLMs to several NZB concentrations (2–30 µM) and found that the composition of
metabolites did not vary within this range. However, the concentration of metabolites increased as the
concentration of NZB increased. Thus, 30 µM was used in all experiments to increase the yield of
metabolites and make their characterization easier. The screening of NZB metabolites was performed
in vitro by incubating NZB (30 µM) with HLMs (1.0 mg ml−1) in phosphate buffer (50 mM at pH 7.4)
and MgCl2 (3.3 mM) for 120 min at 37°C in a shaking water bath. The in vitro metabolization of NZB
was stimulated by the addition of NADPH (1.0 mM) and terminated by the addition of ice-cold
acetonitrile [34,35]. The same HLM incubation experiment was repeated in the presence of potassium
cyanide to capture the reactive intermediates. All reactions were performed in triplicate to verify the 5.5
×104
+TIC product ion (** - > **) NZB HLMs 495.d
+ product ion (37.210 min) (495.0 - >**) NZB HLMs 495.d
×103
0.5
2
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.5
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
58.1
84.0
97.9
164.1
209.3
112.1
287.0
495.2
1
1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
counts versus acquisition time (min)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
(a)
(b)
Figure 2. PI chromatogram of NZB (a) and DIs mass spectrum at 37.21 min (b). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
4 5.5
×104
+TIC product ion (** - > **) NZB HLMs 495.d
0.5
2
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
1
1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
(a)
royalsocietypublishing.org/journal/rsos
4 4 (a) royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 3.1. Fragmentation analysis of NZB The chemical structure of NZB contains five building blocks (isonicotinamide, benzimidazole, azepane,
tertiary dimethyl amine and butenoyl). The fragmentation of the NZB PI generated qualitative DIs that
were used to identify the metabolic changes in the NZB structure. The NZB PI peak eluted at 37.21 min
(figure 2a). The fragmentation of the PI at m/z 495 generated six DIs at m/z 287, m/z 209, m/z 164, m/z 112,
m/z 84 and m/z 58 (figure 2b). The DI at m/z 287 was used to trace any changes on the isonicotinamide and
benzimidazole groups. The DIs at m/z 209 and m/z 164 were used to trace any changes on the azepane
ring. The DIs at m/z 112 and m/z 84 were used to trace any changes on the butenoyl group. The DI at m/z
58 was used to trace any changes on the dimethyl amine group (scheme 1). nazartinib
benzimidazole
isonicotinamide
azepane
butenoyl
tertiary
nitrogen
m/z: 495
DIs
m/z: 112
m/z: 209
m/z: 164
m/z: 287
m/z: 58
m/z: 84
Scheme 1. Fragmentation behaviour of NZB. Asterisk stands for reactive centre. DIs, daughter ions. royalsocietypublishing.org/journal/rso
5 5 isonicotinamide m/z: 112
m/z: 58
m/z: 84 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 m/z: 209
m/z: 164 butenoyl benzimidazole
DIs Scheme 1. Fragmentation behaviour of NZB. Asterisk stands for reactive centre. DIs, daughter ions. Table 1. In vitro phase I and reactive metabolites of NZB. MS, mass spectrometry; NZB, nazartinib; RT, retention time. ble 1. In vitro phase I and reactive metabolites of NZB. MS, mass spectrometry; NZB, nazartinib; RT, retention time. Table 1. In vitro phase I and reactive metabolites of NZB. MS, mass spectrometry; NZB, nazartinib; RT, retention time. 3.2.1. Identification of the NZB481 phase I metabolite The NZB481 PI peak eluted at 36.12 min (figure 3a). The fragmentation of the PI at m/z 481 generated four
DIs at m/z 287, m/z 195, m/z 98 and m/z 44 (figure 3b). In comparison with the NZB fragmentation pattern,
the DI at m/z 287 revealed no metabolic change on the isonicotinamide and benzimidazole groups. The
DIs at m/z 195, m/z 98 and m/z 44 exhibited decreases of 14 m/z units. Thus, the DIs at m/z 98 and m/z 44
indicated that an N-demethylation metabolic change occurred on the dimethyl amine group (scheme 2). 3.1. Fragmentation analysis of NZB molecule
MS scan
most abundant fragment ions (m/z)
RT (min)
metabolic reaction
original drug
NZB
495
287, 209, 164, 112, 84, 58
37.21
no reaction
phase I metabolites
NZB481
481
287, 195, 98, 44
36.12
N-demethylation
NZB509a
509
301, 209, 112, 84
30.19
oxidation at the methyl
attached to the isonicotinamide group
NZB509b
509
287, 233, 126
36.72
α-oxidation of the dimethyl amine group
NZB509c
509
450, 353, 164, 120, 58
43.49
α-oxidation at the azepane ring
NZB511a
511
287, 225, 180, 112
32.76
α-hydroxylation at the azepane ring
NZB511b
511
303, 209, 112, 84
34.14
hydroxylation at the methyl attached to
the isonicotinamide group
reactive metabolites
NZB520
520
493, 207, 164, 83, 57
47.66
cyano addition at the bioactivated carbon
NZB506
506
287, 220, 120, 98
48.95
N-demethylation and cyano addition at
the bioactivated carbon
R. Soc. open sci. 6: 190852 2. Characterization of phase I nazartinib metabolites and reactive intermediates 3.2. Characterization of phase I nazartinib metabolites and reactive intermediates Phase I metabolic reactions (hydroxylation, oxidation and N-demethylation) produced six metabolites. In
addition, we detected two reactive intermediates as cyano adducts (table 1). Phase I metabolic reactions (hydroxylation, oxidation and N-demethylation) produced six metabolites. In
addition, we detected two reactive intermediates as cyano adducts (table 1). Phase I metabolic reactions (hydroxylation, oxidation and N-demethylation) produced six metabolites. In
addition, we detected two reactive intermediates as cyano adducts (table 1). 3.2.2. Identification of the NZB509a and NZB509b phase I metabolites 6: 190852
6 20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
2
1
1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+TIC product ion (** - > **) NZB HLMs 481.d
+ product ion (36.115 min) (481.0 - >**) NZB HLMs 481.d
×103
×103
7
1
0
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
3
4
5
6
98.3
44.3
195.3
286.7
481.2
(a)
(b) 6 (a) +TIC product ion (** - > **) NZB HLMs 481.d +TIC product ion (** - > **) NZB HLMs 481.d
×103
(a) royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 2
1
1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+TIC product ion (** - > **) NZB HLMs 481.d
×103
7
1
2
3
4
5
6
(b)
royalsocietypublishing.org/journal/rsos (b) 20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (36.115 min) (481.0 - >**) NZB HLMs 481.d
×103
0
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
98.3
44.3
195.3
286.7
481.2
(b) counts versus mass-to-charge (m/z) Figure 3. DI chromatogram of NZB481 (a) and DI mass spectrum at 36.12 min (b). Figure 3. DI chromatogram of NZB481 (a) and DI mass spectrum at 36.12 min (b). Figure 3. DI chromatogram of NZB481 (a) and DI mass spectrum at 36.12 min (b). NZB481
m/z: 481
DIs
m/z: 98
m/z: 195
m/z: 44
m/z: 287
Scheme 2. Fragmentation behaviour of NZB481. DIs, daughter ions. m/z: 195 m/z: 98
m/z: 195
m/z: 44
m/z: 287 Scheme 2. Fragmentation behaviour of NZB481. DIs, daughter ions. The fragmentation of NZB509b resulted in three DIs at m/z 287, m/z 223 and m/z 126 (figure 4c). 3.2.2. Identification of the NZB509a and NZB509b phase I metabolites In
comparison with the NZB fragmentation pattern, the DI at m/z 287 indicated the absence of any
metabolic change on the isonicotinamide and benzimidazole groups. The DIs at m/z 223 and m/z 126
showed increases of 14 m/z units. Thus, the DI at m/z 126 indicated that an oxidation metabolic
reaction occurred on the carbon α of the dimethyl amine group (scheme 4). y
g
p
The fragmentation of NZB509c resulted in five DIs at m/z 450, m/z 353, m/z 164, m/z 120 and m/z 58
(figure 4d). In comparison with the NZB fragmentation pattern, the DI at m/z 58 indicated that no
metabolic change occurred on the dimethyl amine group. The DIs at m/z 450 and m/z 353 (resulting
from a retro-Diels–Alder reaction) revealed the oxidation of the azepane ring, in agreement with the
other DIs at m/z 120 and m/z 58 (scheme 5). 3.2.2. Identification of the NZB509a and NZB509b phase I metabolites The PI peaks of NZB509a, NZB509b and NZB509c eluted at 30.19, 36.72 and 43.49 min, respectively
(figure 4a). The fragmentation of the PI at m/z 509 generated several DIs (figure 4b–d). The fragmentation of NZB509a resulted in four DIs at m/z 301, m/z 209, m/z 112 and m/z 84 (figure 4b). In comparison with the NZB fragmentation pattern, the DIs at m/z 209, m/z 112 and m/z 84 revealed no
metabolic change on the azepane ring, dimethyl tertiary amine group and butenoyl group. The DI at m/z
301 showed an increase of 14 m/z units, indicating that the methyl attached to the isonicotinamide group
was oxidized during metabolism (scheme 3). 20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
2
1
1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+TIC product ion (** - > **) NZB HLMs 481.d
+ product ion (36.115 min) (481.0 - >**) NZB HLMs 481.d
×103
×103
7
1
0
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
3
4
5
6
98.3
44.3
195.3
286.7
481.2
(a)
(b)
Figure 3. DI chromatogram of NZB481 (a) and DI mass spectrum at 36.12 min (b). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 3.2.3. Identification of the NZB511a and NZB511b phase I metabolites DI chromatogram of NZB509 metabolites (a) and DI mass spectra at 30.19 min (b), 36.72 min (c) and 43.49 min ( 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min) counts versus acquisition time (min) 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 ×103
(c)
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
58.1
98.1
120.0
164.2
353.1
450.4
509.3
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (36.718 min) (509.0 - >**) NZB HLMs 509.d (d)
64.7
125.9
286.8
233.1
335.0
509.1
91.0
46.1
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (43.497 min) (509.0 - >**) NZB HLMs 509.d
9
0
1
2
3
4
5
6
7
8
×10
Fi
4 DI h
t
f NZB509
t b lit
( )
d DI
t
t 30 19
i (b) 36 72
i ( )
d 43 49
i ( 20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
0 4. DI chromatogram of NZB509 metabolites (a) and DI mass spectra at 30.19 min (b), 36.72 min (c) and 43.49 min ( hromatogram of NZB509 metabolites (a) and DI mass spectra at 30.19 min (b), 36.72 min (c) and 43.49 min (d). Figure 4. DI chromatogram of NZB509 metabolites (a) and DI mass spectra at 30.19 min (b), 36.72 min (c) and The fragmentation of NZB511a at m/z 511 resulted in four DIs at m/z 287, m/z 225, m/z 180 and m/z 112
(figure 5b). 3.2.3. Identification of the NZB511a and NZB511b phase I metabolites The NZB511a and NZB511b PI peaks appeared at 32.76 and 34.14 min, respectively (figure 5a). The
fragmentation of the PI at m/z 511 produced various DIs (figure 5b,c). + product ion (30.186 min) (509.0 - >**) NZB HLMs 509.d
×104
×102
×103
1.2
1
1
1.0
0.8
0.6
0.4
0.2
(a)
(b)
(c)
(d)
0
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
84.3
58.1
98.1
120.0
64.7
125.9
286.8
233.1
335.0
509.1
91.0
46.1
164.2
353.1
450.4
509.3
111.9
301.2
509.5
209.1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+TIC product ion (** - > **) NZB HLMs 509.d
+ product ion (36.718 min) (509.0 - >**) NZB HLMs 509.d
+ product ion (43.497 min) (509.0 - >**) NZB HLMs 509.d
9
1
2
3
4
5
6
7
8
×10 + product ion (30.186 min) (509.0 - >**) NZB HLMs 509.d
×104
×102
1.2
1
1
1.0
0.8
0.6
0.4
0.2
(a)
(b)
( )
0
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
84.3
111.9
301.2
509.5
209.1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+TIC product ion (** - > **) NZB HLMs 509.d
royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 3.2.3. Identification of the NZB511a and NZB511b phase I metabolites 6: 190852
7 ×104
1.2
1
1
1.0
0.8
0.6
0.4
0.2
(a)
(b)
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+TIC product ion (** - > **) NZB HLMs 509.d
royalsocietypublishing.org/journal/rsos
7 7 (a) royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
7 The fragmentation of NZB511a at m/z 511 resulted in four DIs at m/z 287, m/z 225, m/z 180 and m/z 1
figure 5b). In comparison with the NZB fragmentation pattern, the DI at m/z 287 revealed the absence
+ product ion (30.186 min) (509.0 - >**) NZB HLMs 509.d
×102
×103
0.4
0.2
(b)
(c)
(d)
0
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
2
84.3
58.1
98.1
120.0
64.7
125.9
286.8
233.1
335.0
509.1
91.0
46.1
164.2
353.1
450.4
509.3
111.9
301.2
509.5
209.1
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (36.718 min) (509.0 - >**) NZB HLMs 509.d
+ product ion (43.497 min) (509.0 - >**) NZB HLMs 509.d
9
0
1
2
3
4
5
6
7
8
×10
Figure 4. 3.2.3. Identification of the NZB511a and NZB511b phase I metabolites In comparison with the NZB fragmentation pattern, the DI at m/z 287 revealed the absence of
any metabolic reaction at the isonicotinamide and benzimidazole groups, and the DI at m/z 112 indicated The fragmentation of NZB511a at m/z 511 resulted in four DIs at m/z 287, m/z 225, m/z 180 and m/z 112
(figure 5b). In comparison with the NZB fragmentation pattern, the DI at m/z 287 revealed the absence of
any metabolic reaction at the isonicotinamide and benzimidazole groups, and the DI at m/z 112 indicated NZB509a
m/z: 509
DIs
m/z: 112
m/z: 209
m/z: 84
m/z: 301
Scheme 3. Fragmentation behaviour of NZB509a. DIs, daughter ions. royalsocietypublishing.org/journal/rsos
8 n the butenoyl group. The DIs at m/z 225 and m/z 180 showed
t hydroxylation occurred on the azepane ring (scheme 6). ted in four DIs at m/z 303, m/z 209, m/z 112 and m/z 84 (figure 5c). tion pattern, the DIs at m/z 209, m/z 112 and m/z 84 indicated the
he azepane ring, the dimethyl amine group and the butenoyl
DIs
m/z: 112
m/z: 209
m/z: 84
m/z: 301
DIs, daughter ions. DIs
m/z: 126
m/z: 223
m/z: 287
. DIs, daughter ions. m/z: 164
m/z: 58
m/z: 450
m/z: 120
m/z: 353
DIs, daughter ions. royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
8 The DIs at m/z 225 and m/z 180 showed
d on the azepane ring (scheme 6). 3, m/z 209, m/z 112 and m/z 84 (figure 5c). m/z 209, m/z 112 and m/z 84 indicated the
imethyl amine group and the butenoyl
indicating hydroxylation on the methyl
m/z: 112
m/z: 209
m/z: 84
m/z: 126
m/z: 223
/z: 287
m/z: 164
m/z: 58
m/z: 120
m/z: 353
royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
8 58
royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
8 m/z: 112
m/z: 209
m/z: 84
m/z: 301
royalsocietypublishing.org/journa
8 8 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852 DIs Scheme 3. Fragmentation behaviour of NZB509a. DIs, daughter ions. sos
R. Soc. open sci. 6: 190852 NZB509b
m/z: 509
DIs
m/z: 126
m/z: 223
m/z: 287
Scheme 4. Fragmentation behaviour of NZB509b. DIs, daughter ions. DIs m/z: 126 Scheme 4. Fragmentation behaviour of NZB509b. DIs, daughter ions. NZB509c
m/z: 509
DIs
m/z: 164
m/z: 58
m/z: 450
m/z: 120
m/z: 353
Scheme 5. Fragmentation behaviour of NZB509c. DIs, daughter ions. Scheme 5. Fragmentation behaviour of NZB509c. DIs, daughter ions. 3.2.3. Identification of the NZB511a and NZB511b phase I metabolites the absence of any metabolic reaction on the butenoyl group. The DIs at m/z 225 and m/z 180 showed
increases of 16 m/z units, indicating that hydroxylation occurred on the azepane ring (scheme 6). the absence of any metabolic reaction on the butenoyl group. The DIs at m/z 225 and m/z 180 showed
increases of 16 m/z units, indicating that hydroxylation occurred on the azepane ring (scheme 6). The fragmentation of NZB511b resulted in four DIs at m/z 303, m/z 209, m/z 112 and m/z 84 (figure 5c). In comparison with the NZB fragmentation pattern, the DIs at m/z 209, m/z 112 and m/z 84 indicated the
absence of any metabolic reaction on the azepane ring, the dimethyl amine group and the butenoyl
group. The DI at m/z 303 showed an increase of 16 m/z units, indicating hydroxylation on the methyl
attached to the isonicotinamide group (scheme 7). 3.3. Reactive metabolites 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
4.0
4.5
5.5
×103
5.0
1.0
1.5
2.0
2.5
3.0
3.5
+TIC product ion (** - > **) NZB HLMs 511.d
1
1
(a)
(b)
royalsocietypublishing.org/journal/rsos
9 2
4
6
8
10
70.0
95.8
84.0 111.9
180.1
225.2
195.1
286.8
382.2
511.3
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
4.0
4.5
5.5
×103
×102
5.0
1.0
1.5
2.0
2.5
3.0
3.5
0
1.0
1.1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
+TIC product ion (** - > **) NZB HLMs 511.d
1
1
+ product ion (32.670 min) (511.0 - >**) NZB HLMs 511.d
(a)
(b)
( ) 9 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
9 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min) 70.0
95.8
84.0 111.9
180.1
225.2
195.1
286.8
382.2
511.3
q
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
×102
0
1.0
1.1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
+ product ion (32.670 min) (511.0 - >**) NZB HLMs 511.d
(b) 83.9
41.9
97.7
163.8
209.3
136.1
303.0
400.0
466.1
511.3
111.9
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
×102
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
+ product ion (34.139 min) (511.0 - >**) NZB HLMs 511.d
(c)
Figure 5. DI chromatogram of NZB511 metabolites (a) and DI mass spectra at 32.76 min (b) and 34.14 min (c). Figure 5. 3.3. Reactive metabolites In addition to the metabolites described above, two cyano adducts were characterized, indicating the
generation of reactive intermediates in NZB metabolism by HLMs. 2
4
6
8
10
70.0
95.8
83.9
41.9
97.7
163.8
209.3
136.1
303.0
400.0
466.1
511.3
84.0 111.9
111.9
180.1
225.2
195.1
286.8
382.2
511.3
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
20 40 60 80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
4.0
4.5
5.5
×103
×102
×102
5.0
1.0
1.5
2.0
2.5
3.0
3.5
0
1.0
1.1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
4.0
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
+TIC product ion (** - > **) NZB HLMs 511.d
1
1
+ product ion (32.670 min) (511.0 - >**) NZB HLMs 511.d
+ product ion (34.139 min) (511.0 - >**) NZB HLMs 511.d
(a)
(b)
(c)
gure 5. DI chromatogram of NZB511 metabolites (a) and DI mass spectra at 32.76 min (b) and 34.14 min (c). 3.3. Reactive metabolites DI chromatogram of NZB511 metabolites (a) and DI mass spectra at 32.76 min (b) and 34.14 min (c). 5. DI chromatogram of NZB511 metabolites (a) and DI mass spectra at 32.76 min (b) and 34.14 min (c). NZB511a
m/z: 511
DIs
m/z: 112
m/z: 225
m/z: 287
m/z: 180
Scheme 6. Fragmentation behaviour of NZB511a. DIs, daughter ions. Scheme 6. Fragmentation behaviour of NZB511a. DIs, daughter ions. NZB511b
m/z: 511
DIs
m/z: 112
m/z: 209
m/z: 84
m/z: 303
Scheme 7. Fragmentation behaviour of NZB511b. DIs, daughter ions. 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60
56.9
81.9
98.1
110.2
137.3
164.0
207.4
287.0
450.0
493.3
80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (47.664 min) (520.0 - >**) NZB HLMs KCN 520.d
+ EIC product ion (** - > 493.0) NZB HLMs KCN 520.d
1
1
0
0.5
1.0
1.5
2.0
2.5
3.0
0
1.4
1.5
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
×102
×102
(a)
(b)
Figure 6. DI chromatogram of NZB520 (a) and DI mass spectrum at 47.66 min (b). NZB511b
m/z: 511
DIs
m/z: 112
m/z: 209
m/z: 84
m/z: 303
Scheme 7. Fragmentation behaviour of NZB511b. DIs, daughter ions. royalsocietypublishing.org/journal/rsos
10 m/z: 112
m/z: 209
m/z: 84
m/z: 303
royalsocietypublishing.org/journa
10 10 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
10 Scheme 7. Fragmentation behaviour of NZB511b. DIs, daughter ions. Scheme 7. Fragmentation behaviour of NZB511b. DIs, daughter ions. 3.3.1. Identification of the NZB520 cyano adduct The NZB520 PI peak eluted at 47.66 min (figure 6a). The fragmentation of the PI at m/z 520 produced
five DIs at m/z 493, m/z 207, m/z 164, m/z 83 and m/z 57 (figure 6b). The DI at m/z 493 indicated the
loss of 27 m/z units, representing the neutral loss of a hydrogen cyanide molecule. The DI at m/z 164
revealed the absence of any metabolic reaction on the azepane ring. The DIs at m/z 137 and m/z 83
confirmed that cyanide ion addition occurred on the bioactivated carbon α of the terminal tertiary N
atom (dimethyl amine) (scheme 8). 3.3. Reactive metabolites The fragmentation of the PI at m/z 520 produced
five DIs at m/z 493, m/z 207, m/z 164, m/z 83 and m/z 57 (figure 6b). The DI at m/z 493 indicated the
loss of 27 m/z units, representing the neutral loss of a hydrogen cyanide molecule. The DI at m/z 164
revealed the absence of any metabolic reaction on the azepane ring. The DIs at m/z 137 and m/z 83
confirmed that cyanide ion addition occurred on the bioactivated carbon α of the terminal tertiary N
atom (dimethyl amine) (scheme 8). 3.3.2. Identification of the NZB506 cyano adduct
0
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
1.0
1.2
1.4
1.6
1.8
0.8
0.6
0.4
0.2
0.1
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100
81.2
119.9
97.9
220.2
287.2
506.3
120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ EIC product ion (** - > 220.0) NZB HLMs KCN 506.d
1
1
×102
×102 + product ion (48.950 min) (506.0 - >**) NZB HLMs KCN 506.d
(a)
(b)
Figure 7. DI chromatogram of NZB506 (a) and DI mass spectrum at 48.95 min (b). NZB506
m/z: 506
DIs
m/z: 220
m/z: 287
m/z: 98
m/z: 120
Scheme 9. Fragmentation behaviour of NZB506. DIs, daughter ions. 0
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0
1.0
1.2
1.4
1.6
1.8
0.8
0.6
0.4
0.2
0.1
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60 80 100
81.2
119.9
97.9
220.2
287.2
506.3
120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ EIC product ion (** - > 220.0) NZB HLMs KCN 506.d
1
1
×102
×102 + product ion (48.950 min) (506.0 - >**) NZB HLMs KCN 506.d
(a)
(b)
Figure 7. 3.3. Reactive metabolites DI chromatogram of NZB506 (a) and DI mass spectrum at 48.95 min (b). royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
11 0
1.0
1.2
1.4
1.6
1.8
0.8
0.6
0.4
0.2
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+ EIC product ion (** - > 220.0) NZB HLMs KCN 506.d
1
1
×102
(a)
royalsocietypublishing.org/journal/rsos
11 11 (a) royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
11 4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min) 0
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
counts versus acquisition time (min)
20 40 60 80 100
81.2
119.9
97.9
220.2
287.2
506.3
120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
×102 + product ion (48.950 min) (506.0 - >**) NZB HLMs KCN 506.d
(b)
Fi
7 DI h
f NZB506 ( )
d DI
48 95
i (b) (b) 0 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 Figure 7. DI chromatogram of NZB506 (a) and DI mass spectrum at 48.95 min (b). ure 7. DI chromatogram of NZB506 (a) and DI mass spectrum at 48.95 min (b). Figure 7. DI chromatogram of NZB506 (a) and DI mass spectrum at 48.95 min (b). NZB506
m/z: 506
DIs
m/z: 220
m/z: 287
m/z: 98
m/z: 120
Scheme 9. Fragmentation behaviour of NZB506. DIs, daughter ions. DIs
m/z: 220
m/z: 287
m/z: 98
m/z: 120 Scheme 9. Fragmentation behaviour of NZB506. DIs, daughter ions. 3.3. Reactive metabolites 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
20 40 60
56.9
81.9
98.1
110.2
137.3
164.0
207.4
287.0
450.0
493.3
80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (47.664 min) (520.0 - >**) NZB HLMs KCN 520.d
+ EIC product ion (** - > 493.0) NZB HLMs KCN 520.d
1
1
0
0.5
1.0
1.5
2.0
2.5
3.0
0
1.4
1.5
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
×102
×102
(a)
(b)
Figure 6. DI chromatogram of NZB520 (a) and DI mass spectrum at 47.66 min (b). 2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
counts versus acquisition time (min)
+ EIC product ion (** - > 493.0) NZB HLMs KCN 520.d
1
1
0
0.5
1.0
1.5
2.0
2.5
3.0
×102
(a) (b) 20 40 60
56.9
81.9
98.1
110.2
137.3
164.0
207.4
287.0
450.0
493.3
80 100 120 140 160 180 200 220 240 260 280
counts versus mass-to-charge (m/z)
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
+ product ion (47.664 min) (520.0 - >**) NZB HLMs KCN 520.d
0
1.4
1.5
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
×102
(b)
Figure 6. DI chromatogram of NZB520 (a) and DI mass spectrum at 47.66 min (b). Figure 6. DI chromatogram of NZB520 (a) and DI mass spectrum at 47.66 min (b). m/z: 137 NZB520
m/z: 520
DIs
m/z: 207
m/z: 83
m/z: 164
m/z: 57
m/z: 493
m/z: 137
Scheme 8. Fragmentation behaviour of NZB520. DIs, daughter ions. m/z: 164 m/z: 164 m/z: 207 m/z: 207 m/z: 493 m/z: 493 m/z: 57 Scheme 8. Fragmentation behaviour of NZB520. DIs, daughter ions. 3.3.1. Identification of the NZB520 cyano adduct
The NZB520 PI peak eluted at 47.66 min (figure 6a). 3.3.2. Identification of the NZB506 cyano adduct The NZB506 PI peak eluted at 48.95 min (figure 7a). The fragmentation of the PI at m/z 506 generated four
DIs at m/z 287, m/z 220, m/z 120 and m/z 98 (figure 7b). The DI at m/z 287 indicated the absence of any
metabolic reaction on the isonicotinamide and benzimidazole groups. The DIs at m/z 220 and m/z 98 m/z: 520
HLMs
KCN
KCN
demethylation
hydroxylation
dehydration
iminium ion
intermediates
NZB520
m/z: 506
NZB506
nazartinib
azepane
butenone
tertiary
nitrogen
BIOACTIVATED
CARBON
Scheme 10. Proposed pathway of bioactivation during NZB metabolism by human liver microsomes and the cyanide trapping
strategy. 12 royalsocietypublishing.org/journal/rsos
R. Soc. open sci. 6: 190852
12 dehydration Scheme 10. Proposed pathway of bioactivation during NZB metabolism by human liver microsomes and the cyanide trapping
strategy. confirmed the addition of a cyanide ion on the activated carbon α of the terminal tertiary N atom
(dimethyl amine) and N-demethylation of the dimethyl amine group (scheme 9). 3.4. Bioactivation mechanism of NZB The characterization of the NZB506 and NZB520 cyano adducts revealed the generation of reactive
iminium intermediates in NZB metabolism. The hydroxylation of the bioactivated carbon in NZB
followed by dehydration resulted in the generation of reactive iminium electrophiles that were
captured by a cyanide nucleophile to form a stable cyano adduct (scheme 10). The bioactivation
pathway for the formation of reactive intermediates has been previously studied using drugs
containing cyclic tertiary amines. However, herein, the reactive intermediates were generated by
bioactivation of an aliphatic noncyclic carbon attached to a tertiary amine rather than by azepane
bioactivation [39–44]. References 1. Siegel RL, Miller KD, Jemal A. 2016 Cancer
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mutation for lung cancer. Transl. 4. Conclusion The current study provided experimental evidence to support further work on NZB toxicity. Six in vitro
NZB phase I metabolites and two cyano adducts were identified (figure 8) and bioactivation mechanisms hydroxylation
or oxidation
N-demethylation
cyano
addition
hydroxylation
or oxidation
oxidation
nazartinib
Figure 8. Chemical structure of NZB showing the sites of phase I metabolic reactions responsible for the generation of the detected
metabolites. The main bioactive centre is indicated by an asterisk. royalsocietypublishing.org/journal/rsos
R. 13 13 hydroxylation
or oxidation N-demethylation hydroxylation
or oxidation Figure 8. Chemical structure of NZB showing the sites of phase I metabolic reactions responsible for the generation of the detected
metabolites. The main bioactive centre is indicated by an asterisk. were proposed. The knowledge on bioactivation mechanisms is crucial for determining the chemical
groups involved in bioactivation. This information may be used for the development of new
molecules containing small sterically hindering groups, isosteric replacement or a spacer to prevent
NZB bioactivation; inhibiting the generation of reactive species in this way would result in reduced
toxicity. The data obtained in this study will contribute towards the development of new drugs with
enhanced safety profiles. Ethics. The study’s design (in vitro assays using commercially available liver microsomes) exempts it from the approval
by Ethics Committees. Data accessibility. The data supporting the results in this article can be accessed at the Dryad Digital Repository: https://
doi.org/10.5061/dryad.j5m8h10 [45]. Data accessibility. The data supporting the results in this article can be accessed at the Dryad Digital Repository: https://
doi.org/10.5061/dryad.j5m8h10 [45]. Authors’ contributions. A.A.K. and A.S.A. designed and supervised the study. A.S.A., M.W.A. and A.A.K. performed the
optimization for the experimental steps and protocol. M.W.A. conducted the experiments and drafted the manuscript. All authors revised and approved the final version of the manuscript. All authors agreed with the submission of the
manuscript to Royal Society Open Science Journal. Authors’ contributions. A.A.K. and A.S.A. designed and supervised the study. A.S.A., M.W.A. and A.A.K. performed the
optimization for the experimental steps and protocol. M.W.A. conducted the experiments and drafted the manuscript. All authors revised and approved the final version of the manuscript. All authors agreed with the submission of the
manuscript to Royal Society Open Science Journal. Competing interests. The authors declare no competing interests. Funding. The authors thank the Deanship of Scientific Research at King Saud University for funding this work through
Research Group Project no. RG-1435-025. royalsocietypublishing.org/journal/rsos
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The financial burden from non-communicable diseases in low- and middle-income countries: a literature review
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REVIEW Open Access Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 © 2013 Kankeu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. The financial burden from non-communicable
diseases in low- and middle-income countries:
a literature review Hyacinthe Tchewonpi Kankeu1*, Priyanka Saksena2, Ke Xu3 and David B Evans4 Hyacinthe Tchewonpi Kankeu1*, Priyanka Saksena2, Ke Xu3 and David B Evans4 Abstract Non-communicable diseases (NCDs) were previously considered to only affect high-income countries. However,
they now account for a very large burden in terms of both mortality and morbidity in low- and middle-income
countries (LMICs), although little is known about the impact these diseases have on households in these countries. In this paper, we present a literature review on the costs imposed by NCDs on households in LMICs. We examine
both the costs of obtaining medical care and the costs associated with being unable to work, while discussing the
methodological issues of particular studies. The results suggest that NCDs pose a heavy financial burden on many
affected households; poor households are the most financially affected when they seek care. Medicines are usually
the largest component of costs and the use of originator brand medicines leads to higher than necessary expenses. In particular, in the treatment of diabetes, insulin – when required – represents an important source of spending for
patients and their families. These financial costs deter many people suffering from NCDs from seeking the care they
need. The limited health insurance coverage for NCDs is reflected in the low proportions of patients claiming
reimbursement and the low reimbursement rates in existing insurance schemes. The costs associated with lost
income-earning opportunities are also significant for many households. Therefore, NCDs impose a substantial
financial burden on many households, including the poor in low-income countries. The financial costs of obtaining
care also impose insurmountable barriers to access for some people, which illustrates the urgency of improving
financial risk protection in health in LMIC settings and ensuring that NCDs are taken into account in these systems. In this paper, we identify areas where further research is needed to have a better view of the costs incurred by
households because of NCDs; namely, the extension of the geographical scope, the inclusion of certain diseases
hitherto little studied, the introduction of a time dimension, and more comparisons with acute illnesses. Keywords: Financial burden, Low- and middle-income countries, Non-communicable diseases, Rev * Correspondence: hyacinthe.kankeu@inserm.fr
1Aix-Marseille University (Aix-Marseille School of Economics), CNRS & EHESS,
Centre de la Vieille Charité, 2 Rue de la Charité, 13236 Marseille, Cedex 2,
France
Full list of author information is available at the end of the article Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 the following key words: “Non-communicable disease”,
“chronic illness”, “diabetes”, “cardiovascular disease”,
“cancer”, and “chronic respiratory disease” with “cost”,
“impoverish”, “financial burden”, “health expenditure”,
“expense”, “out-of-pocket”, “health spending”, “catastro-
phic expenditure”, “catastrophic expense”, and “catas-
trophic spending”. A total of 8,966 results (including
duplicates) were obtained. After duplicate removal, titles
and abstracts of the remaining papers were reviewed to
assess their relevance according to the following inclu-
sion criteria: i) papers in English or French; ii) from
1990 onwards; iii) covering at least one low-, lower-
middle-
or
upper-middle-income
countrya
[11];
iv)
measuring the household or individual financial costs; v)
of one condition (or more) falling under the definition
of “chronic diseases” [12] or classified in “Group II dis-
eases” according to the ICD-10 code [8]. This screening
led to the selection 43 articles and a secondary literature NCDs in low- and middle-income settings as well [8]. Indeed, the impact is expected to differ because there is
little financial risk protection in many LMICs and thus
financial costs are largely borne by households them-
selves rather than governments or insurance schemes
[9]. The framework presented in Figure 1 describes the
channels through which NCDs can affect the economic
welfare of households. We conducted a literature review to present existing
evidence on the financial burden from NCDs in low-
and middle-income settings, at the individual and house-
hold level. The aim is to provide accurate and relevant
information on this important issue to policymakers,
and determine where further research is needed. Background exceed communicable diseases as the major cause of dis-
ease burden [1]. Even in the remaining countries where
infectious diseases are the main health problem, NCDs
are growing rapidly. NCDs are expected to exceed com-
municable, puerperal, prenatal and food diseases on the
list of leading causes of death in all countries by
2020. The increasing importance of NCDs has caused
them to no longer be viewed simply as a health issue
but rather as a development issue worthy of discus-
sion at a High-level Meeting of the 66th General As-
sembly of United Nations [2]. The 2010 WHO Global Status report on non-commu-
nicable diseases (NCDs) showed that they are now the
most important cause of mortality worldwide. Indeed,
more than 36 million people died from NCDs in 2008,
mainly cardiovascular diseases (48%), cancers (21%),
chronic respiratory diseases (12%), and diabetes (3%). Nearly 80% of these deaths occurred in low- and middle-
income countries (LMICs), where, on average, they now * Correspondence: hyacinthe.kankeu@inserm.fr
1Aix-Marseille University (Aix-Marseille School of Economics), CNRS & EHESS,
Centre de la Vieille Charité, 2 Rue de la Charité, 13236 Marseille, Cedex 2,
France Considerable literature exists on the impact of NCDs
on households in high-income countries [3-7]; resear-
chers are now beginning to examine the implications of Page 2 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Methods We performed a literature search with Cabdirect, Scien-
cedirect and Web of Knowledge, using combinations of Illness
experience
Treatment
seeking
behaviour
Economic
consequences
Coping strategies &
social resources
Reduced well-
being and
increased financial
vulnerability for
individuals &
households
Intra- & inter-
household labour
substitution
Hiring other labour
& other strategies
Use of savings
Reducing/delaying
consumption of non-
health goods &
services (food,
education, electricity,
leisure, etc.)
Sale of assets
Borrowing
Delaying
investments
Other strategies to
cope with financial
costs (assistance
from others, etc.)
Non-
communica
ble disease
Indirect
costs
Seek
care
No
Yes
Directs
costs
Loss of working time
of person who is ill &
caregivers
Loss of income of
person who is ill &
caregivers (due to
absenteeism, missing
business
appointments, etc.)
Financial costs of
health care
(consultation,
medicines, laboratory,
hospitalization, etc.)
Other financial costs
related to seeking
care (transportation,
special dietary
regimes, etc.)
Figure 1 Framework for the analysis of economic impacts of NCDs on households (modified from McIntyre et al. [10]). Loss of working time
of person who is ill &
caregivers Intra- & inter-
household labour
substitution Indirect
costs costs Hiring other labour
& other strategies Loss of income of
person who is ill &
caregivers (due to
absenteeism, missing
business
appointments, etc.) Directs
costs Framework for the analysis of economic impacts of NCDs on households (modified from McIntyre et al. [10]). Page 3 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Page 3 of 12 discussions and key informant interviews to comple-
ment their analyses [18,32,37-39]. search was performed using the references cited in these
selected papers. Finally, a total of 49 papers were identi-
fied, whose full-length versions were obtained for this re-
view. Each of these studies was examined for information
on disease(s), study population, analysis methods and
findings. These details are presented in Additional file 1:
Table S1. In the studies looking at NCDs in general, the term
“chronic diseases” is frequently used, and even if the
major NCDs are usually taken into account, the defini-
tions vary from one study to another. For example, Shi
et al. defined a chronic ailment as an ailment that lasts
or is expected to last for at least 12 months, resulting in
functional limitations or the need for ongoing medical
services, and includes disability [15]. In Kenya, Chuma
et al. Overview of the methods used in the literature The studies found in the literature reflect the diversity of
methods used to assess household financial burden from
NCDs. The methodological differences in the studies in-
herently prevent a formal meta-analysis from being
performed. However, at the same time, these differences
offer opportunities to explore results through the lens of
different techniques. In this section, we present a discus-
sion on the methodologies used. Some studies look at a specific NCD (e.g., diabetes,
cancers, cardiovascular diseases), while a majority con-
sider NCDs in general or a combination of two or more
specific NCDs. We found only one previous literature
review which included studies on multiple NCDs, but it
includes studies from only a few countries and did not
include any studies from Africa and Latin America [13]. Irrespective of the diseases considered, many studies
assessing the direct costs incurred by households for the
treatment of NCDs also focus on impoverishment and
catastrophic health expenditure due to these expenses. Impoverishment occurs when a respondent would have
had a net income above the poverty line in the absence
of the expenditure on the disease, but below it after. Dif-
ferent poverty lines are used across studies – US$ 1 per
day, US$ 1.08 per day, US$ 1.25 per day and US$ 2 per
day [28,35,39,41,42]. y
The original studies found also differed according to
data sources and sample sizes. Some authors conducted
their own surveys for the purpose of the studies, while
others used data from existing surveys carried out by an-
other entity (e.g., National Institute of Statistics, Ministry
of Health, Health Insurance Plans). In these surveys,
households and individuals were generally chosen ran-
domly, through simple, stratified or cluster sampling
[14-22]. However, many studies used convenient samples
of patients suffering from a specific illness in health care
facilities, something that we report when presenting the
results [23-31]. Additionally, studies looking at specific
diseases generally used relatively small samples, while
those considering a broad set of diseases usually relied
on bigger samples. For the assessment of diabetes costs,
for example, some studies selected a small number of
diabetic patients: 50 in North India, 53 in Cape Town
(South Africa) and 77 in Ghana [23,25,32]. Similarly, in a
study in Enugu (Nigeria), Obi and Ozumba used a sam-
ple of 95 patients suffering from cervical cancer [27]. Overview of the methods used in the literature On
the other hand, up to 206,700 individuals from 48,600
households were included in a study on chronic diseases
in Mexico [33]. In terms of internal validity of findings,
some studies used hospital registries or insurance reim-
bursement records to verify the information reported by
patients and/or their relatives during face-to-face in-
terviews [34-36]; a majority of studies, however, sim-
ply accepted the answers of the respondents as being
valid. Finally, some studies use data from focus group Catastrophic heath expenditure occurs when people
spend a disproportionate amount of their income (some-
times non-food expenditure) on the condition, as de-
scribed in Xu et al. [43]. However, a great variety of
specific definitions for catastrophic health expenditure
were used in the studies presented here. The thresholds
for determining a disproportionate level of expenditure
vary from 10% to 60%; some studies deviated from this
more standard approach. For example, Mukherjee et al. used the concept of “high health care expenditure” in-
stead of catastrophic health payments [44]. In this study,
a household was identified as having incurred high out-
of-pocket expenditure on health care if its annual health
care expenditure was high in comparison to those of other
households within the same caste group in India [44]. Methods defined chronic illnesses as those reported to have
lasted three months or more [38], while for Goudge
et al., any illness that had persisted for longer than a
month was defined as chronic [37]. Mondal et al. con-
sidered that a chronic illness is a condition that lasts
more than three weeks, which needs to be managed on
a long-term basis [40]. However, many of these studies
provide the list of diseases they considered as chronic,
and thus it was possible to know whether NCDs were
included along with some communicable diseases (for
example, HIV/AIDS). In these cases, we report results
related only to chronic NCDs. Nevertheless, in some
studies it was not possible to be sure that the focus was
limited to only chronic NCDs. Diabetes Diabetes is a leading NCD and 16 studies included in
this review looked at the direct costs incurred for both
outpatient and inpatient services. All studies, except
one, relied on convenience samples, so the results need
to be interpreted carefully. Overall, the studies found
that varying shares of household income are allocated to
paying for diabetes care. This ranges from as low as 5%
of income for a rural low-income population in India to
up to 24.5% for a low-income group in Madras (India)
[34,36,45]. Spending can also differ between richer and
poorer households and studies found that poorer house-
holds spend a higher proportion of their income on care
for diabetes than richer households. These differences
can be quite striking – one study from India found that
in urban areas, the share of income spent on diabetes
care in the poorest households was seven times that of
the richest households [45]. Spending on diabetes can
also be a considerable share of overall household health
spending. A study in Sudan reported that on average
65% of household health expenditure was spent on ca-
ring for a child with diabetes [46]. The presence of complications and the duration of the
illness are usually associated with an increase of the dir-
ect costs. For example, Khowaja et al. found that in
Pakistan, the direct cost for patients with co-morbidities
was 45% higher than the direct cost for patients without
co-morbidities [50]. Similarly, in India, those without
complications were found to have an 18% lower cost
compared to the mean annual cost for outpatient care
for all patients with diabetes, while those with three
or more complications had a 48% higher cost [51]. Similar results were found in India, China, Thailand
and Malaysia [34,36,45,48]. These studies also high-
light the fact that treatment at an early stage is much
cheaper for households than treatment at a later stage
with complications. Some studies looked at coping strategies used by
households to pay for these direct costs. In India, the
majority of patients (89%) used their household income
to fund the monitoring and treatment of their diabetes,
while household savings were used by 22% of retired pa-
tients and by 19% of those in the lowest income bracket. When faced with hospitalization, 56% of patients had to
dip into their savings or borrow in order to fund the
costs [51]. The evidence on the direct costs from non-communicable
illnesses Many of the studies assessed direct costs, which include
all costs incurred by individuals and households for the Page 4 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 treatment of NCDs. In theory, these costs should be net
of any reimbursement from insurance. We present evi-
dence on these direct costs organized by disease. versus 3% and 19%, respectively, if the lowest priced
generic product was purchased [41]. Laboratory and
transportation costs were generally the second largest
component of expenditure. Some studies also document
expenditure related to special dietary regimes (up to 20%
of the direct costs in North India [23]). Diabetes Additionally, very few households are reim-
bursed by insurance. In India, Kapur found that only 1%
of patients claimed the costs of treatment on insurance
[51], while Ramachandran et al. observed that medical
reimbursement was obtained by 14.2% of urban pa-
tients but by only 3.2% of rural patients [45]. More-
over, Khowaja et al. found that in Pakistan, none of the
persons with diabetes indicated that their cost was borne
by an insurance company or their employer [50]. Medications are frequently found to be the largest
component of expenditure on diabetes [47]. Spending
on medications represented from 32% to 62% of total ex-
penditure on diabetes care in various setting such as
India, Mexico, Pakistan and Sudan (Table 1). In rural
Ghana, spending on insulin alone represents around
60% of the monthly income of those on the minimum
daily wage [32]. Using originator-brand medication re-
sulted in much higher spending in the only diabetes
study that used random sampling rather than conveni-
ence samples. This study found that in Yemen and Mali,
purchasing an originator brand medicine for gliben-
clamide (a medicine used to treat type II diabetes) in the
private sector was found to potentially impoverish an
additional 22% and 29% of the population, respectively, Cardiovascular diseases Five studies examined spending on cardiovascular dis-
eases. In a study using data from a household survey in
Kazakhstan, people with cardiac problems were found to
pay on average 24% more for health care than people
with other health problems [22]. As with diabetes, stud-
ies from Congo and Uganda also found that the use of
originator brand drugs increases spending on cardiovas-
cular diseases [24,41]. Once again, there was only one
cardiovascular disease study that did not use a conveni-
ence sample [41]. Table 1 Shares of diabetes expenditure spent on
medications
Authors*
Countries
Spending on medicines
as a percentage of
total expenditure on
diabetes (%)
Rayappa et al. [48]
India
32
Elrayah et al. [46]
Sudan
36 (only insulin)
Villarreal-Ríos et al. [49]
Mexico
37
Khowaja et al. [50]
Pakistan
46
Grover et al. [23]
India
62
*All these papers are based on convenience samples. Table 1 Shares of diabetes expenditure spent on
medications Out-of-pocket payments for the treatment of cardio-
vascular diseases also lead to significant costs for house-
holds. Up to 71% of patients who had experienced
an acute stroke were found to face catastrophic health *All these papers are based on convenience samples. Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Page 5 of 12 expenditureb in China, while 37% of them fell below the
poverty line of US$ 1 per day after paying for their
health care [35]. The study of Heeley et al. also found
that catastrophic payments and impoverishment due to
cardiovascular diseases are more common in people
with no health insurance than in those with health insu-
rance [35]. [54]. Using a focus group, Russell and Gilson document
the case of an individual suffering from asthma, who in-
curred a direct cost representing 15% of his monthly
wage when seeking care for a sore chest in a private
clinic and pharmacy [39]. Multiple laboratory tests and
the presence of complications were also found to cause
high expenses for a convenience sample of patients suf-
fering from cirrhosis in Brazzaville (Congo) [26]. In a study covering 35 states and union territories in
India, Rao et al. Non-communicable diseases combined Non-communicable diseases combined
We found a large number of studies – all based on ran-
domized household surveys – looking at NCDs in gen-
eral, instead of focusing on specific illnesses. Some
studies highlight the association of having a household
member suffering from a chronic disease with a signifi-
cant increase in health care expenditure and a higher
risk of impoverishment. In Russia, for example, each
additional case of chronic disease in a household was
found to increase the probability of incurring health care
expenditure by 8% and the amount of healthcare ex-
penditure by 6.2% [19]. Similarly, in Uganda, households
with a member suffering from a chronic illness were
found to be three times more likely to incur costs for
health care than other households [18]. In Kazakhstan,
people with chronic illness were found to pay on average
18% more than people with other health problems, while
in Georgia, the mean cost for outpatient care in case of
chronic illness was almost two times higher than in case
of acute illness [21,22]. On the other hand, a study from
India found that the relative importance of chronic dis-
eases for spending may be lower – the mean annual per
capita health expenditure for a chronic episode was 11%
lower than for an acute one [44]. Two studies using convenience samples also shed
some light on components of spending on cancer care. Indeed, Zhou et al. found that health insurance facili-
tates the financial access of treatment for patients suffer-
ing from oesophageal cancer in China, particularly for
purchasing drugs [31]. Meanwhile, transportation, mul-
tiple
investigations,
radiotherapy
and
chemotherapy
were the main components of direct costs for cervical
cancer in Nigeria [27]. Cardiovascular diseases investigated the coping strategies used
by households to deal with expenses incurred for hospi-
talizations due to cardiovascular diseases [52]; 57% of
these expenses were paid from household savings, 35%
from borrowings, and 8% from the sale of assets. In the
poorest group, up to 55% of out-of-pocket spending was
financed through borrowings, and only 38% through
savings [52]. Coping strategies used to pay for care associated with
these NCDs are similar to those used to cope with more
documented NCDs. In Pakistan for example, Mahmood
and Ali Mubashir using a random sample found that
22.9% of patients with circulatory diseases (heart dis-
eases, rheumatic fever and blood pressure) who visited
private
doctors/clinics
for
treatment
financed
care
through unsecured loans, while 8.8% relied on assistance
from others [53]. Among those who did not visit any fa-
cility, 67.4% reported financial constraints as the reason
for not seeking care. Cancer Cancers also represent an emerging health problem in
LMICs and seeking health care for these diseases can
have a significant effect on families’ welfare. We found
three papers which focus specifically on the direct cost
from cancers. In a study using data from a randomized
household survey in Pakistan, 27.1% of those who sought
care for cancer at private facilities were found to finance
their care through unsecured loans, while 7.1% relied on
assistance from others [53]. Other non-communicable diseases **For households which had catastrophic health care expenditure. **
health expenditure between 30% and 40% of their capacity to pay. As with diabetes, when households are covered by
health insurance, the reimbursement rates for chronic
diseases are relatively low. In Shandong and Ningxia in
China, for example, only 11.16% and 8.67%, respectively,
of overall medical expenditure for chronic diseases was
reimbursed by the NCMS [16]. However, another study
from Western China found that health insurance pro-
vided protection against impoverishment due to ex-
penses for chronic diseases [42]. Government subsidies
for medicines were also found to lower the expenses for
many chronic diseases in Vietnam [29]. health care expenditure in urban areas and 5.73% in
rural areas of West Bengal in India; however, it was up
to 11% in Vietnam and 32% in Maharashtra, Bihar and
Tamil Nadu states of India, with a higher share for
hospitalization and drugs [20,40,55]. All these studies
used a random sample. Another proxy of households’
capacity to pay used in the literature is their non-food
expenditure. Sun et al. found that in China, the average
proportion of chronic disease expenditure to annual
non-food expenditure was about 27% in Shandong
Province and 35% in Ningxia province for patients
covered by New Cooperative Medical Scheme (NCMS), a
public health insurance scheme for rural residents [16]. For non-NCMS members, these proportions were 47%
and 42%, respectively. Coping strategies documented in the literature com-
bining chronic diseases are similar to those described in
the studies on specific NCDs. In Georgia, when house-
holds were lacking financial means, the most dominant
strategy was to borrow from a friend or relative (70%),
followed by selling household valuables (10%) and/or
household goods/products (10%) [21]. In several studies, the presence of household members
with chronic ailments was also found to lead to cata-
strophic health expenditure and impoverishment. The
probability of catastrophic expenditure was then 4.4
times higher among households having incurred ex-
penses for treating chronically ill persons in Georgia,
and up to 7.8 times higher in Burkina-Faso [17,56]. Si-
milar results were found in West Bengal (India), in
Lebanon and in China [15,40,57,58]. Up to 11.6% of
households in Western and Central China were pushed
under the US$ 1.08 poverty line after incurring out-
patient expenses associated with chronic diseases [42]. Moreover, Shi et al. Other non-communicable diseases found the incidence of medical im-
poverishment to reach 19.6% in households where more
than 50% of members had a chronic illness [16]. Other non-communicable diseases The financial burden from other NCDs, such as epi-
lepsy, cirrhosis, chronic obstructive pulmonary disease
(COPD), rhinitis and depressive disorders, is also esti-
mated in some studies. Even if they are not as studied
as the major NCDs presented previously, these types of
illnesses can also exert a considerable pressure on
household finances. For example, a study from Mumbai
(India) based on a random sample of households found
that the share of the annual personal income spent on
outpatient care for allergic rhinitis was 1.7% when treat-
ment was sought in public facilities. Similarly, care for
COPD represented 13.3% of annual personal income
among those using private facilities. With hospitalization
at public facilities, out-of-pocket payments for COPD
represented up to 62.3% of the annual personal income
compared to 50.7% for hospitalization in private facilities Undeniably, expenses incurred when seeking health
care for chronic diseases represent an important finan-
cial burden for households as presented in Table 2. In
fact, the costs of health care for chronic illnesses were
found to represent from 5.0% of household income in
rural Kenya to up to 30–50% of monthly income for vul-
nerable households in South Africa, where care for these
illnesses were unaffordable without gifts from social net-
works [37,38]. Similarly, household spending on chronic
illness represented 4.14% of household’s total annual Page 6 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Table 2 Expenditure on chronic diseases
Authors#
Countries
Spending on chronic
illnesses as a percentage
of household income (%)
Spending on chronic
illnesses as a percentage
of household total health
expenditure (%)
Spending on chronic
illnesses as a percentage
of household non-food
expenditure (%)
Chuma et al. [38]
Kenya (rural)
Urban: 5.7
Rural: 5
Goudge et al. [37]
South Africa (Vulnerable households)
30–50
Mondal et al. [40]
India (West Bengal)
Urban: 4.14
Rural: 5.73
Thuan et al. [20]
Vietnam
27.7* (curative)
11.1**
58.6***
Dror et al. [55]
India (Maharashtra, Bihar and
Tamil Nadu states )
32
Sun et al. [16]
China (Shandong province)
NCMS: 27
Non-NCMS: 47
Sun et al. [16]
China (Ningxia province)
NCMS: 35
Non-NCMS: 42
#All these studies used randomized samples. *For all households. **For households which had catastrophic health care expenditure. ***For households having
health expenditure between 30% and 40% of their capacity to pay. Table 2 Expenditure on chronic diseases #All these studies used randomized samples. *For all households. Other forms of indirect costs
h
f
f
d Some other forms of indirect costs due to NCDs were
found in the literature; these generally concern house-
holds’ livelihood and welfare. The study on cervical
cancer in Buenos Aires (Argentina) by Arrossi et al. exa-
mined these and also found that due to a loss of income,
there were delays in payments for essential services such
as telephone or electricity and as a result 43% of house-
holds had the service cut [28]. There were also significant effects on self-reported
daily food consumption, which was reduced in 37% of
households, while 38% of households reported that they
sold property or used savings to offset income loss. Some impacts on education were found and school ab-
sences were more prevalent in 28% of households. There
were also problems to pay for education in 23% of
households. Furthermore, 45% of patients were cared for
by one or more informal caregivers that did not live with
them and one-third of these caregivers’ households re-
duced their daily consumption of food and 26% had de-
lays in payments of essential services such as electricity
or telephone services. It should be noted that these are
the types of welfare losses have shaped the concept of
catastrophic health expenditure. There were also direct impacts on employment and at
least one member stopped working in 28% of house-
holds affected by cervical cancer. Several interviewees
who stopped working expressed the hope of going back
to their jobs after treatment, fearing at the same time
that this would no longer be possible. Similarly, a study
from Bangalore (India) by Rayappa et al. found that only
33.4% of diabetes patients worked and among those
working, 23% experienced problems at their job, affec-
ting their productivity and at times requiring changing
work to a less strenuous job (5.9%) or giving up the job Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 vary from 2.8 ± 1.7 hours per visit for diabetes in
Pakistan to 58 ± 105 days per year for epilepsy in India
[30,50]. Episodes of respiratory diseases can also cause
important losses of working time as shown in a case
study in Colombo (Sri Lanka) where Russell and Gilson
found a patient suffering from asthma took two days
off work for a sore chest, losing 6% of his monthly
wage [39]. [59]. Nonetheless, in this section, we present the avail-
able evidence on the indirect costs of NCDs as reported
in the literature. This constitutes findings from 11 stu-
dies, which mainly use convenience samples, on loss of
income, loss of time and other forms of financial loss re-
lated to these illnesses. We discuss possible limitations
of these findings in the discussion section. Loss of income
I
I di However, time costs are not limited to patients, but
also affect caregivers. In Buenos Aires (Argentina) for
example, Arrossi et al. found that in 45% of households
with a member suffering from cervical cancer, at least
one member reduced his/her working hours [28]. For
diabetes patients in Thailand, caregivers were found to
spend on average 42.21 ±39.94 hours per month on health
care activities – e.g., giving medicines – and 21.87 ± 31.81
hours on activities of daily living – e.g., helping with eating
and dressing [61]. In India, one study suggests that the indirect cost for
diabetes patients and their caregivers was 28.76% of the
total treatment cost. It was claimed that loss of income
of the patient comprised the greatest portion of indirect
costs (60.54%), followed by loss of income of caregivers
(39.46%) [23]. Rayappa et al. found that in Bangalore
(India), 30.9% of respondents suffering from diabetes
reported a change in personal income, and on average,
they faced a reduction of 20.9% of their personal income
[48]. In addition, 20.8% of the respondents reported
a change in family income, with a mean reduction of
17.4%. Similarly, Arrossi et al. found that in Argentina,
39% of households with a member suffering from cer-
vical cancer lost family income, partially or totally [28]. Among households that lost income, 47% lost less than
25% of family income, 34% lost 25–50% and 19% lost
50% or more of their income. As a result of the reported
loss of income, it was estimated that the proportion of
patient’s households living in poverty increased from
45% to 53%. Likewise, Obi and Ozumba found that in
Nigeria, all patients suffering from cervical cancer and
their relatives lost income from workplaces due to absen-
teeism, disengagement from work and missing business
appointments [27]. In a study covering 19 countries, one
of the two studies using randomized household survey
data documenting indirect costs, Levinson et al. found
that serious mental illness was associated with a potential
reduction in earnings of 10.9% of average national earn-
ings in LMICs [60]. The second study using randomized
household survey data was from Russia and found that
labour income decreased by 4.8% per additional case of
chronic disease in the household [19]. Some studies only
estimate the NCDs-related indirect costs for patients and
their families in absolute value (local currencies or US$)
[50,51,61]. Literature on the indirect costs due to non-communicable
diseases in low- and middle-income countries Households and individuals also bear indirect costs
when they are affected by NCDs. These costs mainly in-
clude time and productivity loss by patients and care-
givers because of the illness as well as income lost by
patients and family members. Whereas there is no doubt
that these indirect costs can pose a substantial burden
on households, there are numerous methodological chal-
lenges in measuring this burden adequately; these chal-
lenges have been discussed in detail in a previous study Page 7 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Page 7 of 12 Discussion This literature review has presented the available evi-
dence on the household financial burden related to
NCDs in LMICs. However, before discussing its most
important results, it is important to highlight some of
the methodological issues in many of the studies that
were included. First, the heavy reliance on convenience
samples
taken
from
people
who
are
seeking
and
obtaining treatment, often at hospitals, will almost cer-
tainly result in an upward bias in costs for the average
person with the condition. The people who do not seek
treatment or who seek treatment at a lower level of care,
implying lower costs, have no chance of being selected. Again not surprisingly, complications related to the se-
verity of illness were found to increase the household fi-
nancial burden, both for the patient and for caregivers. Health promotion, prevention and early treatment would
reduce some of these costs although each country would
need to choose the appropriate mix of prevention and
treatment according to their relative costs and impact. We also found strong evidence that costs could be re-
duced by more rational use of medications for NCDs. The costs of medication for all the different types of
NCDs considered here accounted for the highest pro-
portion of the direct costs; where addressed, originator
brand medicines were frequently used instead of avail-
able generics and costs were then substantially higher
than they needed to be. While many LMICs already have
strategies to promote the rational use of medicines, there
is still some way to go particularly in promoting the use
of lower cost generics. Second, self-reported costs, even from random sam-
ples of patients, are likely to be biased upwards when
there are no controls. Some of the people with the con-
dition would have incurred some health expenses in any
case and this can only be captured by including controls
without the condition [59,62]. In other words, it is likely
that part of the costs reported by patients with NCDs
were not directly associated with those conditions. The weakness or non-existence of mechanisms to pro-
tect households financially from the burden of NCDs is,
however, probably the most important finding in this
study. In the studies that considered insurance and pro-
vided information on reimbursement rates, NCD-related
treatment is generally uncommon and frequently pa-
tients and their relatives do not report that they claimed
any reimbursement from insurance or employers. Discussion Like-
wise, none of the studies we reviewed reported a system
of social security that provides compensation for loss of
income incurred by patients and their families because
of NCDs. Poor households are more likely to suffer dis-
proportionally from the financial effects of this lack of
social protection. To meet the costs, households re-
ported taking unsecure loans, using savings or selling
household assets, all of which can lead to longer-term
problems for the household. For example, the wider lit-
erature suggests that many of the loans taken by house-
holds for health expenses are at very high interest rates
that can take generations to repay [64]. This is part of a
bigger problem in LMICs, many of which rely exten-
sively on direct out-of-pocket payments to fund health
services. Recently, many have recognized the need to
modify the way they raise funds and more generally to
modify their health financing systems so as to improve
financial risk protection and ensure greater access to
needed health services [65]; it is important to note that This issue is particularly important when considering
indirect costs. It is clear that the method of asking
people how many days they could not work overesti-
mates the true loss in work time from a disease because
many of the people, particularly in low-income coun-
tries, would not have been working on those days, or for
all of those days, in the absence of the disease [59]. Nor
do the studies consider whether absent workers are re-
placed by other family members in family enterprises or
farms. For example, frequently other family members fill
in for a sick person during the planting season in agri-
culture so that the same area of land is planted despite
the illness [63]. This does not, of course, mean that there
are no opportunity costs associated with the illness, but
that the measured production from the family enterprise
is not altered as much. In general, therefore, we expect
that the costs from studies with no controls to be over-
estimates of both direct and indirect costs. The substantial variations in study designs and defini-
tions described earlier also make comparisons tricky and
meta-analysis infeasible. There is considerable hetero-
geneity in objectives and the methodologies used in the
papers. Loss of working time The loss of income borne by patients suffering from
NCDs is mainly due to self-reported absenteeism from
usual economic activity. In fact, the treatment of NCDs
usually requires repetitive visits to health facilities in
addition to the inability to work due to their poor health. This can lead to additional losses of working time both
for patients and caregivers. In the literature, the mean
loss of working time reported by patients was found to Page 8 of 12 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 (14.7%) [48]. Considering NCDs in general, Abegunde
and Stanciole found that in Russia, chronic illnesses,
which included NCDs, impose a reduction of 5% in
household
consumption
of
non-health-related
items
[19]. the methodological issues highlighted here and in earlier
sections, we can still conclude that NCDs already im-
pose substantial financial costs on some of their sufferers
in lower-income countries. As a result, the cost of
obtaining treatment for NCDs is also becoming a cause
of impoverishment and financial catastrophe in these
countries. While this is not particularly surprising given
the growing burden of disease associated with these con-
ditions, it has not been documented before. Conclusions
Th li The literature on the social, financial and economic con-
sequences of NCDs in developing countries has not kept
pace with the epidemiological evidence. It has been
known for some time that the burden of disease associ-
ated with NCDs and injuries is already higher than that
associated with the health conditions included in the
Millennium Development Goals (HIV/AIDS, tubercu-
losis, malaria, and maternal, child and reproductive
health), even in developing countries. Moreover, it has
been well documented that the share of NCDs in the
overall disease burden will continue to increase globally. Indeed, the UNs’ 2011 conference on NCDs stressed the
importance of these diseases as a development issue. The literature we reviewed sheds some light on the fi-
nancial consequences of NCDs on households in LMICs. Nonetheless, there are limitations to generalization of
these findings due to methodological challenges. Valid
estimates of the average costs of NCDs will require ran-
dom samples with controls to account for people who
have costly and less costly treatments, and what would
have happened in the absence of the diseases. Panel data
would be ideal although these studies are more expen-
sive than cross-sectional designs. However, importantly,
this review suggests that it is equally as important to
focus on people who could not seek care for NCDs due
to financial reasons. Little is known about the subse-
quent development of disease, impacts on these people’s
health and the financial, social and other consequences
associated with foregone treatment. Through this review, we are also able to identify areas
where further research is needed. Among the four major
NCDs, the financial costs from chronic respiratory dis-
eases are very poorly documented, although they cause
four times more deaths than for example diabetes, which
has been researched more [1,78]. According to the
WHO, almost 90% of COPD deaths occur in LMICs and
the highest prevalence of smoking – the primary cause
of COPD – among men is in these countries [1,78,79]. It
would therefore be interesting to have more assessments
of the financial costs of these diseases in future studies. Additionally, while all studies reviewed here used cross-
sectional data, panel data will be very useful in assessing
the evolution of costs incurred by households because of
NCDs. Conclusions
Th li The comparison of the relative importance of the
cost of NCDs with that of acute illnesses is also of a
great interest here, as according to the papers reviewed,
there is no clear trend. Indeed, some studies show that
NCDs are more costly for households, while others
observe the opposite. Sometimes in the same country,
different results are found depending on the area (urban
vs. rural), the type of health care (outpatient vs. in-
patient) and household socioeconomic status (poor vs. better-off) [17,21,38,39,44,55]. More studies – introdu-
cing for example a time dimension and a distinction be-
tween private and public providers – are therefore
needed to shed more light on this issue. It may also be The push to develop health-financing systems that im-
prove financial risk protection and help achieve universal
health coverage in LMICs is promising. However, poli-
cymakers need to ensure that the health as well as the fi-
nancial burden from NCDs is adequately addressed in
future reforms, while at the same time improve access
and financial protection for all other health services
needed by the population. Discussion While we have more confidence in the studies
relying on randomized samples, we present more details
about each study in file 1: Table S1 to give readers fur-
ther information and to allow them to consider possible
generalizations of the results. Taking into consideration Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Kankeu et al. Health Research Policy and Systems 2013, 11:31
http://www.health-policy-systems.com/content/11/1/31 Page 9 of 12 it will be increasingly necessary to include NCDs in
whatever type of financial risk protection strategy is de-
veloped. This is particularly important for poor families
because NCDs no longer affect only the more affluent
people in society [1,8,13,66,67]. important to expand the geographical outlook in future
research to be more representative of a wider group of
developing countries. This is true even after accounting
for the influence of the languages used in this review. Of
the 49 studies found, most were from Asia, as compared
to only a handful from Latin America or Eastern Europe,
and 10 studies from Africa. While we think that the financial costs reported in this
review will overestimate the costs of a typical patient
with NCDs, such that the numbers cannot be used to
extrapolate the costs of NCDs to a country, they high-
light the other consequences of the lack of financial risk
protection in LMICs. In the random sample studies,
many people with NCDs reported that they did not seek
care at all because of financial reasons (Additional file 2). Many of their conditions are likely to become more severe
in the absence of treatment, leading to early death and
greater problems for caregivers and households. The ef-
fects of not seeking care for poorer households is of par-
ticular concern given that the ability to work is one of the
most important poverty escape routes [68-72]. Strategies
to improve financial risk protection will also lead to in-
creased financial access to health services while demand
side responses, such as cash transfers, can help reduce
some of the financial barriers to seeking care, such as
transport costs. Nevertheless, demand-side approaches in
LMICs are, to our knowledge, limited largely to maternal
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non-communicable diseases in low- and middle-income countries: a
literature review. Health Research Policy and Systems 2013 11:31. 76. UNICEF: Child Poverty: A Role for Cash Transfers in West and Central Africa? Briefing Paper. Dakar: UNICEF West and Central Africa Regional Office; 2009. 77. England R: Experience of Contracting with the Private Sector: A Selective
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Cite this article as: Kankeu et al.: The financial burden from
non-communicable diseases in low- and middle-income countries: a
literature review. Health Research Policy and Systems 2013 11:31. doi:10.1186/1478-4505-11-31
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Evaluation of sparing the pronator quadratus for volar plating of distal radius fractures: a retrospective clinical study
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The First Affiliated Hospital of Xinjiang Medical University
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The First Affiliated Hospital of Xinjiang Medical University
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The First Affiliated Hospital of Xinjiang Medical University The First Affiliated Hospital of Xinjiang Medical University
Xiaoxia Huang Evaluation of sparing the pronator quadratus for volar plating of
distal radius fractures: a retrospective clinical study Yan Zhao
(
zhaoyan0325@163.com
) Yan Zhao
(
zhaoyan0325@163.com
) Background Fractures of the distal radius are common and account for approximately 17% of fractures[1]. They are among the most common
orthopedic injuries seen in the emergency room[2] .This observation is partly due to increased life expectancy, leading to more
osteoporotic fractures and an increasing variety of contact sports leading to high energy trauma in the younger population[3] . Volar locking plate fixation has become the standard surgical procedure for treating unstable distal radius fractures[4–6]. Johnson et
al[7] first reported the function of the pronator quadratus muscle to stabilize the distal ulnar radial joint in 1976. The most commonly
used approach in distal radius fracture surgery is the traditional Henry approach, which requires slicing open the PQ muscle. There is
great controversy regarding the significance of PQ muscle repair and whether the postoperative wrist function and outcomes are
affected. Previous studies showed that even in patients with repaired PQ muscle[8], there is still a significant loss of strength in pronation
after surgery, which may be related to tissue damage and edema or poor repair. Therefore, this study investigates the patient outcomes
following intraoperative PQ muscle preservation with a posteriorly-placed plate. Clinical data were collected from 76 patients with distal radius fractures who underwent open reduction and plate internal fixation. Patients were grouped based on whether the PQ muscle would be spared during the procedure or not. Intraoperative index, postoperative
efficacy, and complications of the patients were evaluated. This retrospective comparative study aimed to investigate the effect of
preserving the PQ muscle on wrist function in patients. Research Article Keywords: Sparing the pronator quadratus, Distal radius fracture, Henry approach, Volar plating
Posted Date: March 31st, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1479023/v1
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This work is licensed under a Creative Commons Attribution 4.0 International
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This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/11 Page 1/11 Abstract Background: The most commonly used approach for distal radius fractures is the traditional Henry approach. However, it requires an
intraoperative incision of the pronator quadratus (PQ) muscle, which results in a series of complications if the repair of the PQ fails. Aim: The objective of this study was to investigate the efficacy of sparing the pronator quadratus for volar plating of the distal radius
fractures. Methods: Seventy-six patients who suffered from distal radius fractures of types 23-B, 23-C1, and 23-C2 as per the AO Foundation and
Orthopaedic Trauma Association (AO/OTA) classification were treated with volar locking plate fixation using either the PQ muscle
incision and repair (group A, n = 39) or the PQ muscle preservation approach (group B, n = 37). Intraoperative index, postoperative
efficacy, and complications of the patients were evaluated. Results: All patients were followed up for more than one year after surgery. All fractures achieved union. There were significant
differences in mean operative time, mean intraoperative blood loss, and mean fracture healing time between the two groups. Still, there
were no significant differences in limb function scores between the two groups at the 12-month postoperative follow-up. Outcomes
assessed at 1 week, 1 month, and 3 months after surgery demonstrated significant differences in the mean range of motion and the
visual analog scale (VAS) between the two groups. As the range of motion and grip strength increased, the VAS scores decreased, and
there was no significant difference between the two groups at 12 months postoperatively. Although tendon irritation and delayed carpal
tunnel syndrome were more common in group A than in group B (7.6% vs. 0% and 5.1% vs. 0%, respectively), the differences were not
statistically significant. Conclusion: The modified Henry approach with sparing pronator quadratus muscle has no significant advantage in the range of wrist
motion and upper limb function in the late stage. Nevertheless, the intraoperative placement of the plate under the pronator quadratus
muscle can shorten the operation time, reduce intraoperative bleeding, reduce early postoperative pain, promote early activity, and
improve the patient's quality of life. It is recommended that the pronator be preserved at the time of surgery. Methods After the Institutional Review Board's written approval, a retrospective study was conducted between January 2019 and October 2020. 76
patients with distal radius fractures who underwent open reduction and plate internal fixation at the First Affiliated Hospital of Xinjiang
Medical University were enrolled in the study. The PQ muscle was cut open in group A, while group B spared the PQ muscle. The inclusion
criteria were: (1) age ≥ 18 years; (2) Unilateral displaced and unstable distal radius fractures; (3) Failure of manual reduction; and (4) AO
classification 23-B, 23-C1, and 23-C2. The following patients were excluded: (1) ipsilateral or contralateral upper limb fractures and/or
dislocation; (2) open fractures; (3) pathological fractures or metabolic bone disease; (4) Fractures greater than 3 weeks in duration; (5) Page 2/11 Page 2/11 associated nerve or vascular injury requiring repair; (6) Previous history of distal radius fracture on the affected side; (7) mental illness;
(8) Poor compliance and lost follow-up. The same medical team carried out all surgical procedures. The patient demographics and fracture characteristics are displayed in
Table 1. There was no significant difference in the pre-operative variables between the two groups. The same medical team carried out all surgical procedures. The patient demographics and fracture characteristics are displayed in
Table 1. There was no significant difference in the pre-operative variables between the two groups. Table 1
Baseline characteristics of the two groups. Group A (39)
Group B (37)
P
Mean age (years)
56(41, 64)
55(49, 58.5)
0.396
Sex
Male
17
12
0.317
Female
22
25
Side of hand
Left
19
17
0.809
Right
20
20
AO classification (n)
B1
9
9
0.871
B2
9
7
B3
5
8
C1
8
7
C2
8
6
Cause of injury (n)
Fall injury
23
21
0.871
Falling injury from height
13
14
Traffic trauma
3
2
Mean interval from injury to surgery (days)
4(3, 5)
4(3, 5)
0.947 Baseline characteristics of the two groups. Surgical procedures Patients were placed supine under general or regional anesthesia, and the surgery was performed. The Henry approach through the flexor
carpi radialis (FCR) tendon sheath was applied for the Group A patients. The tendon was traction to the radial edge of the incision. After
dissecting the FCR tendon sheath, the PQ muscle was exposed and an L-shaped incision was performed along the radial border of the
radius to the radial malleolus, and the PQ was then stripped off the radius. After the fragments were repositioned, fluoroscopic
confirmation was obtained and a plate was inserted for internal fixation. The PQ was sutured with 2 − 0 absorbable sutures. The repair
was abandoned if the PQ muscle was severely destroyed or edematous. For patients in Group B, if there was no primary injury to the PQ
muscle, the fracture was repositioned without dissecting the PQ muscle by modifying the traditional Henry approach. After completion of
the repositioning, a blunt separation between the PQ muscle and the periosteum was performed with a periosteal stripper to establish a
tunnel posterior to the PQ muscle, through which the plate was placed. When the fracture was well repositioned and the plate was well
positioned with X-ray fluoroscopy, the PQ muscle was bluntly separated with a mosquito clamp to reveal the screw holes and the screws
were placed (Fig. 1, 2). Postoperative management Following the surgery, all patients were on routine prophylactic antibiotics for 3 days and were not immobilized in plaster or brace. Patients were allowed to move their fingers immediately after surgery in both groups. Motion at the wrist joint was initiated 3 weeks po ollowing the surgery, all patients were on routine prophylactic antibiotics for 3 days and were not immobilized in plaster or brace. Following the surgery, all patients were on routine prophylactic antibiotics for 3 days and were not immobilized in plaster or brace. Patients were allowed to move their fingers immediately after surgery in both groups. Motion at the wrist joint was initiated 3 weeks post- were on routine prophylactic antibiotics for 3 days and were not immobilized in plaster or brace. Page 3/11 Page 3/11 surgery. At 6 weeks postoperatively, the patient's movement progressed to full active motion tolerated by each patient. Patients were
allowed to resume full activity and weight-bearing after radiographic confirmation of fracture healing. surgery. At 6 weeks postoperatively, the patient's movement progressed to full active motion tolerated by each patient. Patients were
allowed to resume full activity and weight-bearing after radiographic confirmation of fracture healing. The patient was observed post-operatively for internal fixation loosening and complications such as flexor tendon irritation, traumatic
arthritis, joint stiffness and carpal tunnel syndrome. The visual analog scale scores of postoperative wrist pain and the forearm rotation
angle between the two groups were compared. The Dienst score was used to assess the wrist function, and the imaging indexes (radial
height, volar tilt, ulnar inclination) were used to evaluate the efficacy of the surgery. Statistical analysis SPSS Statistics software version 26.0 was used for the statistical analysis. All data were analyzed using a normal distribution test. Mean ± SD was used to represent measurement data with normal distribution. The student's t-test was used to compare measurement
data of normal distribution. Conversely, a nonparametric Mann-Whitney U test was applied to compare two groups if the data did not
display normal distribution, with data expressed as median and interquartile range. The count variables were analyzed by the Chi-square
or Fisher's test, expressed as a number. P < 0.05 was considered statistically significant. No intraoperative vascular injury wound complications, postoperative re-displacement, tendon rupture, or hardware failure were observed
in either group. Moreover, there were no statistically significant differences in the postoperative imaging indexes (radial height, volar tilt,
ulnar inclination) compared between the two groups (Table 3). Three patients (7.6%) in the B group suffered tendon irritation (two were
not repaired due to severe PQ muscle damage and intraoperative repair difficulties), which was relieved after one year when the plate was
removed in stage II. None of the sparing PQ group patients developed flexor tendon irritation after surgery. Two patients (5.1%) in Group
B complained of delayed carpal tunnel syndrome. Results Seventy-six patients who underwent palmar plating for distal radius fractures were enrolled in the study, 39 in Group A and 37 in Group B. All patients were followed up for more than one year after surgery (Fig. 1, 2). There was a successful union of all fractures. Significant
differences were found between the two groups' mean operative time, mean intraoperative blood loss, and mean fracture healing time (P
= 0. 0, and 0.034, respectively). Still, there were no significant differences in limb function scores between the two groups at the 12-month
postoperative follow-up (P = 0.362) (Table 2). Table 2 Table 2 Table 2
Details of intra- and post-operative variables in the two groups. Group A (39)
Group B (37)
P
Mean operative time (min)
75(70, 80)
55(50, 60)
0.00
The mean operative blood loss(ml)
35(30, 45)
20(20, 25)
0.00
Mean bone union time (weeks)
12(11, 12)
11(11, 12)
0.034
Dienst score (12 months)
Excellent
23
25
0.362
Good
10
10
Fair
6
2 No intraoperative vascular injury wound complications, postoperative re-displacement, tendon rupture, or hardware failure were observed
in either group. Moreover, there were no statistically significant differences in the postoperative imaging indexes (radial height, volar tilt,
ulnar inclination) compared between the two groups (Table 3). Three patients (7.6%) in the B group suffered tendon irritation (two were
not repaired due to severe PQ muscle damage and intraoperative repair difficulties), which was relieved after one year when the plate was
removed in stage II. None of the sparing PQ group patients developed flexor tendon irritation after surgery. Two patients (5.1%) in Group
B complained of delayed carpal tunnel syndrome. Page 4/11 Table 3 Table 3
Postoperative imaging indexes
3 days
1 month
3 months
Group
A
B
P
A
B
P
A
B
P
Radial
height
11.36(11.30,
12.55)
12.14(11.43,
12.55)
0.451
11.80(11.31,
12.56)
12.20(11.45,
12.50)
0.673
11.80(11.20,
12.58)
12.10(11.40,
12.25)
0.480
Volar tilt
12.50(11.60,
13.20)
12.25(11.50,
12.65)
0.095
12.40(11.80,
13.30)
12.16(11.60,
12.65)
0.100
12.50(11.90,
13.30)
12.30(11.50,
12.64)
0.143
Ulnar
inclination
23.10(22.60,
23.60)
23.50(22.91,
23.70)
0.260
23.30(22.58,
23.60)
23.46(22.88,
23.65)
0.188
23.20(22.60,
23.56)
23.55(22.91,
23.60)
0.252
The range of motion measurements for each interval is shown in Fig. 4. Outcomes assessed at 1 week, 1 month, and 3 months after
surgery demonstrated significant differences in the mean range of motion and VAS scores between the two groups. The mean values for
all variables gradually improved over the year as the range of motion and grip increased and VAS scores decreased (Fig. 3, 4). There was
no significant difference between the two groups at 12 months postoperatively. Postoperative imaging indexes The range of motion measurements for each interval is shown in Fig. 4. Outcomes assessed at 1 week, 1 month, and 3 months after
surgery demonstrated significant differences in the mean range of motion and VAS scores between the two groups. The mean values for
all variables gradually improved over the year as the range of motion and grip increased and VAS scores decreased (Fig. 3, 4). Discussion There is controversy about whether to spare the PQ muscle intraoperatively in patients with distal radius fractures[9–13]. Even the repair
of the PQ muscle after fracture reduction remains a topic of debate in the current literature. Some surgeons believe that repairing the PQ
muscle only relieves the patient's postoperative pain at an early stage[14]. In addition, some surgeons consider that whether or not the PQ
muscle is repaired intraoperatively is not significantly related to the patient's postoperative functional recovery of the wrist[15, 16]. Despite these statements, 83% of American hand surgeons repair the PQ muscle following volar plate fixation[17]. However, sometimes
we are faced with the situation that the PQ muscle is hard to restore. We found that intraoperatively, the placement of the plate and the
suturing of the PQ muscle were often difficult due to muscle tissue lesion, bleeding, and edema following incision of the PQ muscle. A
clinical case observation study by Swigart et al. revealed that the failure rate of PQ muscle repair was 4%, with 1 in 24 patients
failing[18]. It is now being proposed that maximum preservation of the PQ muscle can decrease intraoperative bleeding to enhance the
repairable anterior rotator muscle[19, 20]. At the same time, some scholars recommend sparing the PQ muscle[21, 22] or completely
repairing the PQ muscle at the end of surgery[22]. However, it is generally difficult to repair the PQ muscle at the radial margin of the
radius because the muscle fascia is not strong enough to hold the sutures in place. Many recent studies have demonstrated that
complete sparing of the PQ muscle relieved postoperative pain, improved rotational function, and facilitated the early functional recovery
of the patient's wrist[23–26]. Similarly, this study confirmed that among 76 patients with unstable distal radius fractures, the 37 cases
undergoing PQ muscle-sparing surgery demonstrated shorter operative time, less bleeding, fewer complications, less postoperative pain,
and early functional wrist exercise, which facilitated the early functional recovery of the affected limb. The use of volar locking plates for distal radius fractures has become increasingly popular in the last few years[27, 28]. Several studies
have reported that patients achieve better functional scores and fewer complications postoperatively compared to other surgical
techniques[29–31]. Despite the benefits of volar plating, complications are common, especially in the flexor tendons of the fingers and
thumb (tenosynovitis, adhesions, ruptures) due to mechanical irritation between the volar plating and the tendons[32–34]. Table 2 There was
no significant difference between the two groups at 12 months postoperatively. Discussion This technique
can diminish these complications since the PQ muscle protects the flexor tendon and allows space for tendon gliding. Jung et al.[35]measured the anatomy of the cadaveric specimen with CT imaging and concluded that the distal fracture of the radius
was fixed using a small incision approach. The distal fracture fragment was large enough to place the distal row of screws without
opening the PQ muscle, and the plate was appropriately placed. The PQ muscle covering the implant's surface prevents risks such as
tendon abrasion, which indicates that the role of the PQ muscle in sheathing the implant cannot be ignored. Carpal tunnel syndrome is a severe complication after surgical treatment of distal radius fractures[36–38]. Kashir et al.[39] first described
an approach for splitting the brachioradialis muscle. By following this approach, they concluded that the integrity of the PQ was
preserved, and all distal radius fractures requiring a volar plating could be treated without further incision. They reported no nerve injury
or subluxation. However, it was found that this approach involves the radial artery during surgery, and the latter is highly susceptible to
damage. In the current literature[40, 41], the incisions used in the MIPO technology for distal radius fractures are adjacent to the median
nerve throughout the operation, and unfamiliarity with the procedure, repeated pulling of the incision, and placement of screws during the Page 5/11 Page 5/11 surgery may easily irritate the median nerve and cause nerve injury. Therefore, it was believed that adequate surgical field exposure is a
prerequisite to avoid complications. surgery may easily irritate the median nerve and cause nerve injury. Therefore, it was believed that adequate surgical field exposure is a
prerequisite to avoid complications. The typical anatomical structure of the PQ muscle is the basis of its function[42]. It was found that the PQ muscles were heavily scarred,
significantly atrophied, and adhered to the surrounding tissue after suturing. Muscle function is mainly impaired due to the distortion of
its anatomy. This impairment is probably due to the disruption of the blood supply from the radial artery to the muscle after the latter has
been severed. Hence, affecting the healing of the muscles. Discussion Therefore, it is recommended to avoid excessive forearm rotation for 3 weeks after surgery and wait for the scar repair
of the PQ muscle to stabilize before active exercise, which is one of the reasons for the poor recovery of early forearm rotation. It was previously believed that the PQ muscle was less critical, and that the anterior rotator function was limited; however, this is not the
case. Jesper Sonntag et al[43] concluded in a study that with or without repair of the PQ muscle after incision, ultrasound results showed
that both were shorter than the healthy side. Still, the shortening was more significant in the unrepaired group. The function of the PQ
muscle depends on its normal structure, and it is believed that incision of the PQ muscle with or without repair produces scarring that
further affects forearm rotation function. Early forearm rotation is likely to cause a re-tear of the PQ muscle, leading to poor forearm
function recovery. Therefore, it is recommended to avoid excessive forearm rotation for 3 weeks after surgery and wait for the scar repair
of the PQ muscle to stabilize before active exercise, which is one of the reasons for the poor recovery of early forearm rotation. Nevertheless, it does not mean that the forearm rotation function is significantly limited at the later stage when the PQ muscle is
dissected intraoperatively. This study showed no significant difference in the ROM of forearm rotation between the two groups at 12
months postoperatively. Hence, the sparing of the PQ muscle intraoperatively did not demonstrate a significant advantage in the long-
term forearm rotation function. However, performing PQ muscle preservation is strongly recommended as it decreases the risk of muscle
re-tear in rehabilitation, dramatically reduces the patient's pain, and encourages exercise. Nevertheless, it does not mean that the forearm rotation function is significantly limited at the later stage when the PQ muscle is
dissected intraoperatively. This study showed no significant difference in the ROM of forearm rotation between the two groups at 12
months postoperatively. Hence, the sparing of the PQ muscle intraoperatively did not demonstrate a significant advantage in the long-
term forearm rotation function. However, performing PQ muscle preservation is strongly recommended as it decreases the risk of muscle
re-tear in rehabilitation, dramatically reduces the patient's pain, and encourages exercise. Discussion Moreover, the PQ muscle is also a vital blood supply to the periosteum, with its provision from the anterior interosseous artery and the PQ
muscle branches of the radial and ulnar arteries, as well as the posterior interosseous artery playing a crucial role in the healing of distal
radius fractures. In this study, the mean bone healing time in the sparing PQ group was 11 weeks, lower than the 12 weeks in the
conventional incision group. In addition, PQ muscle is a brittle piece of muscle, which is challenging to suture and often tears apart after
suturing. The local soft tissue tension increases significantly, especially after the insertion of the plate, making it difficult to pull the
muscle together. Many studies have been reported on the treatment of distal radius fractures with various sparing of the PQ muscle, most of which
analyzed the patients' near future postoperative functional recovery of the wrist. However, there are fewer studies on the long-term
forearm rotation angles. In this study, we compared the long-term postoperative forearm and wrist function between two groups of
patients with or without complete preservation of the anterior rotator muscle, and also focused on the postoperative pain scores of the
patients. Satisfactory fracture reduction and good wrist function can be obtained with the palmar approach no matter whether the PQ
muscle is preserved or not. Improved patient outcomes are observed when the respective surgical indications are mastered, and the
articular surface reduction is adequate. However, preserving the PQ muscle to rehabilitate the forearm rotation is more advantageous in
the short term. It was previously believed that the PQ muscle was less critical, and that the anterior rotator function was limited; however, this is not the
case. Jesper Sonntag et al[43] concluded in a study that with or without repair of the PQ muscle after incision, ultrasound results showed
that both were shorter than the healthy side. Still, the shortening was more significant in the unrepaired group. The function of the PQ
muscle depends on its normal structure, and it is believed that incision of the PQ muscle with or without repair produces scarring that
further affects forearm rotation function. Early forearm rotation is likely to cause a re-tear of the PQ muscle, leading to poor forearm
function recovery. Conclusion The modified Henry approach with sparing pronator quadratus muscle has no significant advantage in the range of wrist motion and
upper limb function in the long term. However, the intraoperative placement of the plate under the pronator quadratus muscle can shorten
the operation time, reduce intraoperative bleeding, may reduce early postoperative pain, promote early activity, and improve patients'
quality of life. It is recommended that the pronator be preserved at the time of surgery. Discussion (5) Repeated muscle traction during the procedure should be avoided to prevent
secondary injury to nerves, blood vessels, and tendons. (6) Maintaining the wrist in the flexed position during the procedure facilitates
reduction and fixation. (7) This approach does not reveal the carpal cavity, and further management of the cavity requires a combination
of arthroscopic carpal techniques. (8) If intraoperative exposure and fixation reduction are problematic, traditional surgical fixation
should be chosen as soon as possible. With the development of the modern economy and the increasing aging population, the proportion of distal radius fractures presenting in
an emergency is high; and people's demand for quality of life is increasing. The collective goal that doctors and patients pursue is
achieving optimal recovery, reducing pain, and returning to society early with minimal trauma and tissue damage. In this study, the
efficacy of sparing the PQ muscle in treating unstable distal radius fractures was investigated. It concluded that sparing the PQ muscle
with a modified Henry approach is feasible for treating intra-articular fractures. The physiological structure of the PQ muscle is preserved,
and the contact between the plate and the tendon and nerve is better isolated, which avoids stimulation of the nerve and tendon and
reduces the possibility of tendon abrasion. Furthermore, the risk of tissue bleeding and edema after incision of the PQ muscle and
eventually adhesions with the surrounding tissue is avoided. The disadvantage is that sparing the PQ muscle may complicate fracture
reduction. It then requires fixation with the aid of multiple Kirschner wires or a small dorsal incision. It is not very operable for AO-type C3
distal radius fractures with serious comminution. Several limitations existed in this study. First, this was a single-center study that enrolled only a small number of patients. High-quality
randomized controlled trials with a larger sample size are still needed to reinforce these results. Secondly, this study did not include distal
radius C3 type fracture cases. A larger sample size containing more fracture patterns would be helpful in a future study. Acknowledgments Not applicable. Discussion In this surgical technique, it was found that bleeding before skin closure and after the release of the tourniquet were often minimal and
sometimes did not even require any postoperative drainage system. It can be explained by the intact PQ muscle and by the cushioning
effect of the muscle on the volar plate. The short duration of the procedure significantly decreases the risk of iatrogenic infection. The AO classification and Fernandez classification are used to classify distal radius fractures, and the AO classification is more popular
to guide treatment and determine prognosis. This study included the AO classification of distal radius fractures type B and C1 and C2. In
addition, type A fractures are recommended to be treated with manual reduction and plaster fixation or external fixation combined with
Kirschner wire since the fracture line is located at the level of the PQ muscle and the PQ muscle itself is a partial injury preoperatively. Type C3, on the other hand, is suggested to be fixed with three-column plates of the distal radius through a combined palmar and dorsal
incisions. Furthermore, the following data were collected from the surgical technique of PQ muscle locking plate preservation in the treatment of
distal radius fractures. (1) The distal incision should not exceed the watershed line of the distal radius. Traumatic arthritis causes severe
discomfort. (2) For dorsally displaced fracture blocks, especially AO fracture type B, reduction and fixation may be assisted by Kirschner
wire or small dorsal incisions. (3) Intraoperative temporary fixation of Kirschner wire should be removed promptly after the insertion of
3–4 locking screws to avoid interfering with the implantation of other screws. (4) The implanted screws should not be too long to prevent
damage to the dorsal extensor tendon. The ‘carpal shoot-through view’ can be used to determine whether the screws fixing the Page 6/11 Page 6/11 metaphysis have penetrated the carpal joint cavity[44]. (5) Repeated muscle traction during the procedure should be avoided to prevent
secondary injury to nerves, blood vessels, and tendons. (6) Maintaining the wrist in the flexed position during the procedure facilitates
reduction and fixation. (7) This approach does not reveal the carpal cavity, and further management of the cavity requires a combination
of arthroscopic carpal techniques. (8) If intraoperative exposure and fixation reduction are problematic, traditional surgical fixation
should be chosen as soon as possible. metaphysis have penetrated the carpal joint cavity[44]. Abbreviations PQ: pronator quadratus PQ: pronator quadratus
FCR: the flexor carpi radialis
VAS: The visual analogue scale scores FCR: the flexor carpi radialis Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. Competing interests The authors declare that they have no conflict of interest. Consent for publication Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the
written consent is available for review by the Editor of this journal. Ethics approval and consent to participate This retrospective study was approved by the Ethics Committee of The First Affiliated Hospital of Xinjiang Medical University and carried
out in accordance with the ethical standards set out in the Helsinki Declaration. Informed consent was received from all participating. Competing interests Authors’ contributions XH: Conducted the study. Collected, analyzed, and interpreted the data. Wrote the manuscript. QJ: Designed the study, and interpreted the data, and edited the manuscript. EK: Interpreted the data. CP: Interpreted the data. CP: Interpreted the data. WK: Edited the manuscript, reviewed the manuscript. MT: Edited the manuscript, reviewed the manuscript. Page 7/11 Page 7/11 YH: Edited the manuscript. DF: Edited the manuscript. YZ: Planned the project. Reviewed the manuscript. All authors read and approved the final manuscript. Availability of data and materials Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request. Ethi
l
d
t t
ti i
t The datasets analyzed during the current study are available from the corresponding author on reasonable request. Funding information This study was not funded by any foundation. References 1. Via GG, Roebke AJ, Julka A. Dorsal Approach for Dorsal Impaction Distal Radius Fracture—Visualization, Reduction, and Fixation
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20(1):1-8. 15. Feeney MS, Wentorf F, Putnam MD. Simulation of altered excursion of the pronator quadratus. Journal of wrist surgery. 2014;
3(03):198-202. 16. Nho J, Gong HS, Song CH, Wi SM, Lee YH, Baek GH. Examination of the pronator quadratus muscle during hardware removal
procedures after volar plating for distal radius fractures. Clinics in Orthopedic Surgery. 2014; 6(3):267-272. 17. Bertelli JA, Ghizoni MF. Reconstruction of C5 and C6 brachial plexus avulsion injury by multiple nerve transfers: spinal accessory to
suprascapular, ulnar fascicles to biceps branch, and triceps long or lateral head branch to axillary nerve. The Journal of hand
surgery. 2004; 29(1):131-139. 18. Swigart CR, Badon MA, Bruegel VL, Dodds SD. Assessment of pronator quadratus repair integrity following volar plate fixation for
distal radius fractures: a prospective clinical cohort study. The Journal of hand surgery. 2012; 37(9):1868-1873. 19. Hohendorff B, Unglaub F, Spies CK, Müller LP, Ries C. Refixierung des Musculus pronator quadratus mit einem Teil des M.-
brachioradialis-Ansatzes bei der palmaren Plattenosteosynthese einer distalen Radiusfraktur. Operative Orthopädie und
Traumatologie. 2020; 32(1):82-86. 20. Kashir A, O Donnell T. A brachioradialis splitting approach sparing the pronator quadratus for volar plating of the distal radius. Techniques in Hand & Upper Extremity Surgery. 2015; 19(4):176-181. 21. Dos Remedios C, Nebout J, Benlarbi H, Caremier E, Sam-Wing J, Beya R. Préservation du muscle carré pronateur dans les
ostéosynthèses des fractures de l’extrémité distale du radius par plaque palmaire verrouillée. Technique chirurgicale. Chir Main. 2009; 28(4):224-229. 22. Heidari N, Clement H, Kosuge D, Grechenig W, Tesch NP, Weinberg AM. Is sparing the pronator quadratus muscle possible in volar
plating of the distal radius? Journal of Hand Surgery (European Volume). 2012; 37(5):402-406. 22. References Heidari N, Clement H, Kosuge D, Grechenig W, Tesch NP, Weinberg AM. Is sparing the pronator quadratus muscle possible in volar
plating of the distal radius? Journal of Hand Surgery (European Volume). 2012; 37(5):402-406. 23. Liverneaux PA. The minimally invasive approach for distal radius fractures and malunions. Journal of Hand Surgery (European
Volume). 2018; 43(2):121-130. 23. Liverneaux PA. The minimally invasive approach for distal radius fractures and malunions. Journal of Hand Surgery (European
Volume). 2018; 43(2):121-130. 24. Liverneaux P, Ichihara S, Facca S, Diaz JH. Résultats de l’ostéosynthèse par plaque antérieure et abord mini-invasif (MIPO) des
fractures de l’extrémité distale du radius: mise au point. Hand Surgery and Rehabilitation. 2016; 35:S80-S85. 24. Liverneaux P, Ichihara S, Facca S, Diaz JH. Résultats de l’ostéosynthèse par plaque antérieure et abord mini-invasif (MIPO) des
fractures de l’extrémité distale du radius: mise au point. Hand Surgery and Rehabilitation. 2016; 35:S80-S85. 25. Rey P, Rochet S, Loisel F, Obert L. how to spare the pronator quadratus during MIPO of distal radius fractures by using a mini-volar
plate. Chir Main. 2014; 33(2):95-99. 26. Salgarello M, Visconti G, Barone-Adesi L. Interlocking circumareolar suture with undyed polyamide thread: a personal experience. Aesthet Plast Surg. 2013; 37(5):1061-1062. 27. Huang H, Wang J, Chang M. Repair of pronator quadratus with partial muscle split and distal transfer for volar plating of distal
radius fractures. The Journal of Hand Surgery. 2017; 42(11):931-935. 27. Huang H, Wang J, Chang M. Repair of pronator quadratus with partial muscle split and distal transfer for volar plating of distal
radius fractures. The Journal of Hand Surgery. 2017; 42(11):931-935. 28. Ruchelsman DE, Klugman JA, Madan SS, Chorney GS. Anterior dislocation of the radial head with fractures of the olecranon and
radial neck in a young child: a Monteggia equivalent fracture-dislocation variant. J Orthop Trauma. 2005; 19(6):428-431. 28. Ruchelsman DE, Klugman JA, Madan SS, Chorney GS. Anterior dislocation of the radial head with fractures of the olecranon and
radial neck in a young child: a Monteggia equivalent fracture-dislocation variant. J Orthop Trauma. 2005; 19(6):428-431. 29. Huang H, Wang J, Chang M. Repair of pronator quadratus with partial muscle split and distal transfer for volar plating of distal
radius fractures. The Journal of Hand Surgery. 2017; 42(11):931-935. 29. Huang H, Wang J, Chang M. Repair of pronator quadratus with partial muscle split and distal transfer for volar plating of distal
radius fractures. The Journal of Hand Surgery. 2017; 42(11):931-935. References Liverneaux P, Ichihara S, Facca S, Diaz JH. Résultats de l’ostéosynthèse par plaque antérieure et abord mini-invasif (MIPO) des
fractures de l’extrémité distale du radius: mise au point. Hand Surgery and Rehabilitation. 2016; 35:S80-S85. 40. Liverneaux P, Ichihara S, Facca S, Diaz JH. Résultats de l’ostéosynthèse par plaque antérieure et abord mini-invasif (MIPO) des
fractures de l’extrémité distale du radius: mise au point. Hand Surgery and Rehabilitation. 2016; 35:S80-S85. 41. Salgarello M, Visconti G, Barone-Adesi L. Interlocking circumareolar suture with undyed polyamide thread: a personal experience. Aesthet Plast Surg. 2013; 37(5):1061-1062. 41. Salgarello M, Visconti G, Barone-Adesi L. Interlocking circumareolar suture with undyed polyamide thread: a personal experience. Aesthet Plast Surg. 2013; 37(5):1061-1062. 42. Fang K, Lin X, Liu X, Ke Q, Shi S, Dai Z. Do we need to suture the pronator quadratus muscle when we do open reduction and interna
fixation for fracture of the distal radius. Bmc Musculoskel Dis. 2020; 21(1):1-7. 42. Fang K, Lin X, Liu X, Ke Q, Shi S, Dai Z. Do we need to suture the pronator quadratus muscle when we do open reduction and intern
fixation for fracture of the distal radius. Bmc Musculoskel Dis. 2020; 21(1):1-7. 43. Sonntag J, Hern J, Woythal L, Branner U, Lange KH, Brorson S. The Pronator quadratus muscle after Volar plating: ultrasound
evaluation of anatomical changes correlated to patient-reported clinical outcome. Hand. 2021; 16(1):32-37. 43. Sonntag J, Hern J, Woythal L, Branner U, Lange KH, Brorson S. The Pronator quadratus muscle after Volar plating: ultrasound
evaluation of anatomical changes correlated to patient-reported clinical outcome. Hand. 2021; 16(1):32-37. 44. Marsland D, Hobbs CM, Sauvé PS. Volar locking plate fixation of distal radius fractures: use of an intra-operative ‘carpal shoot
through’view to identify dorsal compartment and distal radioulnar joint screw penetration. Hand. 2014; 9(4):516-521. 44. Marsland D, Hobbs CM, Sauvé PS. Volar locking plate fixation of distal radius fractures: use of an intra-operative ‘carpal shoot
through’view to identify dorsal compartment and distal radioulnar joint screw penetration. Hand. 2014; 9(4):516-521. Figure 1 a 55-year-old woman with a fall injury resulting in a left distal radius fracture of type B2 according to AO classification. b A tunnel was
established under the PQ muscle during the operation. c The plate was placed below the PQ muscle. d Pre-incisional closure
macrophotograph. e-f Preoperative x-ray of the affected limb. g-h Postoperative x-ray of the affected limb. i-l Wrist function at 3 months
after surgery. References 30. Ruchelsman DE, Klugman JA, Madan SS, Chorney GS. Anterior dislocation of the radial head with fractures of the olecranon and
radial neck in a young child: a Monteggia equivalent fracture-dislocation variant. J Orthop Trauma. 2005; 19(6):428-431. 30. Ruchelsman DE, Klugman JA, Madan SS, Chorney GS. Anterior dislocation of the radial head with fractures of the olecranon and
radial neck in a young child: a Monteggia equivalent fracture-dislocation variant. J Orthop Trauma. 2005; 19(6):428-431. 31. Hershman SH, Immerman I, Bechtel C, Lekic N, Paksima N, Egol KA. The effects of pronator quadratus repair on outcomes after vol
plating of distal radius fractures. J Orthop Trauma. 2013; 27(3):130-133. 31. Hershman SH, Immerman I, Bechtel C, Lekic N, Paksima N, Egol KA. The effects of pronator quadratus repair on outcomes after volar
plating of distal radius fractures. J Orthop Trauma. 2013; 27(3):130-133. Page 9/11 32. Arora R, Lutz M, Hennerbichler A, Krappinger D, Espen D, Gabl M. Complications following internal fixation of unstable distal radius
fracture with a palmar locking-plate. J Orthop Trauma. 2007; 21(5):316-322. 33. Duncan SF, Weiland AJ. Delayed rupture of the flexor pollicis longus tendon after routine volar placement of a T-plate on the distal
radius. AMERICAN JOURNAL OF ORTHOPEDICS-BELLE MEAD-. 2007; 36(12):669. Marsico S. Complications of volar plating of distal radius fractures. Acta Orthop Belg. 2007; 73(6): 34. Rampoldi M, Marsico S. Complications of volar plating of distal radius fractures. Acta Orthop B 35. Jung G, Cho C, Kim J. Anatomical study of the pronator quadratus muscle and comparison to fracture sites of the distal radius. Journal of the Korean Orthopaedic Association. 2012; 47(1):48-53. 36. Gradl G, Mielsch N, Wendt M, Falk S, Mittlmeier T, Gierer P, Gradl G. Intramedullary nail versus volar plate fixation of extra-articular
distal radius fractures. Two year results of a prospective randomized trial. Injury. 2014; 45:S3-S8. 37. Gradl G, Falk S, Mittlmeier T, Wendt M, Mielsch N, Gradl G. Fixation of intra-articular fractures of the distal radius using
intramedullary nailing: a randomized trial versus palmar locking plates. Injury. 2016; 47:S25-S30. 38. Pierrart J, Tordjman D, Ikeuchi N, Delgrande D, Gregory T, Masmejean E. Lésions nerveuses associées aux fractures de l’extrémité
distale du radius. Hand Surgery and Rehabilitation. 2016; 35:S75-S79. 39. Kashir A, O Donnell T. A brachioradialis splitting approach sparing the pronator quadratus for volar plating of the distal radius. Techniques in Hand & Upper Extremity Surgery. 2015; 19(4):176-181. 40. Figure 2 A 21-year-old man with a fall injury resulting in a left distal radius fracture of type C1 according to AO classification. a, b Intraoperative
sparing of the PQ muscle in the surgical approach. c-f Preoperative imaging of the affected limb. g-h Postoperative imaging of the
affected limb. i-l Functional recovery of the wrist joint 3 months after surgery. Page 10/11 Page 10/11 Figure 3
One-year trend in VAS scores for groups A and B. Figure 4
One-year trends in forearm range of motion for group A and B. Figure 3
One-year trend in VAS scores for groups A and B. Figure 3
One-year trend in VAS scores for groups A and B. Figure 3 One-year trend in VAS scores for groups A and B. Figure 4
One-year trends in forearm range of motion for group A and B. Figure 4 Figure 4 One-year trends in forearm range of motion for group A and B. Page 11/11
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Prospects of Targeting the Gastrin Releasing Peptide Receptor and Somatostatin Receptor 2 for Nuclear Imaging and Therapy in Metastatic Breast Cancer
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RESEARCH ARTICLE Background The gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2)
are overexpressed on primary breast cancer (BC), making them ideal candidates for recep-
tor-mediated nuclear imaging and therapy. The aim of this study was to determine whether
these receptors are also suitable targets for metastatic BC. Editor: Rajeev Samant, University of Alabama at
Birmingham, UNITED STATES
Received: October 24, 2016
Accepted: January 5, 2017
Published: January 20, 2017 Editor: Rajeev Samant, University of Alabama at
Birmingham, UNITED STATES Prospects of Targeting the Gastrin Releasing
Peptide Receptor and Somatostatin Receptor
2 for Nuclear Imaging and Therapy in
Metastatic Breast Cancer Simone U. Dalm1*, Willemijne A. M. E. Schrijver2, Anieta M. Sieuwerts3, Maxime P. Look3,
Angelique C. J. Ziel - van der Made4, Vanja de Weerd3, John W. Martens3, Paul J. van
Diest2, Marion de Jong1, Carolien H. M. van Deurzen4 1 Department of Radiology & Nuclear Medicine, Erasmus MC, Rotterdam, The Netherlands, 2 Department of
Pathology, University Medical Center Utrecht, Utrecht, The Netherlands, 3 Department of Medical Oncology
and Cancer Genomics Netherlands, Erasmus MC Cancer Institute, Erasmus MC, Rotterdam, The
Netherlands, 4 Department of Pathology, Erasmus MC, Rotterdam, The Netherlands a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * s.dalm@erasmusmc.nl * s.dalm@erasmusmc.nl * s.dalm@erasmusmc.nl Methods mRNA expression of human BC samples were studied by in vitro autoradiography and asso-
ciated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary
BCs and metastases from different sites were measured by quantitative reverse transcrip-
tase polymerase chain reaction. Receptor mRNA expression levels were associated with
clinico-pathological factors and expression levels of primary tumors and corresponding
metastases were compared. Copyright: © 2017 Dalm et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. OPEN ACCESS Citation: Dalm SU, Schrijver WAME, Sieuwerts
AM, Look MP, Ziel - van der Made ACJ, de Weerd
V, et al. (2017) Prospects of Targeting the Gastrin
Releasing Peptide Receptor and Somatostatin
Receptor 2 for Nuclear Imaging and Therapy in
Metastatic Breast Cancer. PLoS ONE 12(1):
e0170536. doi:10.1371/journal.pone.0170536 * s.dalm@erasmusmc.nl GRPR and SSTR2 in Breast Cancer Metastases Conclusion This does not alter our adherence to PLOS ONE
policies on sharing data and materials. Targeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to
improve BC care in primary as well as metastatic disease. Results Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Files. Binding of GRPR and SSTR radioligands to tumor tissue correlated significantly with recep-
tor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen
receptor (ESR1)-positive tumors (p<0.001 for both receptors). There was no significant dif-
ference in GRPR mRNA expression of primary tumors versus paired metastases. Regard-
ing SSTR2 mRNA expression, there was also no significant difference in the majority of
cases, apart from liver and ovarian metastases which showed a significantly lower expres-
sion compared to the corresponding primary tumors (p = 0.02 and p = 0.03, respectively). Funding: This study was supported by the
Erasmus MC grant "The Application of Radiolabeled
Peptides in Breast Cancer". Competing Interests: MdJ is shareholder of
Advanced Accelerator Applications. We don’t have
any other potential conflicts of interest to report. PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 1 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 Introduction Breast cancer (BC) is the second most common cancer found in women and the fifth cause of
cancer related death [1]. The disease is very heterogeneous. Different subtypes with distinctive
morphological and molecular characteristics exist. The four major intrinsic BC subtypes are
luminal A, luminal B, human epidermal growth factor 2 (HER2, ERBB2)-driven and basal-like
BC [2, 3]. Treatment and prognosis of the disease are highly dependent on these subtypes;
luminal A and luminal B tumors have a better prognosis than basal-like BC [2, 3]. Although
multiple therapy options for BC exist, 20–30% of BC patients experience relapse with meta-
static disease [4]. Peptide receptor mediated nuclear imaging and peptide receptor radionuclide therapy are
methods successfully used in the clinic for imaging and treatment of neuroendocrine tumors
[5]. These methods are based on targeting receptors that are overexpressed on cancer cells
using radiolabeled peptide analogs. Regarding BC, multiple studies have demonstrated overex-
pression of the gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2
(SSTR2). In line with this, several pre-clinical as well as clinical studies demonstrated feasibility
of imaging and/or treatment of BC with GRPR and SSTR2 radioligands with promising
results, and indicated specific BC patients groups that can benefit from the application of these
radioligands [6–10]. However, previous studies were solely based on primary BC while BC-related death is
largely caused by metastatic disease. Targeting the GRPR and SSTR2 could thus especially be
advantageous for treatment of metastatic BC. In this study, we examined the GRPR and SSTR2 mRNA expression levels of primary
tumors and paired metastases, in order to evaluate whether nuclear imaging and therapy
might also be beneficial for metastatic BC. RNA isolation and quantitative reverse transcriptase polymerase chain
reaction (RT-qPCR) Ten 10 μm slides were cut from the FFPE and 10×20 μm from the FF primary BCs and paired
metastases. The first and last sections (5 μm) were stained with hematoxylin and eosin to guide
macro-dissection of the tumor cells for RNA extraction. Total RNA was isolated from the
macro-dissected FFPE sections with the AllPrep DNA/RNA FFPE Kit (Qiagen) and from the
FF sections with RNA-B (Campro Scientific) according the manufacturer’s instructions. Nucleic acid concentrations were measured with a Nanodrop 1000 system. cDNA was gener-
ated for 30 min at 48˚C with RevertAid H minus (ThermoFisher Scientific) and gene-specific
pre-amplified with Taqman PreAmp Master mix (ThermoFisher Scientific) for 15 cycles, fol-
lowed by Taqman probe—based real time PCRs according the manufacturer’s instructions in
a MX3000P Real-Time PCR System (Agilent). The following gene expression assays were eval-
uated (all from ThermoFisher Scientific): GRPR, Hs01055872 m1; SSTR2, Hs0099356 m1;
ESR1, Hs00174860_m1; ERBB2, Hs01001580_m1, and quantified relative to the average
expression of GUSB, Hs9999908_m1; HMBS, Hs00609297_m1 and TBP, Hs00427620_m1
using the delta Cq method (dCq = 2ˆ(average Cq reference genes—Cq target gene)). Samples
that resulted in amplifiable products within 25 cycles for this reference gene set at an input of
50 ng total RNA (91.2% of the samples) were considered to be of good quality to reliably deter-
mine RT-qPCR levels. Additional quality and quantity control measurements that were taken
to ensure reliable RT-qPCR data analysis are described in the Supplemental Methods in
S1 File. In this study, we used ESR1 and ERBB2 mRNA expression levels to determine ESR1 and
ERBB2 status (using a cut-off dCq for ESR1>1 and ERBB2>3.5 by optimal binning for n = 92
and n = 87 overlapping samples, respectively (See S1 File). Materials and Methods
Human BC cases Retrospectively, we selected 74 formalin-fixed paraffin-embedded (FFPE) primary BCs and 77
corresponding metastases from an existing database of the University Medical Center Utrecht
and from the pathology archive of the Erasmus Medical Center [11, 12]. Fresh frozen (FF) tis-
sue of 6 paired primary tumors and regional lymph node metastases were also included. Each
specimen was reviewed by a pathologist (CHMvD) to confirm the presence of malignancy and
to determine the percentage of tumor cells (cut-off point of >50% tumor cells). Inclusion crite-
ria were: availability of clinico-pathological data, the presence of enough tumor tissue and
good RNA quality to reliably determine RT-qPCR levels (see below). After applying these
inclusion criteria, 68 primary tumors and 60 metastases remained, resulting in 60 paired pri-
mary BCs and metastases from different sites, including brain (n = 12), regional lymph nodes
(n = 20), liver (n = 10), ovary (n = 5), lung (n = 5) and other sites (n = 8, consisting of bone
(n = 2), uterus (n = 1), gastrointestinal tract (n = 2) and distant lymph node metastases
(n = 3)). Clinico-pathological characteristics included age, primary tumor size, histological PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 2 / 12 GRPR and SSTR2 in Breast Cancer Metastases subtype, histological grade according to Bloom & Richardson [13], estrogen receptor (ER,
ESR1) status, ERBB2 status, and regional lymph node status. Approval for the use of tissue samples was obtained from the Erasmus MC Medical Ethical
Committee (MEC02953) and adhered to the Code of Conduct of the Federation of Medical
Scientific Societies in The Netherlands. PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 In vitro autoradiography Six pairs of primary BCs and regional lymph node metastases (n = 12 samples) with varying
mRNA receptor expression were analyzed for their ability to bind the GRPR radioligand,
111In-JMV4168, and the SSTR2 radioligand, 111In-DOTA-Tyr3-octreotate, using in vitro auto-
radiography. Fig 1A shows the in vitro autoradiography results of the paired samples. From
the six paired samples analyzed, two cases showed specific binding of the GRPR and SSTR2
radioligands in both the primary tumor and the lymph node metastases. In two cases there
was no binding of the GRPR radioligand and in three cases there was no binding of the SSTR2
radioligand in both the primary tumors and the lymph node metastases. In two cases binding
of the GRPR radioligand was observed in the primary tumor but not in the lymph node metas-
tasis, while in one case binding of the SSTR2 radioligand was observed in the lymph node
metastasis, but not in the primary tumor. When the %AD of the radiotracer bound to the FF tumor tissue was correlated with the
mRNA receptor expression of the FF tumor material, a significant positive correlation was
found for both GRPR (Spearman rs = 0.83, p = 0.0008) and SSTR2 (Spearman rs = 0.87,
p = 0.0003) (Fig 1B+1D). Furthermore, correlation analysis of mRNA receptor expression lev-
els quantified in FF and FFPE material of the same tumor, resulted in a significant positive cor-
relation for both GRPR (Spearman rs = 0.77, p = 0.0034) and SSTR2 (Spearman rs = 0.72,
p = 0.0082) (Fig 1C+1E). Radioligands and in vitro autoradiography The radiolabeled GRPR antagonist, JMV4168 [14], and the radiolabeled SSTR2 agonist,
DOTA-Tyr3-octreotate (Mallinckrodt) were radiolabeled with 111In (Covidien) using quench-
ers to prevent radiolysis as previously described [15, 16]. Specific activity was 80 MBq/nmol
for both radiotracers. Radiochemical purity and radiometal incorporation, measured by
instant thin-layer chromatography on silica gel and high-pressure liquid chromatography as
previously described, were >90% [16]. Slides (10 μM) of FF primary BC and paired metastases (n = 6 each) were used for autoradi-
ography experiments. Tissue sections were incubated with 100 μL incubation buffer (167 mM
Tris-HCL, 5 mM MgCl2, 1% BSA) containing 10−9 M of the radiolabeled peptide for 1 h, with
and without 10−6 M unlabeled tracer to determine specificity of binding. Results of the autora-
diography experiments were quantified using Optiquant (Perkin Elmer) and the percentage
added dose (%AD) of the radioligand bound to the tumor tissue was used as an indirect mea-
surement for the level of protein expression. Radioligand binding to primary tumors and
paired metastasis was compared and correlated with the measured GRPR and SSTR2 mRNA
expression levels in corresponding FF tumor material. Furthermore, mRNA receptor expres-
sion measured in FF tumor material was correlated with mRNA receptor expression measured 3 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases from FFPE tumor material of the same tumor. These correlation analyses were performed to
demonstrate that mRNA expression of FFPE material could be used as a surrogate for radio-
tracer binding. The autoradiography experiments and quantification of the results were per-
formed as described in the Supplemental Methods in S1 File. Statistics For the analysis, the STATA statistical package v14.1 and SPSS version 23 were used. Variables
were checked for normality prior to analysis. To compare mean values between two or more
groups, the Student t-test or analysis of variance ANOVA were used. To compare values for
primary and metastatic disease the paired t-test was applied. Pearson and Spearman correla-
tions were calculated when appropriate. P0.05 were considered statistically significant. PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 Association of GRPR and SSTR2 mRNA expression with clinico-
pathological factors Table 1 and S2 File show the patient characteristics, including the association of GRPR and
SSTR2 mRNA expression of primary BCs with clinico-pathological factors. High GRPR
mRNA expression levels were significantly associated with low histologic grade, lobular sub-
type, ESR1-positive and ERBB2-negative tumors. High SSTR2 mRNA expression levels were
also significantly associated with lobular subtype and ESR1-positive tumors. GRPR and SSTR2 mRNA expression levels of the metastases were correlated with ESR1 and
ERBB2 expression of the metastasis itself (Table 2). Similar to the primary tumors, high GRPR
and SSTR2 mRNA levels were significantly associated with ESR1-positive metastases. Further-
more, high GRPR mRNA status was significantly associated with ERBB2-negative metastases. Unlike the primary tumors, high SSTR mRNA levels were significantly associated with ERBB2-
negative metastatic lesions. 4 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases Fig 1. In vitro autoradiography of primary BC and corresponding regional lymph n
Hematoxylin and eosin (H&E) staining and autoradiography results after incubating cells
radioligand, 111In-JMV4168, and the SSTR2 radioligand, 111In-DOTA-Tyr3-octreotate. B+
quantified autoradiography results (% AD) with mRNA expression of fresh frozen (FF) tis
Correlation of mRNA expression of FF and formalin fixed paraffin embedded (FFPE) tiss
tumor. doi:10.1371/journal.pone.0170536.g001 Fig 1. In vitro autoradiography of primary BC and corresponding regional lymph node metastases. A. Hematoxylin and eosin (H&E) staining and autoradiography results after incubating cells with the GRPR
radioligand, 111In-JMV4168, and the SSTR2 radioligand, 111In-DOTA-Tyr3-octreotate. B+D. Correlation of
quantified autoradiography results (% AD) with mRNA expression of fresh frozen (FF) tissue. C+E. Correlation of mRNA expression of FF and formalin fixed paraffin embedded (FFPE) tissue of the same
tumor. 5 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases Table 1. Association of GRPR and SSTR2 mRNA expression with clinico-pathological factors of primary BC a. Characteristic
No of patients
Percentage of patients
GRPR mRNA log2
SSTR2 mRNA log2
Mean
SD
Mean
SD
All patients in this cohort
68
100
-2.90
3.8
-2.51
2.15
Age at surgery (years) b
40
11
16
-1.63
4.02
-2.23
2.13
41–55
27
39
-1.96
3.75
-2.08
2.38
56–70
22
32
-4.96
2.85
-3.07
1.94
> 70
7
10
-2.72
4.32
-2.90
2.04
P
0.08
0.22
Tumor size c
2 cm
25
36
-2.62
3.97
-2.61
2.24
2 5 cm
29
42
-3.47
3.44
-2.32
2.16
> 5 cm
10
14
-2.00
4.22
-2.95
2.09
P
0.51
0.72
Histopathological subtype d
Ductal
55
80
-3.34
3.76
-2.82
2.15
Lobular
11
16
-0.80
3.44
-1.04
1.74
Other
2
3
-2.38
3.72
-2.04
1.26
P
0.04
0.01
Bloom & Richardson grade e
I + II
15
22
-1.23
3.57
-1.80
2.20
III
44
64
-3.66
3.91
-2.88
2.18
P
0.04
0.12
ESR1 status e
Negative
25
36
-6.52
1.68
-3.83
2.09
Positive
42
61
-0.79
3.04
-1.79
1.80
P
<0.001
<0.001
ERBB2 status e
Negative
46
67
-2.12
3.60
-2.44
2.26
Positive
11
16
-5.04
2.62
-2.7
2.23
P
0.006
0.73
Regional lymph node status e
Negative
15
22
-4.40
3.06
-2.96
2.33
Positive
44
64
-2.67
4.02
-2.48
2.01
p
0.13
0.44
a Due to missing values numbers don’t always add up to 68
b R
i
f d
l BC
d l b l
BC
d
i
h
d doi:10.1371/journal.pone.0170536.t001 GRPR and SSTR2 in Breast Cancer Metastases Table 2. Association of receptor mRNA expression with ESR1 and ERBB2 status in BC metastases. ER status a
ERBB2 status a
Negative
Positive
p
Negative
Positive
p
Mean
SD
Mean
SD
Mean
SD
Mean
SD
All metastases
No of patients
24
35
48
11
GRPR
-6.26
2.76
-1.87
3.90
<0.001
-2.67
3.77
-7.98
2.15
<0.001
SSTR2
-3.95
1.64
-2.89
2.23
0.04
-2.91
1.96
-5.11
1.49
<0.001
Regional lymph node metastases
No of patients
4
16
16
4
GRPR
-7.0
1.96
-1.70
3.53
0.003
-1.70
3.54
-7.02
1.84
0.002
SSTR2
-4.03
0.97
-2.92
2.67
0.20
-2.77
2.47
-4.62
1.92
0.16
All distant metastases b
No of patients
20
19
32
7
GRPR
-6.12
2.92
-2.01
4.27
0.001
-3.15
3.84
-8.52
2.26
<0.001
SSTR2
-3.94
1.76
-2.86
1.85
0.07
-2.98
1.69
-5.40
1.26
0.001
a P for student t-test. b Numbers do not add up to 60 because for 1 patient ESR1 and ERBB2 were unknown. doi:10 1371/journal pone 0170536 t002 b Numbers do not add up to 60 because for 1 patient ESR1 and ERBB2 were unknown. doi:10.1371/journal.pone.0170536.t002 and corresponding regional lymph node and distant metastases in the brain, lung, liver and
ovaries. However, in the group of metastases from other sites, GRPR mRNA expression levels
were significantly lower in the metastases compared to the corresponding primary BC
(p = 0.02). Regarding SSTR2 mRNA levels, there were no significant differences in SSTR2 mRNA
expression of the primary tumor and the paired metastasis in regional lymph nodes, brain,
lung and other locations. However, SSTR2 mRNA levels of liver and ovarian metastases were
significantly lower compared to the expression in the corresponding primary BC (p = 0.02 and
p = 0.03, respectively). Next, we compared the receptor mRNA expression levels between distant metastases from
various metastatic sites amongst each other. GRPR mRNA levels were significantly higher in
the ovarian metastases (p = 0.03), while there were no significant differences in SSTR2 mRNA
expression levels in distant metastases from different sites. In some cases studied (n = 11), there was a discordance regarding ESR1 status of primary
BCs and corresponding metastases. When studying the effect of change in ESR1 status on
receptor mRNA expression in primary tumors and paired metastasis, GRPR and SSTR2
mRNA expression changed accordingly (higher GRPR/SSTR2 mRNA expression in ESR1-posi-
tive lesions compared to ESR1-negative lesions) in the majority of the tumors. However, this
difference was only significant (p<0.05) for ESR1-positive primary BCs with corresponding
ESR1-negative metastases (n = 6). Discordance regarding ERBB2 status was seen in 6 paired
samples. In these samples a change in ERBB2 status of primary BCs and corresponding metas-
tases did not have a consistent effect on GRPR mRNA expression levels. GRPR and SSTR2 mRNA expression of primary BC vs. corresponding
metastases Fig 2 shows the box plots of GRPR and SSTR2 mRNA expression in primary tumors and corre-
sponding metastases. Comparison of receptor mRNA expression levels of primary tumors and corresponding
metastases showed no significant difference in GRPR mRNA levels between primary tumors 6 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 Discussion In line with previously published findings [10], we demonstrated that there was a
high correlation between mRNA expression of the receptors and radiotracer binding, which
suggests that mRNA expression levels can be used as a surrogate for radiotracer binding. Next, we determined the GRPR and SSTR2 mRNA expression of the paired primary BCs
and metastases. When we associated receptor mRNA expression levels of primary BCs and
metastases with clinico-pathological factors, we observed a significantly higher GRPR and
SSTR2 expression in both ESR1-positive primary BC and metastases. These findings are in
agreement with our previous findings [10] and findings by Kumar et. al. [17] and Stoykow
et al. [7]. The latter publication describes a clinical study in which the GRPR radioligand,
68Ga-RM2 was successfully used for imaging of BC lesions and imaging success-rate associated
positively with ER and PR status. Furthermore, Prignon et al. [18] demonstrated that 68Ga-
AMBA, a GRPR agonist, was better suited for monitoring response to hormonal treatment
than 18F-FDG PET in an ER-positive BC model. In another study, van den Bossche et al.[19]
published data indicating an estrogen-dependent regulation of SSTR expression in BC cell
lines. Since ER-positive BC accounts for approximately 75% of the BC population, applying
receptor targeted nuclear imaging and/or therapy using GRPR or SSTR2 radioligands could be
beneficial for the majority of the BC population [2]. In paired primary tumors and metastases a change in ESR1 expression from positive to neg-
ative resulted in a significant decrease in GRPR and SSTR2 mRNA levels. This may indicate an
ESR1 dependent expression of GRPR and SSTR2, which is consistent with literature [19, 20]. Difference in ERBB2 status in primary tumors and paired metastasis did not show a clear effect
on GRPR mRNA expression, although these numbers were too small for reliable conclusions. Comparison of GRPR and SSTR2 mRNA levels of primary tumors and corresponding
metastases resulted in similar GRPR mRNA expression in primary tumors and paired regional
lymph nodes and distant metastases of the brain, lung, liver and ovaries. However, GRPR
mRNA expression was significantly higher in primary tumors compared to corresponding
metastases from other sites. Since this group is very diverse, containing metastases from dis-
tant lymph nodes, bone, uterus and metastases from the gastrointestinal tract, it is not possible
to draw solid conclusions. Regarding SSTR2, mRNA expression levels were significantly lower
in liver and ovarian metastases compared to the paired primary BC. Discussion Targeting of GRPR and SSTR2 overexpressed on BC cells with radioligands can offer novel
imaging and therapy options for BC. Previous clinical and preclinical studies reported promis-
ing results. However, these studies were restricted to primary BC, while metastases are the
main cause of BC-related death. In this study, we compared GRPR and SSTR2 mRNA 7 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases expression levels in a unique dataset of primary BC and corresponding metastases to deter-
mine whether receptor-based imaging and/or therapy could also be useful for metastatic BC
For this purpose we selected FFPE material of primary BCs and corresponding metastases
Fig 2. GRPR and SSTR2 mRNA levels in primary BC (PBC) and corresponding metastases (BCM). Significant differences are indicated by *. doi:10.1371/journal.pone.0170536.g002
GRPR and SSTR2 in Breast Cancer Metasta Fig 2. GRPR and SSTR2 mRNA levels in primary BC (PBC) and corresponding metastases (BCM). Significant differences are indicated by *. doi:10.1371/journal.pone.0170536.g002 Fig 2. GRPR and SSTR2 mRNA levels in primary BC (PBC) and corresponding metastases (BCM). Significant differences are indicated by *. doi:10.1371/journal.pone.0170536.g002 doi:10.1371/journal.pone.0170536.g002 expression levels in a unique dataset of primary BC and corresponding metastases to deter-
mine whether receptor-based imaging and/or therapy could also be useful for metastatic BC. For this purpose, we selected FFPE material of primary BCs and corresponding metastases
from different sites, and compared mRNA receptor expression levels of the paired samples. expression levels in a unique dataset of primary BC and corresponding metastases to deter-
mine whether receptor-based imaging and/or therapy could also be useful for metastatic BC. For this purpose, we selected FFPE material of primary BCs and corresponding metastases
from different sites, and compared mRNA receptor expression levels of the paired samples. expression levels in a unique dataset of primary BC and corresponding metastases to deter-
mine whether receptor-based imaging and/or therapy could also be useful for metastatic BC. For this purpose, we selected FFPE material of primary BCs and corresponding metastases
from different sites, and compared mRNA receptor expression levels of the paired samples. 8 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases Prior to this, we evaluated whether mRNA expression levels of tumor tissue properly represent
radioligand binding, by correlating in vitro autoradiography results with mRNA expression
levels of selected primary tumors and corresponding metastases with varying mRNA receptor
expression. PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 Discussion Combining our findings, both GRPR and SSTR2 are promising targets for nuclear imaging
and/or therapy in primary and metastatic ER-positive BC, but GRPR seems more suitable due
to its retained expression in the metastases. This finding is also supported by a previous study
by our group, in which we demonstrated GRPR expression in 48/50 BCs [6], while SSTR2 was
only expressed in 26/53 BCs (S.U. Dalm, C.H.M. van Deurzen, M. Melis, J.W. Martens and M. de Jong, unpublished data, 2014). Since a substantial portion of BC patients experience relapse with metastatic disease, it is
important to develop new treatment options for this late stage of disease. We showed that
receptor mRNA expression levels were similar in primary tumors and corresponding metasta-
ses in the majority of the cases, implying that targeting these receptors for disease monitoring
or therapy might improve BC patient care. Biopsy material or excised tumors can be used to determine receptor expression by immu-
nohistochemistry, RNA in situ, in vitro autoradiography or RT-qPCR [21]. Disease monitor-
ing of receptor-positive tumors can then be performed by single photon emission computed
tomography/computer tomography (SPECT/CT), positron emission tomography (PET)/CT
or PET/magnetic resonance imaging using radioligands targeting these receptors. Also, 9 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases dedicated breast PET cameras can be used. These dedicated cameras have improved sensitivity
and specificity compared to whole body PET, because of a restricted field of view, resulting in
higher cancer detection [22]. Furthermore, tumors can be treated with therapeutic radioli-
gands. Another option is to use GRPR or SSTR2 radioligands for visualization of sentinel node
metastases or as a guide for BC surgery (e.g. preoperative imaging, radioguided surgery) in
patients with receptor positive primary tumors [23, 24]. The next step would be to perform clinical studies to investigate the feasibility of imaging
primary tumors and metastases with radioligands targeting these receptors. One important
aspect is to study physiological uptake of the radioligands in other organs, since this is of great
importance for successful nuclear imaging and treatment. However, previous studies using
radioligands targeting these receptors on other tumor types did not report on any alarming
physiological uptake [5, 7]. Another potential interesting target for receptor mediated nuclear imaging and/or therapy
is chemokine c-x-c motif receptor 4 (CXCR4), since high CXCR4 expression was reported in
BC with high metastatic potential. Discussion Furthermore, radiotracers targeting the CXCR4 are
available and have been tested successfully pre-clinically and clinically. Unfortunately, the
CXCR4-targeted radiotracer available to us (Pentixafor) showed reduced affinity when labeled
with 111In, hampering in vitro autoradiography experiments. We could therefore only analyze
CXCR4 mRNA expression levels as described in S3 File. S2 File. Supplemental table.
(DOCX) S2 File. Supplemental table. (DOCX) S3 File. Chemokine C-X-C motif receptor 4 expression in primary and metastatic breast
cancer. (DOCX) Conclusion The presented data indicates that nuclear based imaging and therapy has the potential to
improve BC patient care in primary as well as in metastatic disease, by targeting GRPR and
SSTR2. Both GRPR and SSTR2 radioligands, but especially GRPR radioligands, are promising
for imaging and treatment of ER-positive primary and metastatic BC. Acknowledgments The authors thank Natalie D. ter Hoeve and Erik de Blois for their technical assistanc Author Contributions Conceptualization: SUD CHMvD AMS MdJ JWM. Formal analysis: SUD MPL. Funding acquisition: MdJ CHMvD JWM. Investigation: SUD VdW ACJZ-vdM AMS WAMES CHMvD. Methodology: SUD CHMvD JWM AMS MdJ. References 1. IACR. GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012:
International Agency for Research on Cancer (http://globocan.iarc.fr/Default.aspx, 29-04-2015); [29-04-
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International Agency for Research on Cancer (http://globocan.iarc.fr/Default.aspx, 29-04-2015); [29-04-
2015]. http://globocan.iarc.fr/Default.aspx. 2. Yersal O, Barutca S. Biological subtypes of breast cancer: Prognostic and therapeutic implications. World J Clin Oncol. 2014; 5(3):412–24. doi: 10.5306/wjco.v5.i3.412 PMID: 25114856 3. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human
breast tumours. Nature. 2000; 406(6797):747–52. doi: 10.1038/35021093 PMID: 10963602 4. Early Breast Cancer Trialists’ Collaborative G. Effects of chemotherapy and hormonal therapy for early
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radionuclide therapy using radiolabeled somatostatin analogs: focus on future developments. Clin
Transl Imaging. 2014; 2:55–66. doi: 10.1007/s40336-014-0054-2 PMID: 24765618 6. Dalm SU, Martens JW, Sieuwerts AM, van Deurzen CH, Koelewijn SJ, de Blois E, et al. In vitro and in
vivo application of radiolabeled gastrin-releasing peptide receptor ligands in breast cancer. J Nucl Med. 2015; 56(5):752–7. doi: 10.2967/jnumed.114.153023 PMID: 25791989 7. Stoykow C, Erbes T, Bulla S, Ma¨cke H, Drendel V, Bronsert P, et al. Gastrin-releasing peptide receptor
expression is associated with estrogen receptor status in breast cancer: Findings of a PET/CT pilot
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111-octreotide scintigraphy, intraoperative gamma-detector localisation and somatostatin receptor
expression in primary human breast cancer. Breast Cancer Res Treat. 2002; 74(2):101–11. PMID:
12186370 9. Van Den Bossche B, Van Belle S, De Winter F, Signore A, Van de Wiele C. Early prediction of endo-
crine therapy effect in advanced breast cancer patients using 99mTc-depreotide scintigraphy. J Nucl
Med. 2006; 47(1):6–13. PMID: 16391181 10. Dalm SU, Sieuwerts AM, Look MP, Melis M, van Deurzen CH, Foekens JA, et al. Clinical Relevance of
Targeting the Gastrin-Releasing Peptide Receptor, Somatostatin Receptor 2, or Chemokine C-X-C
Motif Receptor 4 in Breast Cancer for Imaging and Therapy. J Nucl Med. 2015; 56(10):1487–93. doi:
10.2967/jnumed.115.160739 PMID: 26251419 11. Conceptualization: SUD CHMvD AMS MdJ JWM. 10 / 12 PLOS ONE | DOI:10.1371/journal.pone.0170536
January 20, 2017 GRPR and SSTR2 in Breast Cancer Metastases Project administration: SUD CHMvD AMS. Resources: CHMvD AMS WAMES PJvD SUD MdJ. Supervision: SUD CHMvD MdJ JWM. Validation: SUD CHMvD. Visualization: SUD MPL CHMvD. Writing – original draft: SUD CHMvD. Writing – review & editing: SUD WAMES AMS MPL ACJZ-vdM VdW JWM PJvD
CHMvD. Validation: SUD CHMvD. Writing – review & editing: SUD WAMES AMS MPL ACJZ-vdM VdW JWM PJvD MdJ
CHMvD. Writing – review & editing: SUD WAMES AMS MPL ACJZ-vdM VdW JWM PJvD MdJ
CHMvD. Writing – review & editing: SUD WAMES AMS MPL ACJZ-vdM VdW JWM PJvD MdJ
CHMvD. PLOS ONE | DOI:10.1371/journal.pone.0170536
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dance in ERalpha, PR and HER2 receptor status across different distant breast cancer metastases
within the same patient. Ann Oncol. 2013; 24(12):3017–23. PMID: 24114857 12. Schrijver WA, Jiwa LS, van Diest PJ, Moelans CB. Promoter hypermethylation profiling of distant breast
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Tomography in Breast Cancer. Diagnostics (Basel). 2015; 5(1):61–83. 23. Mariani G, Erba P, Villa G, Gipponi M, Manca G, Boni G, et al. Lymphoscintigraphic and intraoperative
detection of the sentinel lymph node in breast cancer patients: the nuclear medicine perspective. J Surg
Oncol. 2004; 85(3):112–22. doi: 10.1002/jso.20023 PMID: 14991882 24. Hindie E, Groheux D, Brenot-Rossi I, Rubello D, Moretti JL, Espie M. The sentinel node procedure in
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jnumed.110.081711 PMID: 21321267 12 / 12
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English
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Do we need to evaluate diastolic blood pressure in patients with suspected orthostatic hypotension?
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Clinical autonomic research
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cc-by
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Abstract Purpose The contribution of diastolic blood pressure
measurement to the diagnosis of classical orthostatic
hypotension is not known. We aimed to explore the
prevalence of isolated systolic and diastolic orthostatic
hypotension components in patients with syncope and
orthostatic intolerance. Results One hundred eighty-six patients (12.2%) met cur-
rent diagnostic criteria for classical orthostatic hypoten-
sion. Of these, 176 patients (94.6%) met the systolic
criterion and 102 patients (54.8%) met the diastolic crite-
rion. Ninety-two patients (49.5%) met both systolic and
diastolic criteria, whereas ten patients (5.4%) met the
diastolic criterion alone. Of these, three had systolic blood
pressure \90 mmHg during tilt test and were diagnosed
with orthostatic hypotension on the grounds of low stand-
ing blood pressure. Based on patient history and ancillary
test results, causes of orthostatic intolerance and syncope
other than orthostatic hypotension were present in the
remaining seven patients. Methods A total of 1520 patients aged [15 years with
suspected syncope and/or symptoms of orthostatic intol-
erance were investigated in a tertiary center using tilt-
table testing and continuous non-invasive blood pressure
monitoring. Classical orthostatic hypotension was defined
as a decline in systolic blood pressure C20 mmHg and/or 1
Department of Clinical Sciences, Faculty of Medicine,
Clinical Research Center, Lund University, Malmo¨, Sweden Conclusions An abnormal orthostatic fall in diastolic
blood pressure without an abnormal fall in systolic blood
pressure is rare among patients with syncope and ortho-
static intolerance. Approximately 95% of patients with
classical orthostatic hypotension can be identified by sys-
tolic criterion alone. 2
Department of Cardiology, Ska˚ne University Hospital, Inga
Marie Nilssons gata 46, 205 02 Malmo¨, Sweden 3
Department of Medical Imaging and Physiology, Ska˚ne
University Hospital, Malmo¨, Sweden 4
National Heart & Lung Institute, Imperial College, London,
UK Keywords Orthostatic hypotension Blood pressure
Diastolic Syncope Orthostatic intolerance 5
Department of Neurology, Leiden University Medical Centre,
Leiden, The Netherlands 6
Department of Neurology, Harvard Medical School, Beth
Israel Deaconess Medical Center, Boston, USA Clin Auton Res (2017) 27:167–173
DOI 10.1007/s10286-017-0409-7 RESEARCH ARTICLE Artur Fedorowski1,2
• Viktor Hamrefors1,3 • Richard Sutton4 • J. Gert van Dijk5 •
Roy Freeman6 • Jacques WM Lenders7,8 • Wouter Wieling9 Artur Fedorowski1,2
• Viktor Hamrefors1,3 • Richard Sutton4 • J. Gert van Dijk5 •
Roy Freeman6 • Jacques WM Lenders7,8 • Wouter Wieling9 Received: 3 January 2017 / Accepted: 15 February 2017 / Published online: 27 February 2017
The Author(s) 2017. This article is published with open access at Springerlink.com diastolic blood pressure C10 mmHg at 3 min of tilt test. The
prevalence
of
upright
systolic
blood
pressure
\90 mmHg and its overlap with isolated diastolic ortho-
static hypotension was also assessed. Results One hundred eighty-six patients (12.2%) met cur-
rent diagnostic criteria for classical orthostatic hypoten-
sion. Of these, 176 patients (94.6%) met the systolic
criterion and 102 patients (54.8%) met the diastolic crite-
rion. Ninety-two patients (49.5%) met both systolic and
diastolic criteria, whereas ten patients (5.4%) met the
diastolic criterion alone. Of these, three had systolic blood
pressure \90 mmHg during tilt test and were diagnosed
with orthostatic hypotension on the grounds of low stand-
ing blood pressure. Based on patient history and ancillary
test results, causes of orthostatic intolerance and syncope
other than orthostatic hypotension were present in the
remaining seven patients. diastolic blood pressure C10 mmHg at 3 min of tilt test. The
prevalence
of
upright
systolic
blood
pressure
\90 mmHg and its overlap with isolated diastolic ortho-
static hypotension was also assessed. & Artur Fedorowski
Artur.fedorowski@med.lu.se Introduction 7
Department of Internal Medicine, Radboud Medical Centre,
Nijmegen, The Netherlands Classical orthostatic hypotension (OH) is defined as a
sustained reduction of systolic blood pressure (SBP) of at
least 20 mmHg and/or diastolic BP (DBP) of 10 mmHg
within 3 min of standing or head-up tilt to at least 60. These expert-based criteria were originally defined in 1996 8
Department of Medicine III, Technical University Dresden,
Dresden, Germany 9
Department of Internal Medicine, Academic Medical Centre,
University of Amsterdam, Amsterdam, The Netherlands 12 3 Clin Auton Res (2017) 27:167–173 168 drink water at will. Prior to examination, the patients were
asked to complete a questionnaire which explored past
medical history, duration, frequency and features of syn-
cope-related symptoms, smoking status and current phar-
macological treatment. After supine rest for at least
10 min, a standardized 70 head-up tilt test (HUT) was
performed
until
syncope/presyncope
or
pronounced
symptoms of orthostatic intolerance occurred, or for a
maximum of 20 min, followed by optional nitroglycerin
provocation according to the Italian protocol [3]. Prior to
HUT, carotid sinus massage was performed in patients
aged C40 years according to the Newcastle protocol [20],
and the Valsalva maneuver completed HUT [13]. Beat-to-
beat blood pressure (BP) and electrocardiogram (ECG)
were continuously monitored using a validated noninvasive
photoplethysmographic method (Nexfin monitor; BMEYE,
Amsterdam, Netherlands) [4], and subsequently analyzed
offline
using
a dedicated
program provided
by
the
manufacturer. in a consensus statement endorsed by the American
Autonomic Society and the American Academy of Neu-
rology [1, 11], and were later adopted by the European
Society of Cardiology (ESC) syncope guidelines [17]. Although the definition of OH includes separate criteria for
SBP and DBP changes, in daily practice the diastolic cri-
terion seems to be rarely used to diagnose OH. We aimed to examine the contribution of the SBP and
DBP criteria, applied separately and in combination, to the
detection of OH. We hypothesized that only a minority of
OH patients would be identified by DBP criteria alone. Accordingly, we assessed proportions of patients who met
the systolic and diastolic OH criteria from a large database
of patients investigated for unexplained syncope and/or
orthostatic intolerance. Statistical analyses The main characteristics of the study population are pre-
sented as mean and standard deviation for continuous
variables and as percentages for categorical variables. Group differences in continuous variables were compared
using analysis of variance (ANOVA), and dichotomous
variables were compared using Pearson’s chi-square test. All analyses were performed using IBM SPSS Statistics
version 23 software (IBM Corp., Armonk, NY, USA). All
tests were two-sided, if applicable, wherein p \ 0.05 was
considered statistically significant. Data analysis Between September 2008 and May 2016, a total of 1533
patients aged [15 years with suspected syncope and/or
symptoms of orthostatic intolerance unexplained by initial
evaluation [17] were investigated at the Syncope Unit of
Ska˚ne University Hospital, Malmo¨, Sweden. Patients were
recruited through referrals from primary care and from
hospitals in the southern region of Sweden. Prior to
investigation
at
the
Syncope
Unit,
additional
tests
including exercise and continuous 24-h ECG, external and
implantable event recorder, echocardiography, coronary
angiography, brain imaging and EEG were performed to
exclude cardiac and neurological causes of symptoms. We
excluded eight patients with scleroderma and five with
supine SBP \90 mmHg. Patients with scleroderma had
unreliable readings of finger artery blood pressure, usually
falsely
low,
whereas
patients
with
supine
SBP
\90 mmHg were hypotensive at baseline and we decided
to exclude them for the clarity of results interpretation. This yielded 1520 patients with complete data eligible for
the study; of these, 1382 (91%) had a history of suspected
syncope and 1089 (72%) reported orthostatic intolerance. The study protocol was approved by the Regional Ethical
Review Board in Lund, Sweden (ref. no. 82/2008), and all
study participants gave their written informed consent. Written consent on behalf of minors was obtained from
parents. Blood pressure and heart rate (HR) in the supine position
1 min prior to HUT and at 3 min of HUT were calculated
as an average of a 30-s period and recorded in the database. The predefined point for the second hemodynamic assess-
ment at 3 min of HUT was selected to comply with the
current definition of classical orthostatic hypotension [11]. If the syncope occurred within the 3-min HUT period, the
last 30-s period before the onset of prodromal symptoms,
profound hypotension and/or bradycardia was analyzed and
averaged. Systolic OH was defined as SBP decline
C20 mmHg, and diastolic OH as DBP decline C10 mmHg
within 3 min of HUT [11]. In addition, we also assessed
the prevalence of upright SBP\90 mmHg [17], as stated in
the current ESC guidelines, and SBP decline C30 mmHg
in patients with supine SBP C160 mmHg, as proposed by a
previous study of OH in hypertensive and normotensive
patients [5, 29]. Results The characteristics of the study population are presented in
Table 1. There was a slight predominance of women, and
the mean age was 53 years. One-third of patients reported a
history of hypertension and current antihypertensive treat-
ment. Among the total 1520 patients, 186 (12.2%) met the
current diagnostic criteria of OH using combined cutoff
values for either SBP or DBP. Of these, 176/186 (94.6%)
met the systolic criterion. and 102/186 (54.8%) met the
diastolic OH criterion. A total of 92/186 (49.5%) patients
met both systolic and diastolic criteria, whereas 84/186
(45.2%) met only the systolic criterion and 10/186 (5.4%)
met only the diastolic criterion (Fig. 1). Thus, of the total
of 186 patients with OH according to the current consensus
criteria, only one of 19 was classified as having OH on the
grounds of isolated DBP decrease. Fig. 1 Proportions of patients (n = 186) diagnosed with classical
orthostatic hypotension (OH) according to current consensus criteria
stratified into isolated systolic OH (n = 84), systolic and diastolic OH
(n = 92), and isolated diastolic OH (n = 10). SBP systolic blood
pressure, DBP diastolic blood pressure. Systolic OH = orthostatic
SBP decline C20 mmHg; diastolic OH = orthostatic DBP decline
C10 mmHg Patients with systolic OH were older and more likely
men, had higher supine SBP and DBP, and a higher pro-
portion of hypertension, antihypertensive treatment, and
manifest coronary disease compared with non-OH patients
(Table 1, p B 0.001 for all comparisons). One hundred
fifty-nine patients with systolic OH (90.3%) were diag-
nosed with syncope due to OH by the clinicians performing
the tests, whereas in the remaining 17 patients (9.7%),
systolic OH was detected but not found to be decisive for
the syncope etiology. In the supine SBP range equal to or above 160 mmHg
(n = 170/1520, 11.2%), a total of 49 patients met the
current systolic criterion of SBP decline C20 mmHg; of
these, 34 had SBP decline C30 mmHg (p \ 0.001 for
difference between groups). The proportions of patients
classified with abnormal orthostatic BP according to articipants. Data are presented as number and percentage or mean and standard deviation Table 1 Characteristics of study participants. Data are presented as number and percentage or mean and standard deviation
Characteristic
All
(n = 1520)
No OH
(n = 1334)
DSBP B
-20 mmHg
(n = 176)
p value vs. Examination protocol The patients were asked to take their regular medication
and fast for 2 h before the test, but they were allowed to 123 Clin Auton Res (2017) 27:167–173 169 Fig. 1 Proportions of patients (n = 186) diagnosed with classical
orthostatic hypotension (OH) according to current consensus criteria
stratified into isolated systolic OH (n = 84), systolic and diastolic OH
(n = 92), and isolated diastolic OH (n = 10). SBP systolic blood
pressure, DBP diastolic blood pressure. Systolic OH = orthostatic
SBP decline C20 mmHg; diastolic OH = orthostatic DBP decline
C10 mmHg BMI body-mass index, OH orthostatic hypotension, SBP systolic blood pressure, DBP diastolic blood pressure, HUT head-up tilt test, AMI acute
myocardial infarction, CABG coronary artery bypass graft, PCI percutaneous coronary intervention
* Excluding delta SBP B -20 mmHg Results Data are presented as number and percentage or mean and standard 12 170 Clin Auton Res (2017) 27:167–173 Table 2 Proportions of subjects with abnormal orthostatic blood pressure changes according to different criteria in a population of patients
(n = 1520) with unexplained syncope and/or orthostatic intolerance
Diagnostic criteria
n (%)
n (difference)
DSBP B -20 mmHg and/or DDBP B -10 mmHg*
186 (12.2)
Reference
DSBP B -20 mmHg only
176 (11.6)
-10
DSBP B -20 mmHg and/or 3-min HUT SBP \90 mmHga
188 (12.4)
?2
DSBP B -20 mmHg if supine SBP \160 mmHg or DSBP B -30 mmHg if supine SBP C160 mmHg or 3-min
HUT SBP \ 90mmHga
173 (11.4)
-13
SBP systolic blood pressure, DBP diastolic blood pressure, HUT head-up tilt test
* Orthostatic hypotension definition according to the current Autonomic Societies Consensus (2011)
a Definition of orthostatic hypotension according to the current European Society of Cardiology guidelines for active standing test (2009)
Table 3 Hemodynamic data of ten patients with isolated diastolic orthostatic hypotension recorded in the supine position and after 3-min head-
up tilt test (HUT) with the most likely syncope etiology
Patient
Gender/age (years)/AHT
BP supine (mmHg)
BP 3-min HUT (mmHg)
DDBP (mmHg)
Syncope etiology*
M/84/yes
100/62
82/49
-13
OH
M/82/yes
101/62
83/52
-10
OH
M/41/no
103/65
89/55
-10
OH
F/34/no
110/67
96/56
-11
VVS
F/22/no
125/72
109/56
-16
VVS
M/86/yes
134/76
122/62
-14
CSS
F/35/no
135/83
137/56
-27
VVS
F/63/no
143/89
126/78
-11
VVS
F/66/no
150/90
134/79
-11
VVS
F/67/yes
201/92
217/81
-11
VVS
M male, F female, y years, BP blood pressure, DBP diastolic blood pressure, AHT antihypertensive treatment, OH orthostatic hypotension, VVS
vasovagal syncope
* The investigator determined the most likely syncope etiology based on patient’s history, results of additional tests and HUT Table 2 Proportions of subjects with abnormal orthostatic blood pressure changes according to different criteria in a population of patients
(n = 1520) with unexplained syncope and/or orthostatic intolerance
Diagnostic criteria
n (%)
n (difference)
DSBP B -20 mmHg and/or DDBP B -10 mmHg*
186 (12.2)
Reference
DSBP B -20 mmHg only
176 (11.6)
-10
DSBP B -20 mmHg and/or 3-min HUT SBP \90 mmHga
188 (12.4)
?2
DSBP B -20 mmHg if supine SBP \160 mmHg or DSBP B -30 mmHg if supine SBP C160 mmHg or 3-min
HUT SBP \ 90mmHga
173 (11.4)
-13
SBP systolic blood pressure DBP diastolic blood pressure HUT head-up tilt test ts with abnormal orthostatic blood pressure changes according to different criteria in a population of patients
yncope and/or orthostatic intolerance Table 3 Hemodynamic data of ten patients with isolated diastolic orthostatic hypotension recorded in the supine position and after 3-min head-
up tilt test (HUT) with the most likely syncope etiology
Patient
Gender/age (years)/AHT
BP supine (mmHg)
BP 3-min HUT (mmHg)
DDBP (mmHg)
Syncope etiology*
M/84/yes
100/62
82/49
-13
OH
M/82/yes
101/62
83/52
-10
OH
M/41/no
103/65
89/55
-10
OH
F/34/no
110/67
96/56
-11
VVS
F/22/no
125/72
109/56
-16
VVS
M/86/yes
134/76
122/62
-14
CSS
F/35/no
135/83
137/56
-27
VVS
F/63/no
143/89
126/78
-11
VVS
F/66/no
150/90
134/79
-11
VVS
F/67/yes
201/92
217/81
-11
VVS
M male, F female, y years, BP blood pressure, DBP diastolic blood pressure, AHT antihypertensive treatment, OH orthostatic hypotension, VVS
vasovagal syncope
* The investigator determined the most likely syncope etiology based on patient’s history, results of additional tests and HUT p
or determined the most likely syncope etiology based on patient’s history, results of additional tests and HUT g
y
p
* The investigator determined the most likely syncope etiology based on patient’s history, results of additional old woman (#10), had a very high SBP above 200 mmHg
and was diagnosed with vasovagal syncope (VVS) after
nitroglycerine challenge and reproduction of previous
attacks. Results no OH
DDBP B -10 mmHg
only* (n = 10)
p value vs. no OH
Age (years)
53 ± 21
51 ± 21
68 ± 15
\0.001
58 ± 23
0.31
Sex (male)
602 (40)
495 (37)
103 (59)
\0.001
4 (40)
0.85
BMI (kg/m2)
25 ± 5
25 ± 5
25 ± 4
0.17
24 ± 3
0.47
SBP supine (mmHg)
132 ± 22
130 ± 21
146 ± 26
\0.001
130 ± 31
0.98
DBP supine (mmHg)
72 ± 10
71 ± 9
76 ± 12
\0.001
76 ± 12
0.11
Heart rate supine (beats/min)
70 ± 12
70 ± 12
70 ± 12
0.47
71 ± 14
0.89
SBP 3-min HUT (mmHg)
130 ± 24
133 ± 23
109 ± 27
\0.001
120 ± 40
0.62
DBP 3-min HUT (mmHg)
76 ± 12
78 ± 11
65 ± 14
\0.001
63 ± 12
\0.001
Heart rate 3-min HUT (beats/min)
81 ± 16
80 ± 17
79 ± 17
0.33
83 ± 14
0.66
History of hypertension
450 (30)
368 (28)
79 (45)
\0.001
3 (30)
0.71
Current antihypertensive
treatment
529 (35)
437 (33)
88 (50)
\0.001
4 (40)
0.47
History of diabetes mellitus
104 (7)
87 (7)
16 (9)
0.43
1 (10)
0.88
History of coronary heart disease
(AMI/CABG/PCI)
109 (7)
85 (6)
24 (14)
\0.001
0 (0)
0.43
Current smoking
193 (13)
172 (13)
20 (12)
0.50
1 (10)
0.98
BMI body-mass index, OH orthostatic hypotension, SBP systolic blood pressure, DBP diastolic blood pressure, HUT head-up tilt test, AMI acute
myocardial infarction, CABG coronary artery bypass graft, PCI percutaneous coronary intervention
* Excluding delta SBP B -20 mmHg Table 1 Characteristics of study participants. Clinical implications of OH criteria Current diagnostic criteria of OH include both systolic and
diastolic cutoff [11]. These criteria are expert-based. In
1996, a consensus committee of the American Autonomic
Society and the American Academy of Neurology met to
discuss the etiological criteria of OH and to determine how
OH should be diagnosed [1]. Prior to this consensus,
investigators and clinicians used varying numbers to
denote the presence of OH, creating confusion. The com-
bined clinical wisdom of this group of experts (n = 13)
proclaimed that a 20-mmHg systolic and/or a 10-mmHg
diastolic decline from lying to standing within 3 min of
standing should be the standard. The 1996 criteria were
primarily based on a study of 92 male and female normal
subjects aged 17–61 years [24, 25], as epidemiological
data were unavailable at that time. A later study by
Fedorowski et al. in a population-based cohort of 924
subjects confirmed that the older 20/10-mmHg standard for
the definition of OH was an excellent cutoff for nor-
motensive persons [5]. However, in subjects with resting
SBP above 160 mmHg, a fall of 30 mmHg should be used. Moreover, only 10% of subjects who met the diagnostic
criteria of OH (9/88) did so on the grounds of isolated
diastolic OH [5]. Consequently, for a clinically relevant diagnosis of OH
in symptomatic patients, the systolic criteria seem to be
sufficient. They will identify approximately 95% of sub-
jects with OH based on the current consensus and the vast
majority of abnormal orthostatic BP responses. Adding the
absolute SBP threshold of below 90 mmHg on standing
may further expand the systolic OH criteria, with the total
number of cases being almost the same as for the combined
systolic-diastolic criteria. The value of additional systolic
criterion may be justified by the fact that symptomatic
patients with hypotension (SBP \90 mmHg) on standing
may require clinical intervention, and their identification
could be important. This is of relevance in individuals with
low SBP where the current OH criteria may miss a clini-
cally significant fall in cerebral perfusion due to a narrow
range of BP fall. In addition, for resting SBP above 160 mmHg, a higher
diagnostic threshold of SBP decline C30 mmHg, as pre-
viously proposed, could be considered [5, 29]. In patients
with severe hypertension, the natural fluctuations of BP are
greater [16], as was also shown in our study. Results equal to or above 160 mmHg presented as mean ± SD Table 4 Hemodynamic changes after 3-min head-up tilt test in the cohort of 1520 patients with history of syncope and/or symptoms of
orthostatic intolerance stratified according to supine blood pressure below vs. equal to or above 160 mmHg presented as mean ± SD orthostatic intolerance stratified according to supine blood pressure below vs. equal to or above 160 mmHg presented as mean ± SD
Hemodynamic
parameter
All patients
3-min HUT DSBP B -20 mmHg
All
patients
n = 1520
Supine SBP
\160 mmHg
n = 1350
Supine SBP
C160 mmHg
n = 170
All
patients
n = 176
Supine SBP
\160 mmHg
n = 127
Supine SBP
C160 mmHg
n = 49
DSBP (mmHg)
-2 ± 17
-1 ± 15
-12 ± 27*
-37 ± 18
-34 ± 13
-45 ± 24*
DDBP (mmHg)
?5 ± 9
?5 ± 8
?1 ± 13*
-11 ± 11
-10 ± 9
-12 ± 15
D Heart Rate (beats/
min)
10 ± 11
11 ± 11
7 ± 9*
?10 ± 11
?10 ± 12
?9 ± 8
SBP systolic blood pressure, DBP diastolic blood pressure, HUT head-up tilt test
* p \ 0.001 for difference between the groups (supine SBP below vs. equal to or above 160 mmHg) SBP systolic blood pressure, DBP diastolic blood pressure, HUT head-up tilt test
* p \ 0.001 for difference between the groups (supine SBP below vs. equal to or above 160 mmHg) magnitude of changes in SBP is larger than that of DBP,
and is thus much easier to measure. Second, the accuracy
of BP measurements may vary by around 5 mmHg [19]
due to blood pressure oscillations and measurement
imprecision—an amount that is half of the diagnostic
threshold for DBP, engendering greater confidence in the
change in SBP. Finally, an abnormal fall in DBP with a
minor or no fall in SBP will increase pulse pressure. Since
the main determinants of brain blood flow are the absolute
level of arterial pressure and the pulse pressure [27], an
isolated fall in diastolic pressure is not likely to induce
significant hypoperfusion of the brain. It has been shown
that symptoms of orthostatic intolerance such as dizziness
or (pre-)syncope are strongly dependent on SBP and not on
DBP decline [23]. Discussion We report here that an abnormal decrease in diastolic blood
pressure without an abnormal decrease in systolic pressure
is very rare among patients investigated for suspected
syncope and orthostatic intolerance. The overwhelming
majority of patients with OH can be identified by a systolic
criterion. Moreover, the isolated DBP decrease seems not
to be decisive for the final diagnosis of syncope and the
management of patients. Results The six remaining patients (#4–9) were predomi-
nantly younger/middle-aged women (5/6) without antihy-
pertensive treatment who were normotensive on standing
(SBP, 96–137 mmHg), and two had 3-min HUT SBP
below 120 mmHg (Table 3). These patients were diag-
nosed with VVS after reproducing syncope during HUT. The only male patient in this group, an 87-year old (#6),
was diagnosed with syncope due to carotid sinus syndrome. different criteria ranged from 11.4% for modified systolic
criteria including higher systolic threshold in more severe
hypertension, to 12.4% for systolic OH criterion plus
standing SBP \90 mmHg but without isolated diastolic
OH (Table 2). The detailed hemodynamic parameters of the ten
patients with isolated diastolic OH are shown in Table 3. The mean DDBP was -14 ± 7 mmHg (range -27 to
-10 mmHg). None of the basic biometric and clinical
parameters including age, sex, BMI, supine SBP, DBP and
heart rate, history of hypertension, coronary disease or
diabetes, and smoking differed significantly from the rest
of the cohort. Three patients with isolated diastolic OH had
upright SBP below 90 mmHg after 3-min HUT (#1–3), and
were diagnosed with syncope due to OH on the grounds of
patient history and test results. In two of these patients,
symptoms of orthostatic intolerance were considered to be
a side effect of antihypertensive drugs. One of the seven
remaining patients with isolated diastolic OH, a 67-year- Patients withsupineSBP C160 mmHg demonstratedmore
pronounced changes in SBP, DBP and heart rate after 3-min
HUT compared with supine SBP \160 mmHg (Table 4). However, among those who met the systolic OH criterion,
there was a significant difference between the two groups only
in DSBP (-45 ± 24 vs. -34 ± 13 mmHg; p\ 0.001), i.e. those with systolic OH who had higher supine SBP demon-
strated a more pronounced SBP decline during HUT. 123 171 Clin Auton Res (2017) 27:167–173 Table 4 Hemodynamic changes after 3-min head-up tilt test in the cohort of 1520 patients with history of syncope and/or symptoms of
orthostatic intolerance stratified according to supine blood pressure below vs. equal to or above 160 mmHg presented as mean ± SD Table 4 Hemodynamic changes after 3-min head-up tilt test in the cohort of 1520 patients with history of syncope and/or symptoms of
orthostatic intolerance stratified according to supine blood pressure below vs. Study strengths and limitations common in autonomic failure [26]. Thus, the specificity of
OH diagnosis in the more severe hypertension might be
improved by a higher diagnostic SBP threshold to avoid
falsely positive cases due to increased BP variability. The current OH diagnostic criteria are based on expert
opinion and tests performed in small groups of patients
and healthy individuals. The predominant techniques
used at that time were active standing test and inter-
mittent BP measurement using auscultatory or oscillo-
metric methods [18]. The present study is based on a
large sample of symptomatic individuals, a 70 head-up
tilt test, which is a standardized passive orthostatic
challenge method, and non-invasive continuous photo-
plethysmographic technology of hemodynamic monitor-
ing. The study design is therefore generalizable to
typical syncope and autonomic disorder evaluation lab-
oratories. However, our observations should be verified
against similar settings in independent populations, and
compared with conventional BP measurements using a
sphygmomanometer. In addition, patients with neuro-
genic OH due to neurodegenerative diseases such as
Parkinson’s disease, multiple system atrophy and pure
autonomic failure may have been underrepresented in
our study populations. Thus, our conclusions should be
taken with caution in regard to patients with neurogenic
orthostatic hypotension. Further, initial OH was not
assessed in this study, and the contribution of isolated
DBP fall to the diagnosis of initial OH remains unex-
plored [28]. Finally, the current definition of OH is
centered on measurement results and not on the associ-
ated complaints. Thus, we would like to emphasize the
possible discrepancy between OH based on the abnormal
orthostatic BP response observed during diagnostic tests
and its relevance for the patient’s symptoms and the
most likely syncope etiology. Clinical implications of OH criteria Moreover,
pronounced BP swings that lead to an apparent normal-
ization of supine hypertension on standing are very In clinical practice, the diastolic criterion is often
ignored. This may have several reasons: First, the absolute 12 3 172 Clin Auton Res (2017) 27:167–173 Diastolic OH and long-term prognosis Even though diastolic decline in BP during orthostasis may
be less relevant in the clinical diagnosis of OH, its potential
impact on long-term prognosis must be borne in mind. Orthostatic hypotension has been consistently associated
with increased mortality and incidence of cardiovascular
disease in large population-based prospective studies
[2, 22]. Although a significant SBP decrease on standing
demonstrates a similar risk as combined OH criteria [6, 7],
in several studies an independent association between
diastolic (and often asymptomatic) OH and higher inci-
dence
of
myocardial
infarction
has
been
observed
[8, 9, 15]. Moreover, a greater decline in DBP, i.e. equal or
more than 20 mmHg, has been linked with higher mortality
in older patients [14]. We propose that in future epidemi-
ological studies, isolated diastolic OH should continue to
be assessed to clarify this point. Diastolic BP and neurogenic OH A diastolic BP decline within 3 min of standing equal to or
greater than 10 mmHg on at least three separate occasions
has been proposed by Streeten as a characteristic and
obligatory sign of neurogenic OH—i.e. severe autonomic
failure [25]. Streeten postulated that an absence of signif-
icant and consistent diastolic decline would preclude the
diagnosis of neurogenic OH. However, he also observed
that both neurogenic and non-neurogenic patients pre-
sented with significant SBP fall, and practically all these
patients could be identified as having OH on the grounds of
systolic criterion alone. From a clinical point of view, cerebral hypoperfusion is
the most important aspect of OH that must be addressed
and, in highly symptomatic patients, treated [21]. It has
been previously shown that for both symptom generation
and therapy monitoring, the systolic rather than the dias-
tolic (alone or in combination with systolic) hypotension is
the finding that carries the clinical importance [23]. As for
the diagnostic utility of DBP assessment, patients with
suspected neurogenic OH are usually referred to and
evaluated by experts in tertiary centers with access to
reliable diagnostic methods [10, 12, 13]. Thus, the role of
DBP in diagnosis of neurogenic OH is uncertain today and
should be elucidated in well-designed studies performed in
centers with experience in this condition. Conclusions An isolated abnormal orthostatic drop in DBP without a
significant fall in SBP is rare among patients with unex-
plained syncope and orthostatic intolerance. Approxi-
mately 95% of patients with classical OH can be identified
by systolic criteria alone. Our data imply that the systolic
criterion might be used instead of current OH definition
based on both systolic and diastolic criteria. Acknowledgements This study was supported by grants from the
Swedish Heart and Lung Foundation, the Crafoord Foundation, the
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Gestion des ressources humaines et synergie de petites entreprises du secteur informel de l’espace spécialisé Dingɛ ba du quartier Hippodrome de Bamako : Une contribution du système organisationnel des travailleurs et vendeurs de fer au développement du territoire.
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Revue Internationale des Sciences de Gestion
ISSN: 2665-7473
Volume 3 : Numéro 4
Gestion des ressources humaines et synergie de petites entreprises du secteur
informel de l’espace spécialisé Dingɛ ba du quartier Hippodrome de
Bamako : une contribution du système organisationnel des travailleurs et
vendeurs de fer au développement du territoire.
Management of human resources and synergy of small businesses in the
informal sector in the Dingɛ ba specialized space of the Hippodrome district
of Bamako: a contribution of the organizational system of workers and iron
sellers to the development of the territory.
Amara NIMAGA
Enseignant-chercheur
École Normale d’Enseignement Technique et Professionnel (ENETP)
Campus universitaire de Kabala-Mali
Laboratoire de Recherche en Organisation et Processus Sociaux (LAROPS)
République du Mali
nimaga_amara@yahoo.fr
Date de soumission : 21/09/2020
Date d’acceptati on : 26/10/2020
Pour citer cet article :
Nimaga, A (2020) « Gestion des ressources humaines et synergie de petites entreprises du secteur informel de
l’espace spécialisé Dingɛ ba du quartier Hippodrome de Bamako : Une contribution du système organisationnel
des travailleurs et vendeurs de fer au développement du territoire », Revue internationale des sciences de gestion
« Volume 3 : Numéro 4 », pp : 685 - 710.
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Résumé
La recherche s’intéresse au système organisationnel des travailleurs et vendeurs de fer
regroupés dans l’espace spécialisé Dingɛ Ba du quartier Hippodrome de Bamako. La GRH et
les facteurs émergents de la synergie de petites entreprises révèlent une importance particulière
en ce qui concerne leur contribution au développement du territoire. L’approche qualitative
inductive a été adoptée avec comme stratégie d’accès au réel, une étude de cas. Les données
ont été collectées à l’aide d’un entretien semi-directif et traitées au moyen de la technique
d’analyse de contenu. Les résultats montrent que le développement du territoire résulte du rôle
joué par l’activité du fer (employabilité, la qualité de vie des travailleurs, l’attractivité de
l’espace) et par les réseaux socio-professionnels et territoriaux mis en place par les ferrailleurs.
Les réseaux sont très synergiques et permettent à l’espace de bénéficier du partenariat avec les
habitants aux alentours de l’espace, les mairies, les organisations non-gouvernementales
œuvrant pour la formation des acteurs du secteur informel, les partis politiques et les touristes
venus de l’Occident. L’espace trouve dans cette synergie la clarification de son rôle et de ses
responsabilités dans le territoire à partir d’une GRH territoriale réseautée et d’autres facteurs
émergents du regroupement.
Mots clés : GRH ; espace spécialisé ; secteur informel ; développement du territoire ; synergie.
Abstract
The research focuses on the organizational system of workers and iron sellers grouped together
in the Dingɛ Ba specialized space in the Hippodrome district of Bamako. HRM and the
emerging factors of the synergy of small businesses reveal a particular importance as regards
their contribution to the development of the territory. The inductive qualitative approach was
adopted with a case study as a strategy for accessing reality. Data were collected using a semistructured interview and processed using the content analysis technique. The results show that
the development of the territory results from the role played by the activity of iron
(employability, the quality of life of the workers, the attractiveness of the space) and by the
socio-professional and territorial networks set up by the scrap dealers. The networks are very
synergistic and allow the space to benefit from the partnership with the inhabitants around the
space, town halls, non-governmental organizations working for the training of actors in the
informal sector, political parties and tourists from the West. The space finds in this synergy the
clarification of its role and responsibilities in the territory from a networked territorial HRM
and other factors emerging from the grouping.
Keywords: HRM ; specialized space ; informal sector ; development of the territory; synergy.
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Introduction
La réalité de l’économie du secteur informel est matérialisée par la présence d’un ensemble de
très petites entreprises de production, généralement implantées dans les zones urbaines des
pays en voie de développement (Maldonado, 2001). Le secteur informel est caractérisé
aujourd’hui par une disposition géographique dispersée, sans aucune recherche de synergie, ou
au contraire, par un regroupement dans des espaces qualifiables des espaces spécialisés (Salle
et Kane, 2013). Selon Sall et Kane, l’espace spécialisé signifie le regroupement dans un site ou
dans un espace d’un même corps de métier. Cette définition cadre bien avec l’espace des
travailleurs et vendeurs de fer du quartier Hippodrome de Bamako. Dans cet espace, on trouve
les travailleurs et vendeurs de fer de multiples corporations artisanales traditionnelles
regroupés sous des hangars de fortune façonné en tôles et en morceau de fer usés. Les hangars
sont appelés des « Gatta » et les fers usés à base desquels se font les activités sont
communément appelés en langue bambara « nèguɛ kolo ». L’espace spécialisé informel des
travailleurs du fer appelé « dingɛ ba » ou le « grand trou » est une forme territoriale qui se
rapproche du district italien, de la Syllicone Valley (Etats-Unis), de Kompetenznetze
(Allemagne), du Knowledge Clusters (Japon), du système localisé des PME de Ksar Hellal
(Tunisie) et du Douar Berbère (Maroc). Dans cette forme d’espace, les travailleurs sont très
bien organisés (Améziane, 2004) avec des caractéristiques économiques très intéressantes. La
principale caractéristique de l’espace spécialisé qualifié de l’espace économique au sens de
Pyke & al. (1992) est son organisation. Cette organisation a connu un développement
fulgurant, conduisant au succès des unités de production regroupées et en même temps, elle a
relancé l’intérêt conjoint du monde académique, des acteurs du territoire et des pouvoirs
publics qui sont les principaux acteurs du développement du territoire. Dans ce sens, on peut
noter à la suite de Lkhoumsi & Belgaid (2020, p. 351) que le territoire se développe ainsi sur la
base d’un accroissement cumulatif de compétences et évolue le long d’une trajectoire cognitive
prédéterminée par son positionnement initial. De plus, le regroupement sur le territoire instaure
un système et dans ce système où les entreprises sont enclines au regroupement, la place des
ressources humaines dans la chaîne des valeurs, l’organisation des politiques de GRH, leur
élargissement au territoire et la spécificité des relations d’emploi ouvrent de nouvelles
perspectives (Bories-Azeau & al., 2008). Cette nouvelle perspective offre de nouvelles
possibilités d’aborder les petites entreprises informelles regroupées dans un site à volonté ou
non, sous un autre angle que celui de la gestion des ressources humaines classiques. Elle est
indispensable à l’élargissement de la réflexion en dehors de l’organisation pour mieux
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envisager la dynamique des interactions très synergiques dans le territoire. Ainsi, cette voie
permet également d’envisager la capacité des acteurs territoriaux à combiner leurs ressources,
les cultures professionnelles des acteurs, l’animation et les ajustements des mutualisations, la
clarification de certains rôles et les responsabilités dans le territoire. Toutes ces situations
expliquent une fois de plus, l’émergence de la GRH dans le territoire qui se définit comme les
rapports inter-organisationnels et interpersonnels qui dépassent les frontières des unités de
production informelles regroupées en synergie. Elles sont vues, dans ce contexte, comme une
nouvelle forme d’organisation qui demeure une préoccupation majeure : perception de GRH
hors des unités de production, l’apparition des artefacts et leur prise en compte avec les
pratiques GRH territoriale dans l’appréciation du développement du territoire.
Cette
préoccupation trouve sa justification dans le questionnement qui consiste à savoir comment la
GRH et les facteurs non-humains émergents de la synergie des petites unités ont contribué au
développement du territoire des travailleurs et vendeurs du fer de l’espace spécialisé informel
du quartier hippodrome de Bamako ? Cette question majeure a pour objectif de comprendre et
d’expliquer le système par lequel, les travailleurs du fer se sont organisés par rapport à
certaines pratiques de la GRH pour développer leur territoire.
Pour atteindre l’objectif visé, des hypothèses de recherche (propositions de recherche) ont été
créées à partir des constats et des recensions de la documentation pour orienter la recherche : le développement de l’espace résulte de l’employabilité, de la qualité de vie des ferrailleurs et
de l’attractivité de l’espace. – la synergie des ferrailleurs a émergé des réseaux socioprofessionnels dans l’espace qui contribuent au développement du territoire. – l’organisation
synergique des ferrailleurs a permis de clarifier leur rôle et leur responsabilité dans le territoire.
Au regard de ces propositions, il importe donc fort de structurer le travail en revue de
littérature (1), en méthodologie (2), en présentation du cas étudié (3), en analyse des résultats
(4), en discussion des résultats (5) et en implication managériale (6) afin d’avoir une meilleure
compréhension du phénomène étudié.
1. La recension de documentation
La revue de littérature mobilisée est issue des travaux de la gestion des ressources humaines
territoriale (GRH-T), de la gestion des ressources humaines dans les petites et moyennes
entreprises (PME) du secteur informel et du développement des territoires (DT).
1.1. L’espace spécialisé informel comme une composante importante du développement
des territoires
L’espace spécialisé est un territoire économique, différent du territoire en termes de pays et de
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régions. Le territoire selon Ferru (2009 ; cité par Lkhoumsi & Belgaid, 2020, p.350),
correspond à un espace sur lequel s’inscrit un ensemble d’unités productives plus ou moins
cohérentes et constituent de ce fait, comme la firme, une forme d’organisation rassemblant un
pool de compétences spécifiques. Cependant, l’espace dans notre sens, est un petit lopin de
terre, à la dimension d’un terrain ou d’un site, qui abrite un nombre important d’unités de
production, œuvrant dans le même domaine d’activité mais dans de différentes spécialités.
Dans cet ordre, l’espace spécialisé informel est vu au sens de Vidal (2006, p.56) inspiré des
travaux de Mérenne-Schoumaker (2002) comme des « Sites » ou de « Territoire économique ».
Ces sites sont des terrains d’un coin du quartier aménagés par l’Etat et/ou par les collectivités
pour sédentariser les activités économiques.
Ces espaces sont autrement des espaces de redéploiement industriel dans certains endroits des
localités. Ils permettent à la localité d’avoir des économies d’agglomération (Arbour, 2008).
Ces économies d’agglomération sont importantes car, elles représentent de plus en plus des
rentes de localisation et peuvent être très élevées si la capacité entrepreneuriale de la localité
s’élargit.
Selon Marshall (1890), les avantages de l’agglomération résident dans la réduction de coûts qui
sont rendus possibles grâce à l’accès à un bassin de main-d’œuvre qualifiée et partagée, le
partage d’inputs spécialisés et les externalités de connaissances, nommées aussi « knowledge
spillover » (diffusion de la connaissance ou l’externalité des connaissances).
Les structures agglomérées gagnent davantage en se regroupant et les collectivités locales
également (mairies et centres secondaires d’état civil). Ces dernières s’en sortent à travers des
revenus
supplémentaires
d’impôts.
D’où,
l’intérêt
pour
les
autorités
de
miser
sur
l’agglomération des activités territoriales dans les zones urbaines afin de bénéficier de
rendement d’échelles externes (Marsahall, 1890). Le regroupement de petites entreprises créé
de la synergie, c’est-à-dire la coopération entre les acteurs du territoire et développe les projets
émanant des acteurs externes tels que les collectivités, les organismes non gouvernementales
en charges de la promotion des acteurs du secteurs informel. Ces projets peuvent émaner aussi
de l’initiative des acteurs qui se préoccupent du développement de leur territoire. Les projets
initiés sont les projets du développement local font fréquemment référence aux approches top
down et bottum up, considérant que le territoire reste au centre des préoccupations. Ces
démarches conduit les acteurs locaux à agir en relation entre le local, et ses modes de
régulation,
et
les
structures
institutionnelles
d'ordre
gouvernemental.
Ces
interactions
contribuent à assurer une dynamique positive de croissance (Aghaï & Vaesken, 2008). Ce qui
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pourrait soustraire les territoires d’isolement et les inscrire dans le processus les processus de
développement qui se structurent autour d'une démarche endogène, pour laquelle proximité,
synergie et confiance constituent des facteurs de succès (op.cit. p.03).
1.2. La GRH dans les petites entreprises du secteur informel
Dans le secteur informel, la gestion des ressources humaines présente une grande spécificité en
rupture avec son application classique comme nous l’entendons dans les entreprises formelles.
Ses pratiques sont réglementées et se tiennent dans de cadre strictement managérial, légal et
conventionnel. Elles relèvent d’une vision d’ensemble de l’entreprise contrairement aux
entreprises du secteur
informel où sa particularité reste son caractère intuitif, inhérent à la
forme de l’entreprise (Saini & Budhwar, 2008). Malgré l’hétérogénéité des entreprises
informelles de petite taille, la GRH dans le secteur informel est limitée au flair et à la guise du
patron dirigeant qui demeure hétéroclite dans ses attitudes comme le soulignent Messeghem &
Sammut (2011). Les pratiques de GRH telles que le recrutement (par le réseau personnel du
dirigeant), la rémunération (à la tâche, à la pièce, en nature), la formation (initiative du salarié
et sur le tas), la communication (orale) et le leadership sont des pratiques présentes dans les
unités de productions informelles de petite taille comme l’a montré Simen (2017) dans son
étude sur les pratiques de GRH dans les petites entreprises sénégalaises. Ces pratiques dans les
organisation informelles, précisons-le à la suite de Bayad & al. (cités par Chiarello, 2015) sont
la résultante des intentions stratrégiques déterminées du dirigeant propriétaire. C’est le
dirigeant qui détermine et interprète les pratiques RH comme l’ont noté Hambrick & Mason
(1984) dans leur théorie intitulée la théorie des échelons supérieurs. Cette théorie part d’un
double postulat où d’une part, les dirigeants ont des logiques d’action qui sont fonction de leur
interprétation personnelle et d’autre part, leur perception a trait à leurs expériences, leurs
valeurs et leur personnalité. La mobilisation de cette théorie permet de comprendre l’influence
du dirigeant sur les pratiques de la gestion des ressources humaines (GRH) dans les PME.
L’influence des pratiques RH n’est pas seulement démontrée par Hambrick et Mason (1984),
beaucoup d’autres auteurs comme Mahé de Boislande (2015), Torrès & Enrico (2014), Tidjani
(2006) ont montré que les caractéristiques personnelles du dirigeant avaient une influence sur
la GRH en PME, c’est-à-dire que c’est la fonction RH qui est gérée par le dirigeant lui-même.
Dans cette même vision, ce phénomène s’est vu renforcer par la convention discrétionnaire,
associée à la configuration entrepreneuriale, de Pichault & Nizet (2013). Cela a apporté des
éclaircissements sur les pratiques de GRH effectives des PME : peu formalisées, les politiques
RH ne sont pas clairement définies et peu reliées à la stratégie et la relation fusionnelle du
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couple dirigeant-GRH voient son équilibre bousculer. Toutes ces pratiques sont expliquées
davantage dans le tableau de convention discrétionnaire, élaboré par Pichault & Nizet (2013).
Selon ces auteurs, les pratiques opérationnelles de GRH des petites unités de production jouent
un rôle important dans la centralisation du pouvoir, la supervision directe, facilitant la
coordination directe entre les acteurs, le contrôle exercé au quotidien, la formulation de la
stratégie issue de la propre vision du dirigeant. Ces pratiques opérationnelles de GRH
s’inscrivent dans le cadre d’une GRH informelle et spécifique au sein de laquelle, il faut noter
une absence de critères prédéfinis (Adla et al., 2016). Ces pratiques de GRH informelles et
intuitives relevées contribuent à la performance de ces unités de production comme l’ont
attesté Sheehan (2014), Aït Razouk & Bayad (2011).
1.3. Le rôle de la gestion des ressources humaines dans la sédentarité et dans
l’organisation de petites entreprises
L’implantation conduit à la concentration géographique des entreprises qui procure au territoire
un avantage certain en termes d’emplois, de partenariats et de réseaux locaux. Ce phénomène
est connu de par le monde et pour preuve, nous pouvons citer en exemple la petite vallée de la
Bresle en Normandie (rapport du conseil économique et social de la Haute Normandie, 2005)
dans laquelle sont localisés 80% de la production mondiale de flacons de parfum de luxe
(Gosse & Sprimont, 2008), le pôle bancaire d’Hong-Kong (Enright, 2000) dans le domaine de
services, le pôle automobile de Detroit (Porter, 1998) dans le domaine de l’industrie et le
Douar Berbère marocain avec comme base d’étude, la synergie et la solidarité d’acteurs en
zone rurale aride (Aghai & Vasken, 2008). Les structures ancrées dans le Douar développent
une logique cognitive (synergie entre les parties prenantes) et s’appuient sur l’entraide et la
solidarité entre les groupes sociaux au niveau familial, communal et religieux avec l’esprit
d’entreprise et le comportement entrepreneurial comme le notent Touissate & Azdimousa,
(2020) dans leur étude sur l’entrepreneuriat social féminin et l’innovation dans la région du
Moyen-Orient et Afrique du Nord.
Des exemples nombreux ont ancré la problématique de la localisation en management
stratégique comme un choix de première importance pour les entreprises quant à la pertinence
de leur implantation, c’est-à-dire par la qualité de la demande locale, des infrastructures et des
fournisseurs régionaux de même que la proximité des marchés.
De plus, la localisation dans de nombreux cas est aujourd’hui une priorité pour certains acteurs
publics comme les collectivités qui mettent en place les moyens pour offrir des espaces
nécessaires à la domiciliation des entreprises. Les collectivités gagneraient dans cette politique
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à pérenniser les emplois locaux. Elles souhaitent également voir les entreprises créer de la
valeur ajoutée qu’elles gagneraient en partie à travers des prélèvements qu’elles investiront
dans les infrastructures locales.
Il convient de noter que le regroupement des entreprises favorise la proximité des organisations
qui offre une clé d’entrée intéressante pour appréhender les réseaux territoriaux.
Nous
pouvons déjà à ce niveau voir et comprendre, comment le territoire intègre la gestion des
ressources humaines. Cette intégration se comprend par la démarche de Torre & Rallet (2005)
qui, dans leur réflexion, distinguent la proximité géographique et la proximité organisée ; et de
Brechet & Saives (2001) qui notent que ces proximités sont inter-acteurs et entrent alors en jeu,
puis génèrent des apprentissages collectifs. Ces différentes proximités énumérées à la suite de
Boschma (2003), sont des atouts que les entreprises localisées peuvent tirer de l’avantage de
leur agglomération. Sans le regroupement, il est difficile à chacune d’elles, de bénéficier de ces
externalités. Celles-ci donnent du sens à la gestion des ressources humaines, qui est une
stratégie ancrée dans le processus d’implantation.
1.4. Le rôle de la gestion des ressources humaines dans le développement du territoire
La localisation favorise les quartiers périphériques du territoire ou de l’espace en emploi
(Bellone & Maupertuis, 2003), en gains de bien-être, en externalité technologique (Baldwin &
al., 1998) et en externalité pécuniaire (Hirschman, 1958 ; Myrdal, 1957). La localisation
rapproche les structures qui, à leur tour, voient leur proximité se renforcer à travers la
proximité cognitive. Cette dernière représente une dimension importante de pratique RH et elle
est centrale pour la survie et pour la pérennité des entreprises agglomérées. Elle lie les acteurs
et les rend interdépendants, à cause des savoirs partagés qui poussent les acteurs vers une
collaboration et un apprentissage efficace. Les avantages procurés par l’implantation des
entreprises agglomérées conduisent les acteurs à aspirer au développement, c’est-à-dire, ils y
trouveront une marque de l’amélioration ou de bien-être à travers les activités économiques,
initiées dans la localité. Le rôle de l’Etat et des collectivités est central dans ce genre d’activité
et d’économie qualifiée de « Local ». L’Etat et les collectivités ont la capacité de mettre en
place des institutions d’appui à des chartes locales de développement. Ces chartes sont
élaborées par les populations locales, dans le respect de leur identité et de leur expérience
historique de production de liens et de modes d’autocontrôle social (Dorvilier, 2007, p.90).
Le développement territorial s’impose aux acteurs locaux, la mise en place d’une certaine
norme explicite, impliquant tous les membres (Etat et les collectivités territoriales).
Les
normes doivent être issues d’une élaboration collective et par la population locale. Cela, est la
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démarche entreprise par beaucoup d’associations comme les coopératives, le groupement des
artisans et autres. Le développement territorial devient plus important dans le nouvel
environnement d’implantation des entreprises que dans l'ancien (où la notion de territoire
renvoyait principalement au national et par suite au régional). Le développement a pour base le
territoire et repose sur le local qui est un nouveau local. Ce nouveau local est différent de
l'ancien, non seulement du point de vue du territoire, mais du point de vue des liens sociaux. Si
ces deux constats sont corrects, le développement local offre des opportunités intéressantes
pour repenser les rapports entre l'économie et le social (Lévesque, 2006).
1.5. GRH et synergie de petites entreprises regroupées dans le territoire
La disposition des entreprises territorialisées en réseau permet d’appréhender, à travers la
théorie des réseaux sociaux, les groupements des travailleurs cristallisés dans les territoires.
Ces groupements synerques sont favorables au développement du capital social (Bories-Azeau
& al.Ibid. p.02) et à la production des ressources à dominant immatériel (Nahapiet et Ghoshal
1998 ; Alder & Kwon, 2002). Les regroupements favorisent aussi des échanges d’information
(Burt, 1992), le transfert de connaissances et développent le capital intellectuel (Nahapiet &
Ghoshal, 1998 ; Alder & Kwon, 2002, Andew, Inkpen & Tsang, 2005). Ils permettent
également des apprentissages technologiques et relationnels (Ferrary & Pesqueux, 2004).
Defelix & al. (2007) ont présenté un autre dispositif qu’ils appellent le pôle de mobilité. Ce
dispositif émane de leur expérimentation et a été créé en 2007 par des entreprises voulant nouer
un partenariat favorable à l’employabilité de leurs salariés sur un bassin d’emploi. Ce dernier
comme d’autres zones d’emploi fait émerger des pratiques RH communes qui favorisent la
territorialité des unités de production sur une zone géographique telle qu’on peut le constater à
travers le schéma ci-dessous :
Figure n°1 : émergence des pratiques ressources humaines dans le territoire
Regroupement
GRH dans le
te rritoire
- Capital social ;
- Production des RH ;
- Echange des informations ;
- Développement du capital intellectuel ;
- Apprentissage technologique ;
- Apprentissage relationnel.
Mobilité
Territorialité et
développement
du territoire
Employabilité
Réseau
Synergie des acteurs
Pratiques émergentes du territoire
Source : conception de l’auteur.
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Le schéma illustre le modèle de la contribution de la GRH au développement du territoire des
entreprises et comprend les éléments qui renforcent la territorialité des unités de production en
synergie dans un site géographique. Ceci est une structuration issue des sites comme les pôles
de compétitivité, des clusters, des districts industriels avec ou non l’implication des autorités
dans la gestion. Cette structuration est également connue dans les pays du Sud en général et au
Mali en particulier. Dans le cas du Mali, le périmètre d’intervention ou les dispositifs
territoriaux au niveau des espaces spécialisés sont limités aux mairies communales qui ont la
charge de tous les problèmes économiques locaux. Cette délimitation d’actions et l’implication
des représentants du territoire aux problèmes économiques locaux expliquent que le périmètre
de la GRH dépasse l’entreprise (Beaujolin-Bellet, 2008).
Dans le même ordre, Aggeri & Pallez (2002) relèvent les enjeux de l’ingénierie des dispositifs
territoriaux. Selon ces auteurs, tout est à construire et à légitimer. Les personnes sont souvent
plus importantes que les outils au niveau du territoire. C’est ce qui amène Ternaud (in Lamotte,
1998) à dire que l’émergence d’actions collectives suppose : mises en coopération, mécanismes
de coordination et d’adaptation entre agents du territoire. Celle-ci exige des relations sociales
denses (Granovetter, 1992), d’une durée suffisamment longue, d’une circulation efficace
d’informations et d’une proximité territoriale, de combinaison de proximités géographiques,
organisationnelles et institutionnelles.
Ce travail souligne l’importance des acteurs territoriaux car, ils sont au centre de notre
réflexion. Ce sont eux qui façonnent l’économie du territoire et de l’environnement et le
comportement des groupements sociaux. Ils ont au sein de leur territoire, une responsabilité
élargie. Cela va dans le sens de la proposition de Viet (2003) sur les grands principes d’action
pour les mutations économiques d’un territoire. Ces principes ont été identifiés par Aggeri &
Pallez (op.cit.) qui vont au-delà, en proposant la clarification des rôles et missions des
différents acteurs, la responsabilisation accrue au plus près du terrain et la globalisation du
traitement des problèmes : responsabilité élargie et partagée, non-séparabilité des interventions,
contractualisation souple et négociée, continuité.
2. Méthodologie de la recherche
Nous avons adopté une posture épistémologique interprétative avec une approche qualitative
inductive basée sur une étude de cas. Le choix est opéré à la suite de l’exploration qui a permis
de comprendre que les difficultés ne peuvent être saisies qu’à travers une approche qualitative
qui se veut descriptive. Elle se penche à analyser la culture, le comportement humain et le
groupe du point de vue de problématique étudiée. Cette approche permet de comprendre
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complètement ou de façon holistique le contexte social et surtout organisationnel des
travailleurs et vendeurs du fer de l’espace spécialisé qui constitue le cas étudie. Les travailleurs
et vendeurs du fer usé sont de la corporation des forgerons, des marmitons, des coupeurs et
vendeurs de fer, des démonteurs de monteur. Pour approcher ces travailleurs du fer, nous avons
procédé d’abord à une étude exploratoire qui nous a permis de prendre connaissance de
l’espace. Ensuite, nous avons pénétré l’espace à l’aide de contacts tissés au moment de
l’exploration. Enfin, grâce à nos contacts, nous avons pu conduire nos interviews dans
l’espace, au moyen d’un entretien semi-directif. Les entretiens ont été réalisés avec les
travailleurs du fer (11 personnes), les familles aux alentours (4 personnes) et les agents de la
mairie (4 personnes). Les entretiens ont eu lieu en moyenne 2,7 fois par individu avec
l’ensemble de nos interviewés.
Huit hangars (unités de production) des travailleurs et vendeurs de fer ont été retenus dans le
cadre de cette enquête. Dans chacun de ces hangars, nous nous sommes entretenus avec les
patrons, les salariés et les apprentis de tout corps de métier du fer.
Les hangars sont les unités de production qui n’ont pas formellement de nom. Nous leur avons
proposé une dénomination chiffrée par ordre d’entretien et par les initiales (nom et prénom) de
nom de leurs propriétaires. Cette illustration met en évidence l’effectif interrogé en fonction du
métier. Ainsi, les travailleurs et vendeurs de fer ne sont pas retenus en interrogation en fonction
de leur métier, mais en fonction de l’indication qu’ils donnaient à la fin de chaque interview
pour poursuivre les enquêtes (Effet boule de neige : 84,2%, soit 16 individus sur 19 en total).
Quelque rare d’entre eux ont été enquêtés par convenance (15,2%, soit 3 individus sur 19).
Nous avons utilisé la technique d’analyse de contenu thématique comme outil de traitement des
données. L’analyse a porté sur les données qui ont fait l’objet de lectures et de relectures après
la retranscription intégrale et fidèle de ces données. La relecture a permis de se familiariser aux
textes et elle a permis également de condenser et de réduire les informations, voire supprimer
celles qui n’ont pas de sens. A ce niveau se dégagent les thèmes qui se formulent au fur et à
mesure pour donner les sous-catégories desquelles émergent les catégories qui existaient déjà
et qui constituent les axes majeurs des résultats de la présente recherche.
3. Présentation de l’espace spécialisé
L’espace des travailleurs et vendeurs de fer se trouve dans la commune II du district de
Bamako et plus précisément, dans le quartier Hippodrome. Ce quartier est proche des
commerces et son emplacement géographique lui offre des opportunités économiques, à travers
les activités de commerce et surtout de l’artisanat, qui n’a cessé de prendre de l’ampleur.
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Cet espace a depuis toujours occupé une place importante dans la municipalité de
l’hippodrome avec la reconnaissance territoriale dont bénéficient les travailleurs et vendeurs de
fer. L’espace des travailleurs de fer est un espace vaste et emploie une main-d’œuvre
importante avec de nombreuses corporations artisanales. Il est réputé à l’échelon national et
régional dans la vente du fer usé. Les fers usés sont vendus sur place dans l’espace aux
forgerons, marmitons, menuisiers métalliques, soudeurs. Ils sont aussi exportés dans les pays
de la sous-région à destination des pays industriels asiatiques.
L’existence de l’espace spécialisé remonte aux années 1970. Il regroupe les travailleurs venus
pour la plupart des quartiers hors de la commune II. La dynamique de l’espace vient de toutes
les six communes du district de Bamako. Les habitants de la commune II participent moins aux
activités de la ferraille.
Le quartier Hippodrome est un quartier où les habitants aspirent nécessairement à des activités
plus nobles que celles de la ferraille. L’activité de la ferraille selon les impressions des jeunes
de ce quartier, est réservée aux habitants des quartiers défavorisés qui sont aux alentours. Ces
habitants des quartiers défavorisés ont un faible niveau de scolarité. Ce sont eux qui constituent
l’essentiel de la main-d’œuvre de l’espace. A l’issue du constat fait dans l’espace, nous avons
retenu que les travailleurs sont venus des quartiers Baconi (commune I, à l’Est de la commune
II), Sikoroni (Commune I), Faladiè (Commune IV, commune de la rive droite), cercle de Kati
(situé à 15 kilomètres de Bamako). Les populations de la Commune II mènent des activités
économiques très diversifiées selon l’organisation Water aid (2007). Cette économie est
marquée par la prédominance des activités tertiaires.
4. Présentation et analyse des résultats
Les résultats obtenus sont contenus dans le tableau ci-dessous. Ils montrent que le
développement du territoire résulte de deux facteurs, à savoir : les facteurs ressources
humaines et les facteurs non-humains. Ces derniers sont des artefacts qui interviennent dans le
développement du territoire et sont dépendants des facteurs ressources humaines. Les deux
facteurs sont regroupés dans le tableau ci-dessous et expliquent tous deux la contribution au
développement du territoire.
Tableau 1 : récapitulatif des catégories et des sous-catégories
Sous-catégories
Catégories
Dialogue, négociation.
Emplacement.
Regroupement des travailleurs
Diverses provenances.
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Choix et préférence du lieu.
Ressources humaines exogènes
Résolution de chômage (employabilité).
Stratégie de développement.
Développement des activités économiques locales.
Rôle de l’activité du fer
Qualité de vie des acteurs.
Rapport avec la maire.
Rapport avec la population locale.
Rapport avec les structures d’appui.
Rencontres avec les partis politiques.
Construction des rapports sociaux
Rapports clients/fournisseurs.
territoriaux
Réseaux sociaux territoriaux.
Rencontres sociales professionnelles.
Rencontres territoriales.
Source : Enquête de terrain (2019).
Pour mieux comprendre les données du tableau, il convient de proposer une présentation qui
schématise chacun de ces éléments.
Figure 2 : les facteurs RH qui contribuent au développement du territoire.
- regroupement : dialogue, négociation, prestataires externes ;
GRH
- rôle de l’activité de fer : employabilité, qualité de vie des acteurs ;
Développement
du
territoire
- construction des rapports : relations socio-professionnelles, réseaux.
Source : conception de l’auteur.
Figure 3 : les facteurs non-humains qui interviennent et contribuent au développement
- regroupement : provenances, choix et préférences du lieu ;
GRH
- rôle de l’activité de fer : activités économiques locales,
- construction des rapports : rencontre avec les partenaires.
Développement
du
territoire
Source : conception de l’auteur.
Le développement du territoire trouve sa logique d’abord dans le regroupement des travailleurs
et vendeurs de fer. Le regroupement a permis à l’espace de procurer des RH de manière
informelle, en provenance de divers endroits de Bamako et des Régions du Mali comme le note
Ch. C. le menuisier métallique lors de notre troisième entretien. Il soutient que rares sont les
travailleurs qui habitent le quartier Hippodrome. Selon lui, les travailleurs en majorité sont
venus des quartiers environnants et éloignés de l’Hippodrome. L’extrait de son explication est
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le suivant : « A part nous qui avons notre maison à la porte de l’espace, beaucoup de ces
travailleurs sont venus des quartiers qui se trouvent derrière Hippodrome ».
Un autre travailleur du fer est allé dans le même sens que le métallurgiste. Il s’agit du vendeur
de fer Bo. K. Il confirme en précisant le quartier de provenance des travailleurs de fer : « …
Tous les travailleurs de l’espace viennent des quartiers comme : Sikoroni, Bankoni, Bankoni
Ndjenguènebougou Djalakorodi, Titibougou, Kati, Sourouaka, Bougouni… ».
Les trois premiers quartiers sont proches du quartier Hippodrome et sont très défavorisés. Les
autres quartiers sont, selon l’interviewé, loin du quartier Hippodrome.
L’espace est un lieu de rencontre de tous les artisans de Bamako et du Mali. Les travailleurs ne
sont pas des ressources humaines originaires du quartier Hippodrome. Le quartier Hippodrome
ne fournit pas à l’espace ni les ressources matérielles, ni les ressources humaines. Toutes ces
ressources sont exogènes à l’espace.
Le regroupement dans l’espace à favoriser l’implantation des entreprises qui évoluent dans une
grande proximité économique facilitant la mobilité des travailleurs dans l’espace. Dans cette
mobilité, se créer des ressources spécifiques qui bénéficient aux entreprises regroupées dans
l’espace. Ces entreprises bénéficient des expertises et des techniques de certaines d’entre elles
qui aboutissent à la mise en place d’une parfaite collaboration. Selon M. Ko le forgeron
(deuxième entretien, phase 1), c’est dans cette collaboration que naisse l’interaction, dans
laquelle il y a le devenir des entreprises regroupées dans l’espace spécialisé informel.
Ensuite, le rôle de l’activité du fer dans le développement du territoire n’est pas négligeable.
L’activité du fer est une solution à l’emploi comme beaucoup d’autres activités informelles
installées dans les villes et dans les régions des pays africains. Elle génère des possibilités
nécessaires pour occuper l’individu. Elle est source d’incitation à l’esprit entrepreneurial chez
les travailleurs soumis à l’apprentissage. L’entrepreneuriat informel est très développé dans le
métier du fer, car il est pour certains maîtres travailleurs du fer, le moyen permettant à un
apprenti en fin de formation de pouvoir se prendre en charge et surtout de devenir un
travailleur indépendant qui pérennisera la valeur du métier.
L’activité du fer est une stratégie de survie pour les travailleurs de fer et du développement de
la localité. Elle procure de nombreux avantages dans l’espace et dans les communes de
Bamako. Les collectivités (ou les mairies) tirent de nombreux avantages de ces activités surtout
en matière fiscale comme l’a dit l’un de nos interviewés Ad.S., le mécanicien lors de l’ent.
N°12, phase 2 : « L’activité que nous menons dans l’espace profite directement à la maire du
district, car nous lui versons régulièrement la taxe et l’impôt ».
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L’importance de l’activité du fer est telle que personne n’est en marge. On voit certains
habitants aux alentours se livrer aux activités du fer non pas pour produire, comme le font les
forgerons, les soudeurs et autres. Ceux-ci d’après les informations recueillies de notre
interviewé Bo. K le vendeur de fer, se livrent aux petites activités de ramassage des morceaux
de fer. Ils font des tas de fers pour revendre aux travailleurs de fer. L’extrait de ce témoignage
se présente comme suit : « Même les habitants de la localité s’intéressent au métier que nous
faisons ».
Enfin, il y a également les rapports territoriaux qu’il faut comptabiliser dans le développement
du territoire. Ce sont des rapports qui sont établis entre les acteurs pour soutenir et développer
les initiatives des actions sociales comme sociétales territoriales dans le cadre des rencontres
sociales professionnelles et territoriales.
Les rencontres sociales et professionnelles sont établies à partir d’actions sociales qui sont
destinées à la construction des rapports entre les travailleurs de fer. Par contre, les rencontres
sociétales sont fondées à la suite des actions sociétales qui sont orientées vers la protection de
l’environnement dans lequel, se situe l’espace. Cette dernière, concourt à la consolidation de la
relation entre les travailleurs et les habitants de la localité.
Par ailleurs, les réunions organisées dans l’espace entre les travailleurs, entrent également dans
le cadre de la construction des rapports sociaux. Dans l’espace, il y a des rencontres des zones
d’activités. Ces rencontres sont l’occasion d’échanges sur des problèmes des zones d’emploi,
sur des problèmes entre travailleurs et aussi sur des problèmes relatifs aux événements sociaux.
Ces
rencontres développent la solidarité,
favorisent l’engagement et cultivent l’esprit
coopératif.
Ces relations ont contribué à la stabilité des travailleurs du fer dans la localité et ont en plus,
renforcé leur ancrage dans le territoire de l’Hippodrome. Ils ont su capitaliser le lien avec la
collectivité qui leur impose le paiement de la taxe marché et les aide à trouver des partenaires.
La taxe est le premier rapport entre la collectivité et l’espace. Ceci ressort de bon nombre
d’interviews que nous avons eu avec le quatrième adjoint au maire, responsable de l’espace
spécialisé et tous les travailleurs et vendeurs de fer.
Pour stabiliser les relations, les travailleurs font tout pour éviter à la population tout ce qui de
nature peut la mettre, mal à l’aise. Mais la population a tendance à minimiser tout ce que les
travailleurs du fer font dans l’espace et elle apporte son soutien aux travailleurs du fer. C’est ce
que l’un de nos interviewés racontait lors de notre entretien : « Nous avons conscience du bruit
que nous faisons et qui dérange la population locale. Nous avons compris que la population ne
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se plaint pas de nous. Elle sait que nous vivons de cette activité par conséquent, elle nous
supporte à cause de la familiarité qui ne date pas d’aujourd’hui. Nous sommes devenus comme
une famille et dans ce cadre nous partageons ensemble beaucoup de choses ».
Les chefs des hangars de l’espace avec leurs travailleurs ainsi que leur association
s’investissent dans la préservation des rapports, en prenant part à des actions citoyennes dans le
territoire. Cela est une obligation recommandée à toutes les entreprises et surtout à celles
agglomérées dans une localité. Ces actions témoignent de la responsabilité sociale et sociétale
des unités de production, qui est un sujet fort dans le cadre du développeme nt du territoire.
Le développement du territoire est un développement venant en grande partie, des initiatives
des acteurs qui se mobilisent à l’échelle de leurs unités de production localisées dans le
territoire.
Le territoire prend en compte ici le quartier Hippodrome et la commune II du district de
Bamako. Le développement du territoire ici dans ce cas est pris au sens où les actions
impliquent plusieurs acteurs, à savoir : les travailleurs du fer, les institutions locales et les
habitants de la localité de l’espace spécialisé. Les actions de l’espace ont permis de mettre en
place un système de relation sociale et sociétale qui a aujourd’hui un apport économique et
financier indéniable non seulement pour les habitants, mais aussi, pour la mairie et le district de
Bamako. Ce développement est exogène du point de vue des travailleurs. Le quartier
Hippodrome ne produit pas du fer. Il ne pourvoit pas l’espace en ressources humaines. Tous
ces acquis viennent de l’extérieur pour se concentrer dans l’espace de l’Hippodrome. L’espace
est créateur de la valeur et il est le grand réservoir de main-d’œuvre et des ressources
humaines.
5. Discussion des résultats
Le développement du territoire est le résultat organisationnel de l’espace des travailleurs du
fer. Il est structuré en trois catégories, à savoir le regroupement, le rôle de l’activité du fer et la
construction des rapports. L’interpréter du processus de la territorialité de l’activité du fer et du
développement du territoire tourne autour de ces concepts.
Dans le processus du développement du territoire, la GRH occupe une place importante dans la
réflexion au regard de son rôle dans le processus d’implantation des travailleurs du fer. Ce rôle
s’explique par le dialogue, la négociation, l’échange et l’entente entre les autorités et les
travailleurs du fer. Ce sont ces éléments qui ont favorisé la transhumance des travailleurs du fer
de la gare du chemin de fer (situé au centre-ville de Bamako) au quartier Hippodrome.
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La délocalisation des travailleurs et vendeurs de fer répond aux objectifs fixés par l’Etat malien
vis-à-vis du secteur informel. Cette action s’est amplifiée et elle a été appuyée par l’initiative
du bureau international du travail (BIT) qui, en 1978, a mis en place un projet d'appui au
secteur non structuré de Bamako et autres villes secondaires du Mali. L’initiative du BIT
arrivait à point nommé, car le district de Bamako à lui seul était envahi et comptait près de
223 000 unités de production informelle (Union Economique et Monétaire Ouest-africain, 2002).
Cette forte propension du regroupement des unités de production informelles et de l’ancrage
des travailleurs dans l’espace spécialisé est telle que, aujourd’hui, cet espace absorbe une partie
importante des chocs qui ne cessent de produire sur le marché du travail (Cogneau & al. 1996),
où les jeunes diplômés sans emploi et surtout les jeunes enfants à bas âge en déperdition
scolaire sont les plus représentatifs. Il y a afflux des individus en provenance des quartiers
défavorisés, proches du quartier Hippodrome, qui y accèdent pour trouver du travail à en croire
les résultats de l’étude de Lautier (1993, p.13) sur le secteur informel. Ceci, grâce au fait que
les travailleurs de l’espace sont des amis, des parents, des membres d’une même famille ou des
habitants qui logent dans le même quartier. Ces caractéristiques évoquées ont été démontrées
par Tidjani (2006, p.28) dans son étude sur la gestion des ressources humaines dans le secteur
informel en Afrique.
Cette affluence tous azimuts des quartiers défavorisés de Bamako et des régions du Mali a fait
que,
aujourd’hui,
l’espace spécialisé informel de l’Hippodrome est devenu un vaste
« Réservoir de main-d’œuvre » (Savoye, 1997). Nous y trouvons un effectif pléthorique de
travailleurs de toutes les corporations artisanales : forge, marmiton, menuiserie métallique,
soudeur, coupeur de fer, vendeur de fer. Toutes ces activités sont ancrées dans l’espace à cause
des externalités positives qu’elles engendrent et de multiples avantages relevant de l’artisanat
informel.
Le regroupement des travailleurs constitue un aspect très important dans le
processus d’implantation définitive, car les unités de production des ferrailleurs sédentarisés y
ont trouvé un moyen de valoriser leurs structures. Les travailleurs valorisent leurs unités de
production à travers les ressources humaines créées, détenues, formatées et transformées en
actifs stratégiques valorisables qui sont des mécanismes émergés du rassemblement des
entreprises informelles dans une localité.
Les ressources humaines de l’espace ont sensiblement contribué au développement par
l’apprentissage. Les travailleurs de fer ne voient pas leurs carrières dans un seul hangar, mais
plutôt dans ceux d’autres travailleurs et vendeurs de fer de l’espace. Le mouvement effectué
par les travailleurs dans l’espace d’un hangar à un autre, renvoie à la mobilité organisationnelle
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et inter-organisationnelle (Roger & Ventolini, 2005) qui favorise et enrichit les unités de
production en matière de la mutualité (Chabault & Hulin, 2011). L’enrichissement mutuel est
issu du dynamisme des agents et il est très favorable à la construction du réseau dans le
territoire et c’est ce qui traduit véritablement le développement du territoire.
Les enjeux de la construction des relations se situent à l’entrée de l’espace spécialisé et étendu
à toutes les unités de production de l’espace. On trouve dans cette extension un moyen de
cerner la construction des liens dans le territoire (Bories-Azeau & al., 2008) à travers des
actions socio-professionnelles, sociétales et des réseaux créés et mis en place par les
travailleurs de fer.
Le lien existe à cause du fait que les travailleurs intègrent l’espace dès leur enfance et cette
intégration à bas âge dans l’espace se fait par le lien familial ou le capital social (Loup et
Paradas, 2005). Ce lien part de plusieurs considérations telles que l’enseignement à l’esprit
maison, loyalisme, prégnance des cultures de métier traditionnelles, primat des relations
interpersonnelles, etc. (Pichault & Nizet, 2013). Ce sont des valeurs et considérations que les
responsables des hangars souhaitent inculquer aux jeunes enfants en début de carrière. Ces
valeurs sont enseignées avec insistance, car elles influencent grandement les relations et c’est
pourquoi, il est nécessaire de veiller à leur pérennisation pour assurer la continuité et la relève
qui sont des éléments phares du développement du territoire.
Nous clôturons ce point par un mot sur la collectivité (la mairie du quartier Hippodrome et du
quartier Missira) qui a une forte implication dans la territorialité des travailleurs de fer et dans
le développement du territoire. Les actions de la mairie dans le territoire sont très limitées.
Certaines initiatives qu’elle entreprenne, pour s’engager dans des actions en faveur de la
promotion sociale des travailleurs du fer, allant dans le sens des actions préconisées par Croset
(2008) restent à désirer. Elle a œuvré pour inciter davantage les travailleurs de fer au
regroupement et surtout, elle a encouragé l’association des travailleurs de fer mise en place
dans l’espace en lui offrant un bureau au sein d’une de ces antennes qui se trouve dans le
quartier Missira. Ceci pour permettre à la mairie d’être en bon terme avec les travailleurs du fer
et enfin, de contribuer également au développement des activités du fer et au développement
d’une économie locale. C’est d’ailleurs, ce qui a consolidé les rapports entre la maire et les
travailleurs du fer. Cette consolidation de relation est l’expression du développement du
territoire qui prend de l’élan sur des dimensions sociales nées des proximités entre les
entreprises regroupées au niveau local.
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6. Implication managériale
Les résultats de cette recherche ont des implications managériales articulées autour de la
gestion du processus de développement du territoire. L’espace des travailleurs de fer connaît
quelques difficultés dans la gestion du processus de son développement. Ces difficultés sont à
différents niveaux des étapes du processus de développement. On note que le développement
ne s’appuie pas sur les ressources propres au territoire. Les travailleurs de fer, majoritairement
venus de tous les coins de Bamako, voire des régions du Mali. Trois travailleurs sur onze (soit
27,27 %) viennent de la Commune II (le quartier Hippodrome à deux travailleurs et le quartier
Missira à un travailleur). Il est difficile dans ce cas de parler du développement endogène qui,
normalement, devrait puiser ses ressources dans le territoire d’implantation des unités de
production. Malheureusement, ce n’est pas le cas avec la faible représentation des habitants du
quartier Hippodrome dans cet espace de travail. Sur l’effectif de 11 personnes enquêtées, deux
sont du quartier Hippodrome, soit 18,18 %, ce qui n’est pas significatif pour apprécier la
politique du développement local endogène. Ainsi, cette faible représentation s’explique par le
fait que l’espace se retrouve dans un quartier où les habitants, ont un niveau de vie plus ou
moins élevé. On y recense deux types de classe à savoir, la haute classe et la classe moyenne
qui sont, soit dans les activités administratives, soit dans le commerce. Il y a également une
frange importante qui relève de la famille des expatriés. Il s’agit des familles, dans lesquelles
un membre (deux, sinon plus) vit à l’extérieur : en Afrique (Angola, Centrafrique, Congo, etc.),
en Europe (France, Italie, Angleterre, etc.), aux Etats-Unis. Celles-ci préfèrent que leurs
parents (enfants) fréquentent l’école pour exercer un métier bureaucratique noble et propre que
celui du fer.
Ceci étant, l’espace ne fait vivre que la population se trouvant à ses proximités immédiates et
non la majorité des habitants du quartier Hippodrome. La population en majorité, ignore
l’activité et n’en fait pas une préoccupation contrairement à ce que nous avons constaté dans le
Douar Berbère (au Maroc). Dans le Douar, la population locale vit de leurs activités et elle est
très présente avec une communauté de travail très dynamique.
Enfin,
le
dernier aspect de la faible représentativité des travailleurs originaires de
l’Hippodrome dans le processus du développement est celui d’une mauvaise division
administrative et territoriale des quartiers des communes et de la ville de Bamako. Le quartier
Hippodrome est un quartier proche des deux grands marchés du centre-ville et du district de
Bamako vers l’Est. Mais à l’Ouest, son extension se limite aux quartiers Banconi et Sikoroni,
deux quartiers de la commune I qui posent un problème de délimitation géographique. La
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population de ces quartiers est très misérable en termes de niveau de vie et le manque
d’emplois les oblige à se livrer aux petits boulots du secteur informel. Ce qui explique la cause
leur affluence vers l’espace.
Conclusion
La démarche adoptée a permis de comprendre et d’expliquer le système, par lequel les
travailleurs et vendeurs du fer se sont organisés pour développer leur territoire. Le
développement du territoire s’inscrit dans l’organisation et surtout de la synergie des
travailleurs et vendeurs du fer. Ces deux aspects sont les principaux atouts de l’espace
spécialisé informel du quartier Hippodrome de Bamako.
L’agglomération des unités de production dans l’espace spécialisé, la synergie créée par le
regroupement et le réseau socio-professionnel et économique mis en place par les travailleurs
du fer, ont permis de répondre aux besoins locaux d’emplois tant au niveau des employeurs
que celui des demandeurs. L’espace est un grand pourvoyeur d’emplois à cause de
l’interaction, de la coopération et de la concurrence entre les acteurs. Il est garant
d’opportunités aux acteurs et aux parties prenantes. Les opportunités offertes par l’espace se
situent à quatre niveaux : les employeurs font face à une abondance de main-d’œuvre, les
travailleurs acquièrent des qualifications dans un métier et gagnent de revenus de leur travail,
la population locale développe son commerce et enfin, la mairie du district, quant à elle, tire sa
recette de l’impôt payé par les travailleurs et vendeurs de fer. L’espace émerge de multiples
pratiques dans le domaine de la GRH : les modalités d’entrée dans les hangars, les traitements
relatifs au salaire et aux conditions de travail, la relation entre les hangars qui s’intensifie par la
mutualité et la socialisation de l’activité qui engendrent tous, l’employabilité aux travailleurs
en synergie. Ces pratiques en plus des artefacts émergés (externalisation, partenariat, créneau
de nouveaux marchés) se généralisent à tout l’espace et prennent de l’ampleur au niveau de
tous les hangars de l’espace. Elles ont dépassé le stade des organisations et intègrent la
dimension du territoire.
L’extension de ces pratiques est le résultat de l’émergence d’une pratique de GRH territoriale.
Cette extension se trouve être limitée à cause des artefacts découverts dans le processus et qui
influencent aussi le développement du territoire au même titre que les pratiques GRH
émergentes.
L’émergence des pratiques ressources humaines est issue de la volonté et des choix des acteurs
qui ont décidé de rester travailler dans l’espace dans une logique de regroupement formant une
communauté de travail. Ce socle communautaire leur a permis de partager les valeurs autour,
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desquelles tous les acteurs s’identifient. Il est l’expression d’attachement des acteurs à l’espace
spécialisé et au territoire du quartier Hippodrome qui est devenu le centre névralgique des
activités informelles des fers usés. L’activité du fer, menée dans l’espace spécialisé, implique
une dynamique forte avec de multiples corporations de l’artisanat traditionnel qui construit le
territoire, tout en s’ouvrant aux réseaux de relations étendus à tous les travailleurs de l’espace.
Il s’agit d’un système participatif où les acteurs conjuguent leurs forces. La conjugaison des
forces permet de mener des actions d’œuvre sociales et sociétales dont la finalité est de
construire les rapports territoriaux. Elle entraîne aussi les acteurs vers une cohabitation
harmonieuse et durable dans l’espace et dans l’environnement externe de l’espace. Ces actions
constatées, concourent à la préservation des acquis de l’espace dans une logique de
conscientisation des acteurs dans le respect des principes et des valeurs qui sont la base du
fondement de leur environnement de travail. Ceci est l’expression de la contribution des
travailleurs de fer du secteur informel au développement de leur territoire qu’il serait judicieux
de repenser aux autres acteurs et activités informelles regroupées dans les autres communes du
district de Bamako et dans les régions du Mali. Plus précisément, il s’agit de prolonger les
recherches futures à toutes les activités informelles du type regroupé ou isolé dans les quartiers
des communes du district de Bamako et ses environs afin de mieux apprécier la contribution de
ces activités au développement des territoires. Ce prolongement s’inscrit dans le cadre de la
continuité de la réflexion sur l’axe thématique GRH, secteur informel et développement du
territoire. Il permet de convoquer les concepts comme la pérennité et la souffrance au travail
dans le secteur informel, la dynamique des activités informelles, la responsabilité sociale, le
manque d’éthique qui sont des variables nécessaires à mettre en exploration dans le secteur
informel afin d’apprécier leurs impacts sur le développement du district de Bamako.
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Climate Services to Improve Public Health
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Michel Jancloes 1,*, Madeleine Thomson 2,3, María Máñez Costa 4, Chris Hewitt 5,
Carlos Corvalan 6, Tufa Dinku 2, Rachel Lowe 7 and Mary Hayden 8 Michel Jancloes 1,*, Madeleine Thomson 2,3, María Máñez Costa 4, Chris Hewitt 5,
Carlos Corvalan 6, Tufa Dinku 2, Rachel Lowe 7 and Mary Hayden 8 Michel Jancloes 1,*, Madeleine Thomson 2,3, María Máñez Costa 4, Chris Hewitt 5,
Carlos Corvalan 6, Tufa Dinku 2, Rachel Lowe 7 and Mary Hayden 8 1 Health and Climate Foundation, Washington, DC 20005, USA 2 International Research Institute for Climate and Society, Columbia University, Palisades, New York,
NY 10964, USA; E-Mails: mthomson@iri.columbia.edu (M.T.); tufa@iri.columbia.edu (T.D.) 2 International Research Institute for Climate and Society, Columbia University, Palisades, New York,
NY 10964, USA; E-Mails: mthomson@iri.columbia.edu (M.T.); tufa@iri.columbia.edu (T.D.)
3 Mailman School of Public Health, Columbia University, New York, NY 10032, USA
4 Climate Service Center, Fischerstwiete 1, Hamburg 20095, Germany; E-Mail: maria.manez@hzg.de
5 Met Office UK, Exeter, Devon EX1 3PB, UK; E-Mail: chris.hewitt@metoffice.gov.uk
6 Pan American Health Organization, Brasilia CEP 70800-400, Brazil; E-Mail: corvalanc@paho.org
7 Institut Català de Ciènces Del Clima, Barcelona 08005, Spain; E-Mail: rachel.lowe@ic3.cat
8 National Center for Atmospheric Research, Boulder, CO 80301, USA; E-Mail: mhayden@ucar.edu 5 Met Office UK, Exeter, Devon EX1 3PB, UK; E-Mail: chris.hewitt@metoffice.gov.uk
6 Pan American Health Organization, Brasilia CEP 70800-400, Brazil; E-Mail: corvalanc@paho.org
7 Institut Català de Ciènces Del Clima, Barcelona 08005, Spain; E-Mail: rachel.lowe@ic3.cat
8 National Center for Atmospheric Research, Boulder, CO 80301, USA; E-Mail: mhayden@ucar.edu * Author to whom correspondence should be addressed; E-Mail: michel.jancloes@gmail.com;
Tel.: +00-33-683052549. Received: 17 February 2014; in revised form: 16 April 2014 / Accepted: 17 April 2014 /
Published: 25 April 2014 Received: 17 February 2014; in revised form: 16 April 2014 / Accepted: 17 April 2014 /
Published: 25 April 2014 Abstract: A high level expert panel discussed how climate and health services could best
collaborate to improve public health. This was on the agenda of the recent
Third International Climate Services Conference, held in Montego Bay, Jamaica,
4–6 December 2013. Issues and challenges concerning a demand led approach to serve the
health sector needs, were identified and analysed. Important recommendations emerged to
ensure that innovative collaboration between climate and health services assist
decision-making processes and the management of climate-sensitive health risk. Int. J. Environ. Res. Public Health 2014, 11, 4555-4559; doi:10.3390/ijerph110504555 Int. J. Environ. Res. Public Health 2014, 11, 4555-4559; doi:10.3390/ijerph110504555 International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph International Journal of
Environmental Research and
Public Health
ISSN 1660-4601
www.mdpi.com/journal/ijerph Keywords: public health; climate services; weather alert; health policies Keywords: public health; climate services; weather alert; health policies 1. Background Health providers have long understood that knowledge about the climate is relevant to clinical
practice and public health programming. Building on recent advances in our understanding of climate
science and the risks of climate change, national climate services and related institutions have begun to
prioritize the development and provision of global services, to better meet the needs of users and
decision makers in the management of climate-related risks. However, while climate services are being developed for different sectors, such as agriculture and water
resource planning, it is clear that effective demand from the health community in many regions is very low
or non-existent. Health policy makers and practitioners are well aware of the impact of climate on the
dynamics of many diseases and health conditions, such as malaria, emerging infectious diseases,
cardiovascular disease, nutrition deficiencies and food security. Despite this understanding,
climate information is rarely exploited as a means to help prevent and control such health risks. Further, climate service providers are generally absent from the broad public health community of practice. Michel Jancloes 1,*, Madeleine Thomson 2,3, María Máñez Costa 4, Chris Hewitt 5,
Carlos Corvalan 6, Tufa Dinku 2, Rachel Lowe 7 and Mary Hayden 8 Key recommendations included: a move from risk assessment towards risk management;
the engagement of the public health community with both the climate sector and
development sectors, whose decisions impact on health, particularly the most vulnerable;
to increase operational research on the use of policy-relevant climate information to
manage climate-sensitive health risks; and to develop in-country capacities to improve
local knowledge (including collection of epidemiological, climate and socio-economic data),
along with institutional interaction with policy makers. Int. J. Environ. Res. Public Health 2014, 11 4556 Int. J. Environ. Res. Public Health 2014, 11 Int. J. Environ. Res. Public Health 2014, 11 This Communication summarizes the results of the discussion. Significant challenges to the use of
climate information in operational decision-making were observed, including the fact that the health
sector makes up only a fraction of decisions which impact human health. Other significant sectors that
have a big impact on public health include agriculture, water resource management and disaster risk
reduction (which have their own distinct information and decision-making cultures and mechanisms),
as well as education and urban planning. Thus, health may be considered a down-stream outcome of
many sectors—but a priority of only one. An example of this type of disconnect is the historic focus of
agricultural production on yield—with little or no attention to nutrition. It is essential to explicitly
highlight the importance of health deliverables for other sectors. At the same time, the contribution of
health to the performance of critical national development sectors should be emphasized. A Health exemplar, prepared for the GFCS in collaboration with WHO [2], sets the stage for the
engagement of the health sector with GFCS through a “User Interface Platform” but does little to
connect a broader public health agenda that builds on the responses of representatives from other key
sectors. This is a concern that must be addressed at the global and local level. The climate community expressed a clear readiness to provide services for public health authorities
and health related issues, through demand-oriented institutional relationships. However, identifying and
sustaining long-term partnerships with health policy and practice communities can be challenging. As a prerequisite for sustained collaboration, it was recognized that health officials have to be familiar
with the potential use of climate services and information. At the same time, climate service providers
have to understand the epidemiological and operational context of problems for health officials and
solutions for which climate information can influence their decisions. Future requirements include the need for appropriate evidence of the cost/benefit value of climate
services to the health community and the inclusion of climate information in health planning and
programming. While progress has been made through time-limited projects and training events,
the lack of an institutionalized approach to the development of climate services for the health sector
was considered a major barrier to a wider uptake. The technologies capable of providing
“early warning” are rapidly improving. 2. Discussions Recently, international fora have called for a paradigm shift in the development and delivery of
weather and climate services for the health community [1,2]. This builds on innovative “demand led”
approaches, which can be integrated into current decision-making processes for both the formal health
sector and other sectors that impact human health [3]. This solution-driven approach was the subject of a high-level expert panel at the recent
Third International Conference of Climate Services (ICCS3), held in in Montego Bay, Jamaica,
4–6 December 2013. The meeting was developed in response to the Global Framework for
Climate Services (GFCS), launched in Geneva in 2012 by the World Meteorological Congress. According to WMO [4], “The GFCS is a global partnership of governments and organizations that
produce and use climate information and services. It seeks to enable researchers and the producers and
users of information to join forces to improve the quality and quantity of climate services worldwide,
particularly in developing countries” [5]. Health is one of the four priority areas of focus for the GFCS,
the other three being agriculture, water and disaster risk reduction [2]. This panel was organized by the Health and Climate Foundation in collaboration with the
German Climate Service Center. After opening remarks from the organizers, and presentations from
the UK Met Office, the Pan American Health Organization and the International Research Institute for
Climate and Society, an open discussion with workshop participants was held, which addressed
the following questions: Why is there such a gap between climate and health communities? How can we best move towards sustained collaboration? 4557 Int. J. Environ. Res. Public Health 2014, 11 However, ensuring effective response to such warnings is
a challenge and requires political will. Despite the limitations noted, good examples of the development and operationalization of weather
and climate services for health were presented by the panelists with examples from Brazil [6],
Ethiopia [7], Nepal, Niger, United Kingdom and Yemen, among others. References were also made to
existing multidisciplinary networks dealing with meningitis epidemics [8] and human/animal
leptospirosis (the GLEAN Project). A particular challenge observed across initiatives was the gap between the time and space scales
needed by users vs. what can be credibly delivered based on our scientific knowledge and capabilities. There is a rapidly developing capacity (through better modelling and more powerful computers)
to accurately forecast short-term weather events and to provide probabilistic seasonal climate forecasts
and longer-term climate scenarios. Also, participants pointed out the need for reliable epidemiological
data, which are still scarce in many countries, to enable relevant analyses of climate information that
will lead to meaningful findings for decision-making by the health community. Moving forward, priority is being given to opportunities with a high level of pay-off in both a
practical and political perspective. Examples proposed included emergency preparedness and prevention 4558 Int. J. Environ. Res. Public Health 2014, 11 and response to acute events such as epidemics or heat waves [9]. Alternatively, incorporating historical
and “near real time” monitored climate information into routine planning and assessment processes,
to better allocate resources (both in geographic space and for different seasons throughout the year)
was considered a significant opportunity. Experience indicates that while many ministries of health are
motivated to discuss “climate” in the context of “climate change”, the reality on the ground is that they
should prioritize better management of current climate variability, to meet pressing health priorities,
above information on longer time horizons. An applied systems approach, which seeks to address problems of public health significance is required. This can be achieved through the use of new knowledge and tools that bridge research and decision-maker
needs. This applied approach combines hard and soft systems methodology; seeking to optimize the
outcomes of the research at the interface of these two systems (climate services and public health). Acknowledgements This commentary emerged from the dialogue which followed ICCS3 conference panel
“Climate services for better health: moving from risk assessment to risk management” held in
Montego Bay, Jamaica, on the 5 December 2013 and facilitated by Madeleine Thomson. The summary
of the discussion was reported to the participants by María Máñez Costa. The authors acknowledge
the contribution of the discussion participants in stimulating this commentary. 3. Conclusions Following the panel presentations and discussion, a set of key recommendations for better climate
service provision, to ultimately improve public health, emerged: (1) To move from risk assessment towards risk management by clearly identifying climate
information needs for influencing decisions related to climate-sensitive health risk. For example, developing risk maps, warning and alert thresholds and intervention impact analyses. (2) To ensure not only better engagement of the public health community with the climate sector
but also all development sectors whose decisions can have an impact on health, in particular,
the health of the most vulnerable populations. (3) To intensify operational research on the use of policy-relevant climate information—past,
present and future—in the better management of climate-sensitive health risks. (4) To develop in-country capacities for better local knowledge (including collection of
epidemiological, climate and socio-economic data), along with institutional interaction with
policy makers. Author Contributions Michel Jancloes, Madeleine Thomson and María Máñez Costa co-wrote the original text which was
reviewed and revised by Carlos Corvalan, Chris Hewitt, Mary Hayden and Rachel Lowe. Rachel Lowe edited the final version and all authors approved the text. Int. J. Environ. Res. Public Health 2014, 11 4559 Conflicts of Interest The authors declare no conflict of interest. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/). References 1. Connor, S.J.; Omumbo, J.; Green, C.; DaSilva, J.; Mantilla, G.; Delacollette, C.; Hales, S.; Roger, D.;
Thomson, M. Health and climate—Needs. Procedia Environ. Sci. 2010, 1, 27–36. 1. Connor, S.J.; Omumbo, J.; Green, C.; DaSilva, J.; Mantilla, G.; Delacollette, C.; Hales, S.; Roger, D.;
Thomson, M. Health and climate—Needs. Procedia Environ. Sci. 2010, 1, 27–36. 2. Guillemot, J. Health Exemplar: Annex to the Implementation Plan for the Global Framework for
Climate Services; World Meteorological Organization: Geneva, Switzerland, 2013. 2. Guillemot, J. Health Exemplar: Annex to the Implementation Plan for the Global Framework for
Climate Services; World Meteorological Organization: Geneva, Switzerland, 2013. 3. Climate Risk Management in Africa: Learning from Practice; Hellmuth, M.E., Moorhead, A.,
Thomson, M.C., Williams, J., Eds.; International Research Institute for Climate and Society,
Columbia University: New York, NY, USA, 2007. 3. Climate Risk Management in Africa: Learning from Practice; Hellmuth, M.E., Moorhead, A.,
Thomson, M.C., Williams, J., Eds.; International Research Institute for Climate and Society,
Columbia University: New York, NY, USA, 2007. 4. Hewitt, C.; Mason, S.; Walland, D. The global framework for climate services. Nat. Clim. Change
2012, 2, 831–832. 4. Hewitt, C.; Mason, S.; Walland, D. The global framework for climate services. Nat. Clim. Change
2012, 2, 831–832. 5. Global Frame Work for Climate Services. Available online: http://www.gfcs-climate.org
(accessed on 21 April 2014). 5. Global Frame Work for Climate Services. Available online: http://www.gfcs-climate.org
(accessed on 21 April 2014). 6. Lowe, R.; Bailey, T.C.; Stephenson, D.B.; Jupp, T.E.; Graham, R.J.; Barcellos, C.; Carvalho, M.S. The development of an early warning system for climate-sensitive disease risk with a focus on
dengue epidemics in Southeast Brazil. Stat. Med. 2013, 32, 864–883. 7. Dinku, T.; Asefa, K.; Hilemariam, K.; Grimes, D.; Connor, S. Improving availability, access and
use of climate information. WMO Bull. 2011, 60. Available online: http://www.wmo.int/pages/
publications/bulletin_en/archive/60_2_en/60_2_Tufa_en.html (accessed on 17 April 2014). 8. Thomson, M.C.; Firth, E.; Jancloes, M.; Mihretie, A.; Onoda, M.; Nickovic, S.; Broutin, H.;
Sow, S.; Perea, P.; Bertherat, E.; Hugonnet, S. A Climate and Health Partnership to Inform the
Prevention and Control of Meningococcal Meningitis in Sub-Saharan Africa: The MERIT
Initiative. In Priorities in Climate Research, Analysis and Prediction; Hurrell, J., Asrar, G., Eds.;
Springer: Netherlands, 2013. 9. Rogers, D.P.; Tsirkunov, V. Weather and Climate Resilience Effective Preparedness through National
Meteorological and Hydrological Services; The World Bank: Washington, DC, USA, 2013. 9. Rogers, D.P.; Tsirkunov, V. 9.
Rogers, D.P.; Tsirkunov, V. Weather and Climate Resilience Effective Preparedness through National
Meteorological and Hydrological Services; The World Bank: Washington, DC, USA, 2013. References Weather and Climate Resilience Effective Preparedness through National
Meteorological and Hydrological Services; The World Bank: Washington, DC, USA, 2013. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
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doi:10.31261/PS_P.2018.21.21
POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21)
ISSN 1898-1593
AGNIESZKA MADEJA
U n i w e r s y t e t Ś l ą s k i
K a t o w i c e
Pan Cogito w Brazylii
Zbigniew Herbert, 2016, Podróż Pana Cogito. A viagem do Senhor Cogito,
Wydawnictwo Gnome, Katowice, ss. 139 POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21)
ISSN 1898-1593 doi:10.31261/PS_P.2018.21.21 doi:10.31261/PS_P.2018.21.21 AGNIESZKA MADEJA
U n i w e r s y t e t Ś l ą s k i
K a t o w i c e 1 Drugim tomem dwujęzycznej serii była książka wydana z okazji 20-lecia nadania godności
doktora honoris causa Uniwersytetu Śląskiego Stanisławowi Barańczakowi oraz 25-lecia Szkoły
Języka i Kultury Polskiej Uniwersytetu Śląskiego (Barańczak 2015). Zbiór zawiera wiesze pol-
skich poetów: Leszka A. Moczulskiegp, Ryszarda Krynickiego, Ewy Lipskiej, Piotra Somme-
ra, Jana Polkowskiego, Bronisława Maja, Juliana Kornhausera, Stanisława Barańczaka. Utwo-
ry te w języku angielskim zostały przedrukowane za: Polish Poetry of the Last Two Decades of
Communist Rule: Spoiling Cannibal’s Fun, edited by Stanisław Barańczak and Clare Cavanagh,
published 1991 by Northwestern University Press, Evanston, Illinois, USA. Pan Cogito w Brazylii Zbigniew Herbert, 2016, Podróż Pana Cogito. A viagem do Senhor Cogito,
Wydawnictwo Gnome, Katowice, ss. 139 W 2014 roku wydarzeniem literackim, jak nazwał publikację w swojej re-
cenzji Władysław Miodunka (2015), było wprowadzenie na rynek przez
Wydawnictwo Gnome dwujęzycznego tomiku poezji Paula Leminskiego Po-
wróciło moje polskie serce. Meu coração de polaco voltou w wyborze Piotra Kilanow-
skiego oraz w tłumaczeniach Piotra Kilanowskiego i Konrada Szcześniaka. Publikacja jest zwieńczeniem projektu „Poezja buduje mosty – polsko-
-brazylijskie seminaria literacko-kulturowe dla młodzieży polonijnej” reali-
zowanego przez Szkołę Języka i Kultury Polskiej oraz Katedrę Międzynaro-
dowych Studiów Polskich UŚ wspólnie z polonistyką Federalnego Uniwersy-
tetu Parany w Kurytybie (UFPR). Zadanie było dofinansowane przez
Ministerstwo Spraw Zagranicznych w ramach konkursu „Współpraca z Po-
lonią i Polakami za granicą w 2014 roku”. Polsko-portugalski tom wierszy Leminskiego został opublikowany dwu-
krotnie: w Katowicach w Polsce oraz w Kurytybie w Brazylii. Miodunka
pisze, że choć literatura brazylijska, a co dopiero poezja tego kraju nie jest
w Polsce popularna, tom „wierszy Leminskiego buduje znajomość twór-
czości poety w kraju jego pochodzenia” (Miodunka 2015, 273). Autor re-
cenzji tak podkreśla znaczenie publikacji: Dla mnie zorganizowanie takiego projektu i uwieńczenie go publikacją
tomiku wierszy znanego poety brazylijskiego polskiego pochodzenia jest
wydarzeniem samym w sobie. Dotąd podejmowano w Polsce inicjatywy 290 POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21) w celu wydawania u nas prac naukowych polonistów zagranicznych, ni-
gdy jednak nie spotkałem się z inicjatywą wydania tomiku zagranicznego
twórcy polskiego pochodzenia przez współpracujące ze sobą jednostki
uniwersytetu polskiego i zagranicznego. To inicjatywa bardzo oryginalna
i warta naśladowania z tego względu, że za granicą żyło i tworzyło wielu
zasłużonych Polaków i ludzi polskiego pochodzenia, o których w Polsce
nie wie się nic lub prawie nic (Miodunka 2015, 272–273). Na kolejną taką inicjatywę nie trzeba było długo czekać1. Mowa o trzecim
tomiku z dwujęzycznej serii zainaugurowanej zbiorem wierszy Leminskiego,
czyli polsko-portugalskim wydaniu poezji Zbigniewa Herberta. Tym razem to
nie brazylijski twórca polskiego pochodzenia był przybliżany Polakom, a zna-
ny polski poeta został zaprezentowany publiczności portugalskojęzycznej. Podróż Pana Cogito do Brazylii ma swój początek w 2015 roku. We wrze-
śniu tego roku odbyły się w Kurytybie warsztaty polonistyczne zorganizo-
wane przez Stowarzyszenie Sympatyków Szkoły Języka i Kultury Polskiej UŚ
w ramach projektu „Jak i dlaczego warto uczyć się Polski i polskiego – wy-
jazdowe warsztaty języka polskiego” przeprowadzonego przy wsparciu Mi-
nisterstwa Spraw Zagranicznych. Pan Cogito w Brazylii Na Federalnym Uniwersytecie Parany
w Kurytybie seminaria i zajęcia z zakresu polskiej kultury, literatury i języka
polskiego prowadziły Jolanta Tambor, dyrektor Szkoły Języka i Kultury Pol-
skiej UŚ oraz Danuta Opacka-Walasek z Instytutu Nauk o Literaturze Pol-
skiej im. I. Opackiego UŚ. W ramach warsztatów w Casa da Cultura Polônia Brasil miało miejsce
wielogodzinne spotkanie poświęcone poezji Zbigniewa Herberta. Wieczór
poetycki znalazł się w programie ogólnobrazylijskiej, promowanej w mediach,
państwowej imprezy pt. Wiosna Muzeów. Zgromadzonych przywitały i spo-
tkanie otworzyły Shirlei Freder, prezes Casa da Cultura Polônia Brasil oraz
Aleksandra Piasecka-Till z polonistyki UFPR. Noc z poezją poprowadzili
Danuta Opacka-Walasek, wybitna literaturoznawczyni, autorka książek o po-
ezji Herberta i Piotr Kilanowski, tłumacz wierszy poety, wykładowca z Ka- AGNIESZKA MADEJA: Pan Cogito w Brazylii… 291 tedry Polonistyki UFPR. Zainteresowanie poezją Pana Cogito „(jak często
określa się Herberta w Polsce, nazywając go imieniem persony lirycznej, któ-
rą stworzył w swoich wierszach)” (Opacka-Walasek 2016, 6) przerosło ocze-
kiwania organizatorów. W spotkaniu wzięło udział ponad 100 osób, byli to
m.in. przedstawiciele Polonii, nauczyciele i animatorzy kultury polskiej oraz
studenci. Publiczność mogła posłuchać recytacji wierszy Herberta, także ich
portugalskich tłumaczeń, które zaprezentowali m.in. studenci kurytybskiej
polonistyki. Długo też toczyły się rozmowy o emocjach i refleksjach, jakie
wywołuje twórczość poety, o wartościach, jakie przekazuje w swoich wier-
szach. Zaciekawienie poezją i żywa reakcja na twórczość Herberta sprawiły,
iż przedstawiciele obydwu uczelni partnerskich podjęli decyzję o konieczno-
ści przybliżenia dzieł polskiego poety osobom portugalskojęzycznym. Wśród nich są też potomkowie przedstawicieli najstarszej polskiej emigracji. Bariera językowa nie pozwala im poznać dziedzictwa kulturowego tak waż-
nego dla nich kraju przodków, swojej drugiej ojczyzny. Narodził się więc
pomysł wydania wierszy Herberta w tłumaczeniu na język portugalski. Tom Podróż Pana Cogito powstał przy współudziale i współpracy kilku
osób. Przede wszystkim należy wymienić inicjatorkę projektu, Jolantę Tam-
bor, dyrektor Szkoły Języka i Kultury Polskiej UŚ. Wraz z Romualdem Cu-
dakiem, kierownikiem Katedry Międzynarodowych Studiów Polskich byli
osobami prowadzącymi projekt oraz redaktorami naukowymi tomu. Istotną rolę odegrała również Danuta Opacka-Walasek, która opatrzyła
publikację słowem wstępnym. Badaczka, podkreślając ponadnarodowość i po-
nadczasowość Herbertowskich fraz, tak pisze o prezentowanej książce: Jeśli nadzieja wywiedziona z brazylijskiego, wrześniowego spotkania mia-
łaby okazać się ledwie wątłą, delikatną „struną światła” (tak brzmi tytuł
pierwszego tomiku poetyckiego Herberta z 1956 roku, stanowiąc meta-
forę brzemienną w sensy), to na tej jasnej i pełnej poetyckich brzmień
strunie chcemy umieścić kilkanaście jeszcze jego wierszy w przekładzie
portugalskim. Pan Cogito w Brazylii Z dumą – i z nadzieją. Z dumą, że przesłanie w nich za-
warte po raz kolejny spotkało tak żywy oddźwięk, i to wielopokoleniowy,
wielokulturowy (…). Z nadzieją, że brazylijscy odbiorcy poezji Zbignie-
wa Herberta będą mogli tak wiele z niej wziąć dla siebie (…) w wymiarze
artystycznym i tym bardziej ludzkim po prostu: w budowaniu siebie, wła-
snego stosunku do rzeczywistości (Opacka-Walasek 2016, 10–12). Jeśli nadzieja wywiedziona z brazylijskiego, wrześniowego spotkania mia-
łaby okazać się ledwie wątłą, delikatną „struną światła” (tak brzmi tytuł
pierwszego tomiku poetyckiego Herberta z 1956 roku, stanowiąc meta-
forę brzemienną w sensy), to na tej jasnej i pełnej poetyckich brzmień
strunie chcemy umieścić kilkanaście jeszcze jego wierszy w przekładzie
portugalskim. Z dumą – i z nadzieją. Z dumą, że przesłanie w nich za-
warte po raz kolejny spotkało tak żywy oddźwięk, i to wielopokoleniowy,
wielokulturowy (…). Z nadzieją, że brazylijscy odbiorcy poezji Zbignie-
wa Herberta będą mogli tak wiele z niej wziąć dla siebie (…) w wymiarze
artystycznym i tym bardziej ludzkim po prostu: w budowaniu siebie, wła-
snego stosunku do rzeczywistości (Opacka-Walasek 2016, 10–12). Wybór wierszy dokonany przez autorkę wstępu i tłumacza, Piotra Kila-
nowskiego, obejmuje 19 utworów. Wśród nich znaleźć można m.in. takie ty- POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21) 292 tuły: Dwie krople, Nike która się waha, Pan od przyrody, Apollo i Marsjasz, Powrót
prokonsula, Przesłanie Pana Cogito czy Potęga smaku. Bez nieocenionego autora
przekładu podróż Pana Cogito do Brazylii byłaby niemożliwa. Piotr Kila-
nowski do książki dołączył także epilog, posłowie, w którym dowiadujemy
się, w jaki sposób Pan Cogito granice owe przekracza: Nasza wieloznaczna Podróż Pana Cogito jest więc przekraczaniem granic,
zarówno tych, które znosi poezja w niej zawarta, jak i tych, które narzuca
język, ale też i tych fizycznych. Wszak ta Podróż Pana Cogito odbywa się za
pośrednictwem tłumacza, który również swój udział w przekraczaniu
granic ma. Ba, mówią, że tłumaczenie to przekraczanie granic. A w tej
książeczce wiele z nich przekraczamy – autor przekracza granice i podró-
żuje fizycznie i metaforycznie do dalekiej krainy, tłumacz przekracza gra-
nice i próbuje ułatwić podróż do innego języka, innej kultury (…). Nie-
wątpliwie Podróż Pana Cogito jest podróżą przez wiele granic: między Pol-
ską i Brazylią, ideą i jej realizacją, między dwoma językami i kulturami,
między pojmowaniem rutynowym a poetyckim wyzwoleniem (Kilanow-
ski 2016, 128). W książce Podróż Pana Cogito znajdziemy też teksty czworga studentów
UFPR na temat poezji Herberta. Pan Cogito w Brazylii Są to osoby, które wzięły udział w wieczo-
rze poetyckim w Casa da Cultura Polônia Brasil. To oni po polsku i portu-
galsku interpretowali teksty Herberta, a teraz mogli w kilku słowach opisać
emocje, jakie im wtedy towarzyszyły. Piszą o tym, co ich w twórczości pol-
skiego poety fascynuje, co sprawia im trudność, jak rozumieją jego wiersze. Tłumaczenia tekstów studenckich oraz opracowania graficznego tomu pod-
jął się Konrad Szcześniak z Instytutu Języka Angielskiego UŚ. Na koniec
warto wymienić Eneidę Favre, która przetłumaczyła wstęp i posłowie, a któ-
rej – jak pisze Piotr Kilanowski – „poprawki, nieocenione sugestie, cierpli-
wość i uważność, z jaką czyta moje tłumaczenia, znacznie polepszają ich ja-
kość” (Kilanowski 2016, 134). Należy jeszcze dodać, że tom Podróż Pana Cogito powstał w ramach projek-
tu „Pana Cogito poszukiwania języka ojczystego” prowadzonego przez Sto-
warzyszenie Sympatyków Szkoły Języka i Kultury Polskiej UŚ w ramach
programu Narodowego Centrum Kultury „Ojczysty – dodaj do ulubionych
2015”. Projekt obejmował też cykl spotkań poświęconych twórczości Zbi-
gniewa Herberta. 13 grudnia 2016 roku w Centrum Informacji Naukowej i Bibliotece Aka-
demickiej miała miejsce uroczysta promocja tomu Podróż Pana Cogito. Jolanta AGNIESZKA MADEJA: Pan Cogito w Brazylii… 293 Tambor otworzyła spotkanie i opowiedziała o projekcie, w ramach którego
powstała książka. Wykład przybliżający życie i twórczość wybitnego polskiego
poety wygłosiła Danuta Opacka-Walasek, autorka słowa wstępnego i znawczyni
poezji Herberta. Na uroczystości obecny był Konrad Szcześniak, a dzięki
nowoczesnym mediom goście mogli także połączyć się z Kurytybą i poroz-
mawiać z Piotrem Kilanowskim. Promocji towarzyszyła m.in. prezentacja
wyników sondy ulicznej poświęconej Herbertowi i obecności jego twórczo-
ści w świadomości Polaków (pytania przechodniom zadawali studenci mię-
dzynarodowych studiów polskich). Można też było posłuchać wierszy poety
po polsku, po portugalsku i po chińsku – w tłumaczeniu Zhao Ganga z Pe-
kińskiego Uniwersytetu Języków Obcych, profesora wizytującego w Kate-
drze Międzynarodowych Studiów Polskich. Prawie równolegle miała miejsce uroczysta promocja książki w Brazylii. 6 grudnia w Polônia Sociedade w Rio de Janeiro odbyło się spotkanie popu-
laryzujące tom wierszy Zbigniewa Herberta Podróż Pana Cogito. W uroczystości
wzięły udział Magdalena Bąk z Instytutu Nauk o Literaturze Polskiej im. I. Opackiego oraz Wioletta Hajduk-Gawron z Katedry Międzynarodowych
Studiów Polskich, które prowadziły cykl zajęć polonistycznych w Brazylii. Twórczość poety oraz promowany tom zaprezentowała zebranym Magda-
lena Bąk. Podróż Pana Cogito do Brazylii trwała rok. Tyle czasu upłynęło od po-
mysłu wydania dwujęzycznego tomu poezji Herberta do realizacji tej ini-
cjatywy. Pan Cogito w Brazylii Mamy nadzieję, że ten wybór wierszy Zbigniewa Herberta, który – jak
każdy niewielki wybór – jest tylko prezentacją możliwości poety, okaże
się zapowiedzią pełniejszej obecności jego dzieła w Brazylii. Siłę przesła-
nia utworów, na równi z ich walorami artystycznymi, potwierdzili
uczestnicy brazylijskiej Nocy Poezji (Opacka-Walasek 2016, 18). Herbert Z., 2016, Podróż Pana Cogito. A viagem do Senhor Cogito, wyb. i oprac. Opacka-Walasek
D., Kilanowski P., tłum. Kilanowski P., Katowice. Barańczak S., 2015, Nawet język umie układać się w ustach. Even the tongue knows how to curl in your
mouth. 20 wierszy na 20-lecie: Stanisława Barańczaka i Clare Cavanagh przekłady z polskiej poezji
współczesnej, Katowice. Literatura Barańczak S., 2015, Nawet język umie układać się w ustach. Even the tongue knows how to curl in your
mouth. 20 wierszy na 20-lecie: Stanisława Barańczaka i Clare Cavanagh przekłady z polskiej poezji
współczesnej, Katowice. Barańczak S., 2015, Nawet język umie układać się w ustach. Even the tongue knows how to curl in your
mouth. 20 wierszy na 20-lecie: Stanisława Barańczaka i Clare Cavanagh przekłady z polskiej poezji
współczesnej, Katowice. Herbert Z., 2016, Podróż Pana Cogito. A viagem do Senhor Cogito, wyb. i oprac. Opacka-Walasek
D., Kilanowski P., tłum. Kilanowski P., Katowice. POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21) 294 POSTSCRIPTUM POLONISTYCZNE, 2018 • 1 (21) Kilanowski P., 2016, O podróży i o przekraczaniu granic, w: Herbert Z., Podróż Pana Cogito. A via-
gem do Senhor Cogito, wyb. i oprac. Opacka-Walasek D., Kilanowski P., tłum. Kilanowski P.,
Katowice. Leminski P., 2014, Powróciło moje polskie serce. Meu coração polaco voltou, wyb. Kilanowski P., przeł. Kilanowski P., Szcześniak K., Katowice. Kilanowski P., Szcześniak K., Katowice. Miodunka W.T., 2015, Wydarzenie literackie: Leminski osobny, „Postscriptum Polonistyczne” 1 (15). z
p
Opacka-Walasek D., 2016, Słowo wstępne, w: Herbert Z., Podróż Pana Cogito. A viagem do Senhor
Cogito, wyb. i oprac. Opacka-Walasek D., Kilanowski P., tłum. Kilanowski P., Katowice. Opacka-Walasek D., 2016, Słowo wstępne, w: Herbert Z., Podróż Pana Cogito. A viagem do Senhor
C it
b i pr
Op
k W l
k D Kil n
ki P tł m Kil n
ki P K t
i Walasek D., 2016, Słowo wstępne, w: Herbert Z., Podróż Pana Cogito. A viagem do Senhor ogito, wyb. i oprac. Opacka-Walasek D., Kilanowski P., tłum. Kilanowski P., Katowice.
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W2062297289.txt
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https://zenodo.org/records/2219610/files/article.pdf
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en
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THE VALUE OF POSTURE IN THE AFTER-TREATMENT OF " STIFF-SHOULDER."
|
Lancet
| 1,920
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public-domain
| 2,656
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1266
serum
unclumped state (magnification structures
of the joint, causing pain on movement,
and, what is often very important, the persistence of a,
to the apparent disappearance of trench fever hysterical element, the result of prolonged disuse and
and in the control
2000).
Owing
it has not been possible so far to obtain samples of previous painful experiences.
A disablement of this description which has existed
blood from recent cases in order to ascertain whether
the reaction possesses a diagnostic value. The only for any length of time presents a difficult and often
material available has been derived from old cases of very unsatisfactory subject for treatment; it is accom6-12-18 months’ standing with symptoms of " chronic " panied by an amount of muscle-wasting and loss of
trench fever. For this material I am indebted to function which seems to be out of proportion to the
Dr. J. L. Dimond at the New End Hospital. None of extent of the original injury. The usual history in this
these cases gave a reaction even in 1 in 5 dilution. type of case is that of an accidental injury to the
Further data, however, may be obtained from the shoulder some months previously, treated by flexion
investigation on serological lines of experimentally of the arm, which was bound to the side. Subinfected volunteers, and more especially passage cases sequently difficulty was experienced in getting the
inoculated with infective blood.
arm away from the side and above the head ; this has
persisted in spite of treatment by massage, electricity,
S’icm7izar-.
and movement of the arm under an anaesthetic. The
(1) Experiments (about 50 in all) undertaken with the patient complains that the arm is painful when moveto
of
trench
fever
virus
transmitting
object
laboratory ment is attempted and is practically useless; on inquiry
animals proved in the main negative. A very small it is found
that no abduction arm-splint has been worn,
minority of inoculated rabbits and guinea-pigs, however, and that no attempt has been
made to minimise the
exhibited temperature charts which at least suggested effect of
or gradually to restore function in the
gravity
The
the
virus.
a successful transmission o,
question horizontal posture.
must for the present remain undecided, but it is
The Jlental Element.
probable that a certain small percentage of normal
rabbits and guinea-pigs may be susceptible to the
In many -cases of weakness of the muscles of the
virus.
shoulder-girdle I-have found what one might call a
(2) In the course of the work it was established that definite mental element in the disability; in. some
the rickettsia bodies present in the excreta of lice fed
this is manifested by the adoption of an
on
trench fever patients are agglutinated in the patients
anxious resistive attitude, and in others of a passive
presence of immune serum obtained from animals apathetic one.
In whatever way this condition is
immunised with infective lice excreta.
the result is that the disability is
however,
exemplified,
(3) Trench fever rickettsia are not agglutinated by exaggerated, and the difficulties of treatment
correthe serum of animals immunised with the .excreta of
spondingly increased.
normal uninfected lice.
The treatment of the original injury will necessarily
be surgical, and will probably entail a varying period of
immobilisation on some form of retentive apparatus.
THE VALUE OF POSTURE IN THE AFTER- To facilitate early recovery of abduction and elevation
an abduction rectangular arm-splint of some kind should
TREATMENT OF " STIFF-SHOULDER."
be used whenever possible.
Todd, speaking of the
present after-treatment of dislocations of the shoulder
BY CHARLES MACKAY, M.D. MELB.,
by fixation- of the arm to the side, condemns it as
LATE LIEUTENANT-COLONEL, R.A.M.C. ; LATE OFFICER IN CHARGE,
DEPARTMENT OF MUSCLE RE-EDUCATION, ALDER HEY
being irrational,unscientific, and in actual practice
SPECIAL MILITARY HOSPITAL, LIVERPOOL.
unsuccessful.
In my opinion the best type of abduction arm-splint
is
the one described and illustrated by Dr. W. Colin
WHETHER the disability be caused by a compound or
Mackenzie in the British lleclicccl Journal of Feb. 24th,
of
the
the
a
fracture
in
neighbourhood
simple
joint, by
severe dislocation, by a primary
of muscle 1917, and stated to have been introduced by him in 1908.
splint should be made for the individual patient,
of
the
to
the
deltoid, This
through injury
nerve-supply
and
it
can be adjusted and modified to suit varying
trapezius, serratus magnus, or by secondary muscleof shoulder injury; its obvious advantages are
types
a
not
resultof
to
the
wasting,
infrequent
injury
"
its ease of application and removal, with the constant
shoulder is the so-called stiff-shoulder."
A criterion of the functional result following treat- support which it gives to the muscles of the shoulderment of an injury of this character would be evidenced girdle.. When immobilisation is completed the difficulties
the after-treatment will greatly depend upon whether
by the ability of the patient when standing up, firstly of
a
abduction arm-splint has been worn or not.
suitable
to abduct the arm to a right angle, on ahorizontal
former-case recovery of movement may-be rapid.
In,the
with
exthe
rotate
the
arm
shoulder, then to
plane
easy, ii the latter correspondingly
ternally, and finally to raise it vertically above the head. and comparatively
One of the reasons for this strikingThese movements, so apparently simple in health, are slow and dinicult.
is
difference
that
’the
splint will have prevented - any
very easily lost; their mechanism js delicate and comcontraction and shortening of the adductor and internal
and
a
of
one
of
loss
of
function
its
plicated,
component
elements may be sufficient to nullify the activity of the rotator group of muscles, while at the same time resting
without stretching their important opponents.
remainder.
"
in
this article
The expression " stiff-shoulder is used
Gravity an Important Factor.
to mean one at which the greater part of this normal
in
of
In
voluntary movement is not obtainable ;
treating the original injury and in attempting to
range
which attempts at passive movement apparently regain movement at the shoulder-joint it is all-essential
produce pain, are resented by the patient, and are to recognise the very important part played by gravity.
often voluntarily resisted. It is not intended to apply TeXt-books- do,hot lay sufficient stress on this factor,
to old cases of rheumatoid arthritis, nor to the patho- and it is apt to be forgotten, that the weight of a,
logical conditions subsequent to severe injuries and muscular arm may be from 10 to 141b. If after an
acute inflammatory mischief in the joint itself, with the injury to the joint or its neighbourhood the arm be left
formation of definite articular adhesions and osteo- mainly unsupported as regards its own weight, the
arthritic changes limiting movement. In the type of constant dragging pull;-exerted by gravity will greatly
"
stiff-shoulder" which is here referred to the main aggravate, any reflex atrophy of the shoulder-girdle
causative factors interfering with normal function are muscles;this condition may becbRie so marked in old
considered to be the weakness of the muscles which .cases as to produce h sub-luxation of the humorus. In
abduct, externally rotate, and elevate the arm, and in this connexion it is important to lay stress on the point
a percentage of cases the chronic contraction with’ that muscles, not ligaments, are the primary supporting
shortening of the muscles which adduct and internally structures of a joint such as the shoulder. The deltoid
rotate the limb. Additional factors in some cases are the muscle especially-one of the main supports of the
presence of periarticular adhesions in the surrounding shoulder-joint-has acquired a sinister reputation for
‘
‘
weakness
.,’,
1267
rapid wasting which is unequalled by
major muscle in the body.
that of any other
In all injuries in this region an important point
to remember is the preservation of the insertion
of the deltoid; if this has been lost at the time
of injury the resulting disability, as instanced by
Bland-Sutton, will, in spite of any after-treatment,
necessarily be very great. The deltoid and the trapezius
are here regarded as the essential muscle factors in any
attempt to regain movement at the shoulder-joint by
rest and muscle re-education ; one or other having been
lost the utility of the arm is very greatly diminished.
When the surgical condition of the injury allows of an
attempt at movement the removal ofthe splint should
be very carefully supervised, and the patient then made
to lie flat on a wide couch-30 inches-with a low pillow
under the head and neck.
,
the attempts which are sometimes made in these cases
of stiff-shoulder to diagnose and treat the condition, the
rough handling, the struggle between patient and surgeon,
the abortive attempts at passive movement, the movement under an anaesthetic, and the last condition
remaining sometimes worse than the first. To examine
a wasted shoulder after months or years of massage,
electricity, and passive movements, and to find almost
total absence of voluntary movement with a partially
subluxated head of the humerus is to realise the
limitations in these cases of many of our modern
methods of treatment.
I While drawing attention to what is regarded as a
serious omission in the routine treatment of these
! patients, it is not implied that in skilled hands successful results have not followed the combination of other
methods, such as, for example, the electrical treatment
so strongly advocated by Bristow.
It is suggested,
however, that more rapid and beneficial results would
follow if this positional aid to treatment were more
universally adopted. It would scarcely seem necessary
to point out-if it were not such a frequent occurrence
-that all attempts at after-treatment of a stiff-shoulder
in the sitting or standing-up position with the arm
hanging by the side are contra-indicated as tending to
perpetuate the disability, and to discourage the patient
from attempts at voluntary movement.
The Essential Principle in Treatment.
The line of treatment here advocated is simple, and
its very simplicity perhaps accounts for its comparative
neglect ; with personal modifications it follows the plan
of muscle re-education advised by Dr. Colin Mackenzie
in his recent work on the " Action of Muscles."
Its essential principle is to encourage any possible
amount of voluntary movement and to treat the patient
at the beginning in the lying-down horizontal position.
This position helps considerably toeliminate two
ConcZnsion.
important factors retarding recovery of movement,
gravity-the weight of the arm-and muscular spasm.
During. nearly five years’ war service, and while
It is necessary to obtain muscle relaxation before serving pn medical boards for the Ministry of Pensions,
the attempt at voluntary movement is begun, and I have. seen numerous cases of shoulder injury
in the horizontal position this relaxation can be most whoseafter-treatment had, in my opinion, , been
easily obtained. With’the elbow flexed and the carried out without due attention to anatomical prinforearm supported, the patient is then encouraged ciples -of muscle rest and muscle re-education.
By
to abduct the arm’ farther and farther away from the kindness of Sir Robert Jones I was enabled to see
the side and afterwards gradually to- elevate it. In and treat many cases of this nature while in charge of
order to facilitate movement in difficult cases card- the Muscle Re-education Department at Alder Hey
board owdered with chalk may be’placed under Military Hospital, Liverpool. My belief in the efficacy
the arm to diminish frictional resistance.
These of the method of treatment now formulated was defiattempts to obtain voluntary movement should be nitely strengthened by the results obtained there. It is
carried out once, twice, or three times a day on a firm considered that if voluntary movement in a correct
support, table or couch, and for a variable period anatomical position could have been combined during
depending on the weakness of the muscles and on the the war with the massage, electricity, and passive
ability of the patient to concentrate his attention on movements so extensively practised, the resulting civil
the muscular effort involved. Once full abduction and inefficiency and consequent list of pension recipients
elevation of the arm have been obtained in the lying- would have been considerably reduced.
down horizontal position, it may be definitely stated
That active movement leading directly to a gradual
that it is only a question of time before full movement return of previous full working activities is the ideal
will be obtained in the upright vertical position. This aim of all forms of treatment in this class of case would
result, however, can only be achieved gradually, and sometimes appear to be forgotten; too much is often
progress is accomplished by slowly elevating the head ’, done for the patient, and he is allowed to do too little
and shoulders of the patient either on pillows or by for himself.
Where opposing muscles have become
Each elevation is regarded contracted and shortened, or where peri-articular
means of a special couch.
as a stage in the re-education process, and perfection of
adhesions of a minor degree have occurred in the
movement must be obtained at any-one stage before surrounding structures, the patient’s own vigorous
proceeding to the next highest one. Even if the differ- volitional efforts are the best means of gradually
the contracted opponents or of breaking down
ence in elevation be only a few inches, it may be too
much at first for the weak muscles.
the adhesions. The use of force and an anaesthetic may
It is wise to begin each daily period of exercise in the be essential if time is an element, and if painful passive
horizontal position, and gradually to work upwards to and voluntary movements can afterwards be borne by
the highest elevation previously reached. The muscles the patient ; but I believe that better results will be
are by this means enabled to accommodate themselves
obtained if treatment on the lines above indicated can
to their load, better results ’are obtained, and initial be gradually carried out.
Once movements have been
obtained in the lying down position and gradually
disappointment to the patient is avoided.
increased these may be strengthened, and final comMethods of Dealing with the Disablement.
of the joint obtained, on the lines of the
In the old-standing type of stiff-shoulder the problem plete mobility
active gymnastic exercises ably carried out by Colonel
has to be approached in two ways : firstly, to gain the H. E.
Deane, R.A.M.C., at Croydon.
patient’s confidence that curative, or rather ameliorameans of this paper it is hoped that the value of
By
to
are
tive, procedures
place
possible; and secondly,
of the arm in an abducted position with
the affected muscles during treatment in such a position early splintingof
a recognition
the value of the horizontal posture as
that their work begins at a minimum.
Mackenzie an aid to
form of treatment, may be fully realised,
any
rather aptly calls this the zero position, and Professor
and some little advance be thus made in the treatment
Arthur Keith has named it the minimal load position. of a
notoriously difficult condition. Evolution and the
It may be stated that the patient’s mind has to be
of the
attitude are responsible for
adoption
stimulated as to the value of personal effort, and through more than one of upright
our modern difficulties of treatment.
his mind his muscles have to be re-educated to enable
References.—Thomas: Principles of Treatment of Diseased
them to regain as much of their lost power and Joints.
Hilton : Rest and Pain. Keith : Menders of the Maimed.
coordination as possible. Misdirected effort resulting Mackenzie : The Action of Muscles. Elmslie : After-treatment of
in spasm must be constantly avoided, and in this field Wounds. Bland-Sutton : The British Journal of Surgery, January,
of activity there exists plenty of scope for both physical 1916. Todd : Practitioner, March, 1920. Deane : Gymnastic Treatment for Joint and Muscle Treatment. Bristow : Joint and Muscle
and psychical re-education. It is not pleasant to see Injuries.
stretching
‘
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https://openalex.org/W2467564494
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https://europepmc.org/articles/pmc4941520?pdf=render
|
English
| null |
Enhanced optoelectronic performances of vertically aligned hexagonal boron nitride nanowalls-nanocrystalline diamond heterostructures
|
Scientific reports
| 2,016
|
cc-by
| 8,429
|
Enhanced optoelectronic
performances of vertically
aligned hexagonal boron nitride
nanowalls-nanocrystalline diamond
heterostructures received: 23 March 2016
accepted: 13 June 2016
Published: 11 July 2016 Kamatchi Jothiramalingam Sankaran1,2, Duc Quang Hoang1,2, Srinivasu Kunuku3,
Svetlana Korneychuk4, Stuart Turner4, Paulius Pobedinskas1,2, Sien Drijkoningen1,2,
Marlies K. Van Bael1,2, Jan D’ Haen1,2, Johan Verbeeck4, Keh-Chyang Leou3, I-Nan Lin5 &
Ken Haenen1,2 Field electron emission (FEE) properties of vertically aligned hexagonal boron nitride nanowalls
(hBNNWs) grown on Si have been markedly enhanced through the use of nitrogen doped
nanocrystalline diamond (nNCD) films as an interlayer. The FEE properties of hBNNWs-nNCD
heterostructures show a low turn-on field of 15.2 V/μm, a high FEE current density of 1.48 mA/cm2
and life-time up to a period of 248 min. These values are far superior to those for hBNNWs grown on Si
substrates without the nNCD interlayer, which have a turn-on field of 46.6 V/μm with 0.21 mA/cm2 FEE
current density and life-time of 27 min. Cross-sectional TEM investigation reveals that the utilization of
the diamond interlayer circumvented the formation of amorphous boron nitride prior to the growth of
hexagonal boron nitride. Moreover, incorporation of carbon in hBNNWs improves the conductivity of
hBNNWs. Such a unique combination of materials results in efficient electron transport crossing
nNCD-to-hBNNWs interface and inside the hBNNWs that results in enhanced field emission of
electrons. The prospective application of these materials is manifested by plasma illumination
measurements with lower threshold voltage (370 V) and longer life-time, authorizing the role of
hBNNWs-nNCD heterostructures in the enhancement of electron emission. Field emission electron sources are the vital building blocks in an array of appliances, including microwave
amplifiers, e-beam induced light sources, flat panel displays and travelling wave tubes1–6. In the last few years,
the design, realization and application of a new generation of cold cathodes based on advanced nanomaterials
have been the object of remarkable interest by researchers. The intention has been to find innovative nanoma-
terials possessing the best possible FEE performances in terms of low turn-on field, high FEE current density
and robustness. Recent consideration has been paid to diamond7, aluminum nitride (AlN)8, and boron nitride
(BN)9,10, with negative electron affinity (NEA), and carbon nanotubes11,12 with a large field enhancement factor.i fii
For a high-quality electron field emitter, adequate electrons supply from the substrates (the Si) to the emit-
ting sites (the hBNNWs) is critical, besides the low work function for the emitting surface. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 Enhanced optoelectronic
performances of vertically
aligned hexagonal boron nitride
nanowalls-nanocrystalline diamond
heterostructures Combination of two
chemically different nanostructured materials in a heterostructure is a versatile building method for modern
nanodevices. Compared to single component structures, heterostructures habitually reveal enhanced charac-
teristics, such as emission efficiency13 and high electron mobility14, which are the significant factors for many
device performances15–17. A myriad of heterostructured materials have been developed. Heterostructures such as 1Institute for Materials Research (IMO), Hasselt University, 3590 Diepenbeek, Belgium. 2IMOMEC, IMEC vzw, 3590
Diepenbeek, Belgium. 3Department of Engineering and System Science, National Tsing Hua University, 30013
Hsinchu, Taiwan. 4Electron Microscopy for Materials Science (EMAT), University of Antwerp, 2020 Antwerp, Belgium. 5Department of Physics, Tamkang University, 251 Tamsui, Taiwan. Correspondence and requests for materials should
be addressed to K.J.S. (email: sankaran.kamatchi@uhasselt.be) or K.H. (email: ken.haenen@uhasselt.be) Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 1 www.nature.com/scientificreports/ Figure 1. The plane view SEM micrographs of (a) NCD films, (b) hBNNWs-NCD, (c) nNCD films and
(d) hBNNWs-nNCD. (e) micro-Raman spectra for I. NCD films, II. hBNNWs-NCD, III. nNCD films and IV. hBNNWs-nNCD. hi Figure 1. The plane view SEM micrographs of (a) NCD films, (b) hBNNWs-NCD, (c) nNCD films and
(d) hBNNWs-nNCD. (e) micro-Raman spectra for I. NCD films, II. hBNNWs-NCD, III. nNCD films and IV. hBNNWs-nNCD. single-walled CNTs–CdSe quantum dots18, SnO2-CNTs19, MoS2–WS2
20, CNT-graphene21, gold–ultrananocrys-
talline diamond (UNCD)22, BN–graphene23, CNT–UNCD on Si tips24, ZnO nanopillars–Si25, ZnO nanorods–
UNCD26, graphene-nanodiamond27, InAs–GaAs nanowires28, TiSi2 nanonets–Si nanostructures29 have been
extensively studied. Fabrication of such heterostructures is not only vital for fundamental studies but also for
diverse advanced functional devices, for instance, interconnects and emitters, etc. Being encouraged by a distinctive possibility to merge two nanostructured materials, here we set out to inves-
tigate novel vertically aligned hexagonal boron nitride nanowalls (hBNNWs)-nanocrystalline diamond (NCD)
heterostructures and to examine their FEE and plasma illumination (PI) properties. The bonding structure and
microstructure of hBNNWs-NCD heterostructures were detailed characterized to better understand the possible
mechanism of the growth of hBNNWs on NCD films and the enhanced FEE properties of these heterostructures. Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 Results and Discussionh nNCD films, the hBNNWs formed a more uniform layer that is closely related to the very smooth surfaces of the
nNCD films (Fig. 1d). The hBNNWs were vertically aligned with respect to the plane of the NCD substrates. They
were mutually interconnected forming an extended network. In contrast, a non-uniform and unbranched growth
of hBNNWs was observed when the nanowalls were grown on Si substrates (Fig. S1, Supporting Information). F
h
l
i
f h
l
i d
b
i
R
l
i
l
b of hBNNWs was observed when the nanowalls were grown on Si substrates (Fig. S1, Supporting Information). Further analysis of these samples was carried out by micro-Raman spectroscopy employing a laser beam
with wavelength of 488 nm and spot size of ~1 μm. Spectrum I in Fig. 1e acquired from NCD film on Si shows a
peak at 1334 cm−1, the characteristic F2g band for diamond lattices. Small broad resonance peaks near 1138 cm−1,
1480 cm−1 and 1554 cm−1 are also present; these peaks are associated with disordered carbon32,33. The Raman
spectrum of these films was markedly modified when N2 species were incorporated into the plasma in the growth
of nNCD films. The Raman spectrum of the nNCD films (spectrum III, Fig. 1e) contains ν1-band (1185 cm−1) and
ν3-band (1532 cm−1) resonance peaks, which correspond to the deformation modes of CHx bonds in the nNCD
films34, and D-band (1368 cm−1) and G-band (1562 cm−1) resonance peaks, which correspond to sp2 bonded
carbon, i.e., the disordered carbon and graphite35–38. The diamond (1334 cm−1) resonance peak, which corre-
sponds to the F2g resonance mode of the 3C diamond lattice, is only barely visible. The invisibility of the diamond
resonance peak does not mean that the films comprise no sp3-bonded carbon. It is due to the fact that the Raman
signal is overwhelmingly more sensitive to the sp2-bonded carbon than to the sp3-bonded ones that renders the
diamond peak remains relatively feeble37. On the other hand, hBNNWs-Si materials contain a major peak that
appears at 1368 cm−1, attributed to the high frequency intralayer E2g vibration mode of hBN (spectrum I of Fig. S2,
Supporting Information)39–42. Since the hexagonal boron nitride (hBN) material is porous in nature, only a small
peak corresponding to the hBN signal (indicated by an arrow in spectra II and IV) is observed at 1366 cm−1 for
hBNNWs-NCD and hBNNWs-nNCD samples. Results and Discussionh The fabrication of hBNNWs-NCD heterostructures was a two-step process. In the first step, two types of NCD
films (undoped NCD and nitrogen doped NCD (nNCD)) were grown on mirror polished (100)-oriented silicon
(Si) wafers using an ASTeX 6500 series microwave plasma enhanced chemical vapor deposition (MPECVD)
reactor. Figure 1a shows an SEM image of an undoped NCD film grown on the Si substrate. The entire sur-
face was very rough and uniformly covered with randomly oriented diamond grains with clearly distinguishable
edges and facets and having grain sizes of 0.3–0.8 μm. The SEM image of nNCD films shown in Fig. 1c reveals a
dramatic change in the surface morphology of the films when adding N2 in the CH4/H2 microwave plasma. The
sharp-edged crystal structure is replaced by a roundish granular structure with nano-sized grains. In the second
step, hBNNWs were grown on the NCD films by a home-built unbalanced 13.56 MHz radio frequency (RF)
sputtering technique30,31. Figure 1b,d show the surface morphology of hBNNWs grown on the diamond films. Both NCD film surfaces were uniformly covered with a layer of two dimensional nanostructures (nanowalls)
with a compact and curled morphology. It is to be noted that the SEM morphology of hBNNWs is closely related
with granular structure of the underneath NCD films. For the NCD films, the hBNNWs formed micron-sized
aggregates, which seem to grow on top of each diamond grain of the NCD films separately (Fig. 1b), whereas, for Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 2 www.nature.com/scientificreports/ Figure 2. FTIR spectra for I. hBNNWs-Si, II. hBNNWs-NCD and III. hBNNWs-nNCD. FTIR transmission
spectra were undertaken vertically to the film surface in the range frequency of 400–4000 cm−1 with a resolution
of 2.0 cm−1. Figure 2. FTIR spectra for I. hBNNWs-Si, II. hBNNWs-NCD and III. hBNNWs-nNCD. FTIR transmission
spectra were undertaken vertically to the film surface in the range frequency of 400–4000 cm−1 with a resolution
of 2.0 cm−1. nNCD films, the hBNNWs formed a more uniform layer that is closely related to the very smooth surfaces of the
nNCD films (Fig. 1d). The hBNNWs were vertically aligned with respect to the plane of the NCD substrates. They
were mutually interconnected forming an extended network. In contrast, a non-uniform and unbranched growth
of hBNNWs was observed when the nanowalls were grown on Si substrates (Fig. S1, Supporting Information). Results and Discussionh The literal field enhancement factor Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 3 www.nature.com/scientificreports/ (β) value can be calculated from the F-N ‘slope’ using the relation: β = [−6.8 × 103
of the F N plot From the slopes of the F N plots the β values of these heterostructu
Figure 3. (a) The field electron emission properties (Je-E curves) and (b) the curren
of I. hBNNWs-Si, II. hBNNWs-NCD, III. hBNNWs-nNCD. The inset in “a” shows
Nordheim plots. (c) The plasma current density versus applied voltage with the inse
illumination stability, the life-time, of the microplasma cavities, which were fabricat
hBNNWs-NCD and III. hBNNWs-nNCD as cathode materials. Figure 3. (a) The field electron emission properties (Je-E curves) and (b) the current density versus time curves
of I. hBNNWs-Si, II. hBNNWs-NCD, III. hBNNWs-nNCD. The inset in “a” shows the corresponding Fowler-
Nordheim plots. (c) The plasma current density versus applied voltage with the inset showing the plasma
illumination stability, the life-time, of the microplasma cavities, which were fabricated using I. hBNNWs-Si, II. hBNNWs-NCD and III. hBNNWs-nNCD as cathode materials. Figure 3. (a) The field electron emission properties (Je-E curves) and (b) the current density versus time curves
of I. hBNNWs-Si, II. hBNNWs-NCD, III. hBNNWs-nNCD. The inset in “a” shows the corresponding Fowler-
Nordheim plots. (c) The plasma current density versus applied voltage with the inset showing the plasma
illumination stability, the life-time, of the microplasma cavities, which were fabricated using I. hBNNWs-Si, II. hBNNWs-NCD and III. hBNNWs-nNCD as cathode materials. (β) value can be calculated from the F-N ‘slope’ using the relation: β = [−6.8 × 103 ϕ3/2]/m, where m is the slope
of the F-N plot. From the slopes of the F-N plots the β values of these heterostructures were calculated by taking
the ϕ value of hBN as 6.0 eV51. The hBNNWs-Si exhibits the lowest β value of 560. The β value increases to 2110
for hBNNWs-NCD, which is almost 4 times as much that of the hBNNWs-Si. Upon nitrogen doping, the β value
further increases to 3057. These FEE parameters are listed in Table S1.h h
p
The stability of the FEE current is an imperative parameter associated to the applications of cold cathode mate-
rials. The life-time stability measurements for these films were evaluated by measuring the Je versus time curves of
these heterostructures. Fig. Results and Discussionh The hBN peak is overlapped with the D band (1368 cm−1) of the
diamond films (spectra II and IV, Fig. 1e). i
g
FTIR spectroscopy measurements were performed to further investigate the bonding characteristics of
these heterostructures. Spectrum I of Fig. 2 for hBNNWs-Si shows one sharp absorption peak at 812 cm−1 and
one broad absorption band with the bottom in the range of 1300–1500 cm−1, which was attributed to the A2u
(B-N-B bending vibration mode parallel to the c-axis) and E1u (B-N stretching vibration mode perpendicular
to the c-axis) modes of hBN43–45, respectively, validating their good crystallinity. Spectra II and III of Fig. 2 for
hBNNWs-NCD and hBNNWs-nNCD heterostructures are very alike. Both spectra illustrate the characteristic
peaks of hBN. In addition, the peak at 1238 cm−1 can be consigned to the stretching vibration of boron-rich (or
carbon-rich) B-C bonds46,47. These observations imply that there are some carbon species incorporated into the
hBNNWs films. The absorption band centered at 1238 cm−1 may also be associated to the stretching vibration of
C-N bonds47,48. Furthermore, the formation of sp2 C-N bonds could contribute to the small absorption peaks at
~1542 cm−1 and 1564 cm−1, respectively46,49. Notably, the stretching vibration of C-N bonds are expected to give
an absorption band centered at 1238 cm−1, but it cannot be clearly resolved from the B-C bands.h p
y
The FEE properties of hBNNWs-diamond heterostructures are derived through the FEE current density
(Je)−applied field (E) curves, which are illustrated in Fig. 3a. The Fowler-Nordheim (F-N) theory could very well
describe the FEE phenomenon50. The corresponding F-N curves are plotted as insets. The E0 value for inducing
FEE process decreased from 46.6 V/μm for hBNNWs-Si (curve I, Fig. 3a) to 35.5 V/μm for hBNNWs-NCD (curve
II, Fig. 3a), whereas the Je value increased from 0.21 mA/cm2 at an applied field of 91.6 V/μm for hBNNWs-Si to
a value of 0.46 mA/cm2 at an applied field of 61.3 V/μm for hBNNWs-NCD. The FEE properties are even better
for the hBNNWs-nNCD, which used nNCD films as interlayer, viz., with the smallest E0 value of 15.2 V/μm and
the highest Je value of 1.48 mA/cm2 (at an applied field of 21.3 V/μm, curve III, Fig. 3a). Furthermore, a plot of log
(Je/E2) versus (1/E), the so-called F-N plot inset of Fig. 3a, gives a straight line. www.nature.com/scientificreports/ The comparison on the FEE parameters of hBNNWs-nNCD with other field emit-
ting materials52–59 reported in literature, is summarized in Table 1.h g
p
The challenging developments in the hardiness of the hBNNWs-NCD heterostructures can be better exem-
plified by using these field electron emitters as cathodes for a microplasma device, as the cathodes are subjected
to energetic Ar-ions bombardment in this device, which is considered as the harshest environment for electron
emitters60,61. Figure S3 in Supporting Information shows that the microplasma devices using hBNNWs-NCD
heterostructures as cathode exhibit a superior plasma illumination (PI) behavior to those using hBNNWs-Si
as cathodes. The microplasma devices using the hBNNWs-nNCD as cathode can be triggered by a voltage as
low as 370 V (image series III, Fig. S3 in the Supporting Information), whereas those using hBNNWs-NCD as
cathode need 430 V to ignite the plasma (image series II, Fig. S3 in the Supporting Information). In contrast,
the hBNNWs-Si based microplasma devices need a higher voltage, around 460 V (image series I, Fig. S3 in the
Supporting Information), to trigger the plasma. The plasma intensity enhances monotonically with the applied
voltage. The superior PI characteristics are better illustrated by the variation of the plasma current density (Jpl) ver-
sus the applied voltage. Figure 3c indicates that the Jpl values increased monotonously with the applied voltage and
reached 1.27 mA/cm2 and 2.46 mA/cm2, at an applied voltage of 500 V, for hBNNWs-NCD and hBNNWs-nNCD
based microplasma devices (curves II and III, Fig. 3c), respectively, whereas only a Jpl = 0.57 mA/cm2 at an applied
voltage of 500 V was achieved for the hBNNWs-Si based ones (curve I, Fig. 3c). g
g
To estimate the constancy of the PI performance from hBNNWs-diamond based microplasma devices, the
plasma current was observed over a long period with a constant applied voltage of 500 V. It should be men-
tioned that under this applied voltage, the Jpl-value is 1.45 mA/cm2 for hBNNWs-NCD and is 2.35 mA/cm2 for
hBNNWs-nNCD based microplasma devices. Under this test condition, the PI intensity of the hBNNWs-NCD
and hBNNWs-nNCD based microplasma devices continues to be stable over 163 min and 122 min, respectively,
displaying the high stability of the hBNNWs-diamond based microplasma devices (curves II and III, inset of
Fig. 3c). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Materials
Turn-on field (V/μm)
Field enhancement factor
Life-time stability (min)
Carbon nanotubes52
1.4
4350
33
Boron nitride nanotubes53
32.5
98
—
Carbon nanowalls54
4.7
1399
240
AlN nanocones55
4.8
1561
—
ZnO Nanowires56
3.82
2303
—
MoO3 nanobelts57
8.7
—
120
Ultrananocrystalline diamond nanorods58
2.04
1945
—
Graphene nanoflakes59
1.05
3120
166
hBNNWs-nNCD heterostructuresThis study
15.2
3057
248
Table 1. Comparison of the key field electron emission performance parameters between the presented
hBNNWs-nNCD heterostructures and other field emitters. Table 1. Comparison of the key field electron emission performance parameters between the presented
BNNWs-nNCD heterostructures and other field emitters. of residual gas molecules and diffusion of adsorbed species on the emitter surface, the emission current variations
corresponding to Je of 0.21 mA/cm2 recorded over a period of 284 min for hBNNWs-NCD at a working field of
53.5 V/μm and 248 min for hBNNWs-nNCD corresponding to Je of 0.20 mA/cm2 at a working field of 19.6 V/μm
before the start of degradation (curves II and III, Fig. 3b). In contrast, the hBNNWs-Si (curve I, Fig. 3b) shows the
emission current variations recorded only a period of 27 min at a working field of 90.0 V/μm corresponding to Je
of 0.198 mA/cm2. It is worth noting that even though the hBNNWs-nNCD films show slightly inferior life-time
stability to the hBNNWs-NCD films, the robustness of these films is still much better than the life-time stability
of conventional EFE materials. The comparison on the FEE parameters of hBNNWs-nNCD with other field emit-
ting materials52–59 reported in literature, is summarized in Table 1.h of residual gas molecules and diffusion of adsorbed species on the emitter surface, the emission current variations
corresponding to Je of 0.21 mA/cm2 recorded over a period of 284 min for hBNNWs-NCD at a working field of
53.5 V/μm and 248 min for hBNNWs-nNCD corresponding to Je of 0.20 mA/cm2 at a working field of 19.6 V/μm
before the start of degradation (curves II and III, Fig. 3b). In contrast, the hBNNWs-Si (curve I, Fig. 3b) shows the
emission current variations recorded only a period of 27 min at a working field of 90.0 V/μm corresponding to Je
of 0.198 mA/cm2. It is worth noting that even though the hBNNWs-nNCD films show slightly inferior life-time
stability to the hBNNWs-NCD films, the robustness of these films is still much better than the life-time stability
of conventional EFE materials. Results and Discussionh 3b shows that, ignoring short-term fluctuations owing to adsorption and desorption Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 4 Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 www.nature.com/scientificreports/ Figure 4. A cross-sectional HRTEM image with bright field cross-sectional TEM image as inset of
hBNNWs-Si. These micrographs show the presence of aBN and tBN sequentially prior to the growth of hBN
phase when the hBNNWs were grown directly on Si substrates. A and B of Fig. 5b, clearly showing that region A contains hBN materials and region B contains diamond.h
thickness of the hBNNWs was measured to be around 10 nm (not shown). In order to investigate a possible incorporation of carbon into the hBN, we adopted spatially resolved
STEM-electron energy loss spectroscopy (EELS). In the experiment, the region indicated by the rectangle in
Fig. 6aI was scanned using a fine probe, collecting a core-loss EELS spectrum containing the C-K, B-K and N-K
edges in each point. By integrating the intensity under the C, Band N edges, elemental maps were generated that
are displayed in Fig. 6aII,III,IV, respectively. It is clear from the maps that some carbon has been incorporated into
the hBN structures (indicated by arrows in Fig. 6aII). In Fig. 6b, two summed EELS spectra from the diamond and
the hBN regions (designated in Fig. 6aI) are plotted. The carbon K-edge spectrum acquired from the diamond
region is typical of sp3-carbon, with a strong σ* contribution at 292 eV and deep valley in 302 eV63,64. The EELS
spectrum corresponding to the hBN region of Fig. 6a exhibits two distinct edges; the boron-K 188 eV and the
nitrogen-K at 401 eV65–67. The fine structure of the B-K and N-K edges are typical of the sp2-coordinated layered
BN, indicating that the obtained nanowalls are BN. As mentioned above, in addition to the core-loss K-edges
of B and N, the presence of carbon is also detected through presence of a core-loss carbon-K edge at 284 eV
(π* band). The fine structure of the carbon K-edge is typical of sp2-bonded carbon, confirming that some carbon
is incorporated into the hBN. These EELS results together with the elemental maps (cf. Fig. 6aII–IV) indicate the
presence of C, B and N species in the hBNNWs-NCD heterostructures, which is in accord with the B-C, B-N,
and C-N bonds observed in the FTIR data (cf. Fig. 2). Notably, Leung et al. also observed the diffusion of C in
the interface during the growth of cubic BN on amorphous tetrahedral carbon interlayers68. www.nature.com/scientificreports/ That the nNCD films is less stable than the NCD films can be ascribed to the presence of nanogra-
phitic phase in the former materials that , in turn, resulted in shorter life-time for hBNNWs-nNCD hetero-
structures than that of hBNNWs-NCD heterostructures. In contrast, the Jpl value of 0.54 mA/cm2 (at constantly
applied 500 V) decreased fast after 28 min of plasma ignition for the hBNNWs-Si-based microplasma devices
(curve I, inset of Fig. 3c). Consequently, the better PI performance of the microplasma devices based on the
hBNNWs-diamond heterostructures, as associated with that of hBNNWs-Si based ones, is closely interrelated
with the enhanced FEE properties of the hBNNWs-diamond heterostructures.hi p
p
The microstructure of these heterostructures was examined by annular dark field scanning transmission elec-
tron microscopy (ADF-STEM) and high resolution TEM in order to clarify the basis of the FEE enhancement and
the PI performance of the hBNNWs-diamond heterostructures. The cross-sectional high resolution TEM image
of hBNNWs-Si was shown in Fig. 4 with the corresponding bright field cross-sectional TEM shown as inset in
this figure. These micrographs illustrate that when hBN was directly grown on Si-substrates, it requires the forma-
tion of precursor layers prior to its nucleation. The deposition of hBN on Si first yields an interlayer of amorphous
BN (aBN) followed by the formation of turbostratic BN (tBN) with (002) basal planes almost perpendicular to
the substrate surface62. The edges of the (002) BN basal planes then serve as nucleation sites of hBN. In contrast,
Fig. 5a shows a typical cross-sectional ADF-STEM micrograph of the hBNNWs-NCD heterostructure, in which
the hBNNWs and the diamond regions are clearly marked. Figure 5b shows a high resolution image obtained
from a typical region at the hBN-diamond interface of the hBNNWs-NCD heterostructure. It can be seen that the
hBNNWs grow directly on the diamond surfaces, without the formation of any precursor layers like aBN or tBN
prior to its nucleation. Highly ordered lattice fringes of hBNNWs can be observed, indicating that the hBNNWs
are well crystallized. Fourier transformed diffractograms corresponding to regions A and B are shown in isnets Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. A cross-sectional HRTEM image with bright field cross-sectional TEM image as inset of
hBNNWs-Si. These micrographs show the presence of aBN and tBN sequentially prior to the growth of hBN
phase when the hBNNWs were grown directly on Si substrates. Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 www.nature.com/scientificreports/ The presence of C in
the interface region is possibly induced by carbon incorporation and dynamic recoil ion mixing in an early stage
of boron nitride deposition. This incorporated carbon region then relates to a C-B-N gradient layer, which may
contribute to the interfacial stress relaxation. On the basis of FTIR and STEM-EELS observations, it is obvious
that the hBNNWs nucleated and grew directly on the NCD surface, inhibiting the formation of aBN and disor-
dered BN phases in the interface. Presumably, the possible interaction of B-C-N species at hBNNWs-to-NCD
interface is the main factor, which facilitates the nucleation of hBN clusters without the formation of intermediate
BN phases (aBN or tBN).i p
(
)
As mentioned earlier, for a high-quality electron field emitter, an adequate supply of electrons from the sub-
strate (the Si) to the emitting site (the hBNNWs) is critical, besides the low work function for the emitting sur-
face. In order to enhance the efficiency of the electron supply, the conductivity of both hBN and diamond and
the resistance of the hBN-to-Si interface need to be optimized. The utilization of diamond as intermediate layer,
which was coated onto the Si substrates prior to the growth of hBNNWs, fulfills these critical requirements
simultaneously. As illustrated by the TEM investigations, the use of diamond intermediate layer suppresses the
formation of an aBN phase and tBN. The hBN forms directly on diamond that enhances the transport of elec-
trons crossing the hBN-to-diamond interface. Concerning the mechanism that using nNCD films as interme-
diate layer resulted in better EFE behavior compared with those which used NCD as intermediate layer, the
induction of a sp2 graphite phase in the grain boundaries of the nNCD films (evident from micro-Raman studies,
cf. spectrum III, Fig. 1e) due to the addition of N2 in the plasma69 is presumed to be the main factor. As the sp2
graphitic phase increased the percolative conduction paths in the nNCD films that could be one of the factors Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 6 www.nature.com/scientificreports/ Figure 5. (a) Cross-sectional ADF-STEM image of the hBNNWs-NC
ADF-STEM image of the NCD/hBN interface. The FT pattern from re
the hBN, displaying the (002) reflection. The diamond particle is imag for the improvement in FEE properties for hBNNWs nNCD heteros
Figure 5. (a) Cross-sectional ADF-STEM image of the hBNNWs-N
ADF-STEM image of the NCD/hBN interface. www.nature.com/scientificreports/ The FT pattern from r
the hBN, displaying the (002) reflection. The diamond particle is ima
by the FT pattern taken from region B. Figure 5. (a) Cross-sectional ADF-STEM image of the hBNNWs-NCD heterostructure. (b) High resolution
ADF-STEM image of the NCD/hBN interface. The FT pattern from region A evidences the crystalline nature of
the hBN, displaying the (002) reflection. The diamond particle is imaged along the [011] zone axis, as evidenced
by the FT pattern taken from region B. Figure 5. (a) Cross-sectional ADF-STEM image of the hBNNWs-NCD heterostructure. (b) High resolution
ADF-STEM image of the NCD/hBN interface. The FT pattern from region A evidences the crystalline nature of
the hBN, displaying the (002) reflection. The diamond particle is imaged along the [011] zone axis, as evidenced
by the FT pattern taken from region B. for the improvement in FEE properties for hBNNWs-nNCD heterostructures, as compared to hBNNWs-NCD. Meanwhile, it is important to notice that the graphite phase in the grain boundaries of nNCD films is intrinsically
not resistant to plasma ion bombardment70 that lead to inferior robustness of nNCD-based microplasma devices
compared with that of NCD-based ones. Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 7 www.nature.com/scientificreports/ Figure 6. (a) ADF-STEM micrograph of the hBNNWs-NCD interface together with EELS elemental maps for
carbon, boron and nitrogen taken from the region indicated by the white rectangle (b) Summed EELS core-loss
spectra taken from the diamond and hBN regions in the maps in (a). A significant carbon incorporation into
the hBN is evidenced by the carbon map (arrows) and the strong amorphous carbon peak in the EELS spectrum
taken from the hBN region. Figure 6. (a) ADF-STEM micrograph of the hBNNWs-NCD interface together with EELS elemental maps for
carbon, boron and nitrogen taken from the region indicated by the white rectangle (b) Summed EELS core-loss
spectra taken from the diamond and hBN regions in the maps in (a). A significant carbon incorporation into
the hBN is evidenced by the carbon map (arrows) and the strong amorphous carbon peak in the EELS spectrum
taken from the hBN region. Now, an unresolved question rises on how the unique combination of hBNNWs and nNCD materials assists
in enhancing the FEE properties and what would be the possible field emission mechanism in hBNNWs-nNCD
heterostructures? Basically hBN is of a structure analogous to graphite but exhibits a large bandgap of 5.95 eV71,
viz. Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 Methods
Th
b The Si substrates were kept in an ASTeX 6500 series MPECVD reactor for the growth of diamond films. Prior to
diamond growth, the Si substrates were seeded with a waterbased state-of-the-art colloidal suspension of 5 nm
detonation nanodiamonds74. In the growth of undoped NCD films, a gas mixture of CH4 and H2 with flow rates
of 3 and 297 sccm (CH4/H2 = 1/99), respectively, was excited by 3000 W microwave power. The total pressure in
the chamber was maintained at 20 Torr. The substrates were heated due to the bombardment of the plasma species
and the growth temperature was estimated to be around 675 °C during the growth of undoped NCD films. The
growth temperature was measured using an optical pyrometer. For the growth of nNCD films, a gas mixture of
CH4, H2 and N2 with flow rates of 18, 267 and 15 sccm, respectively (CH4/H2/N2 = 6/89/5), was excited by 3000 W
microwave power, and the total pressure in the chamber was maintained at 20 Torr. The growth temperature dur-
ing the growth of nNCD films was estimated to be around 540 °C.i g
gi
hBNNWs were synthesized on the NCD films by a home-built unbalanced 13.56 MHz RF sputtering tech-
nique. An optimal condition of fabrication was used with a gas mixture Ar(51%)/N2(44%)/H2(5%) and cath-
ode power of 75 W. Herein, a 3 inch-diameter pyrolytic BN ceramic target (Kurt J. Lesker) was used with
material purity and mass-density of 99.99% and 1.96 × 103 kg/m3, respectively. The working pressure and
target-to-substrate distance were 2.1 × 10−2 mbar and 3 cm, respectively. The growth temperature during growth
of hBNNWs was 125 °C31. The samples were characterized by micro-Raman spectroscopy, FTIR spectroscopy, SEM, HRTEM,
ADF-STEM and STEM-EELS using, respectively, a Horiba Jobin-Yuan T64000 spectrometer, FTIR NICOLET
8700 spectrometer, and FEI Quanta 200 FEG microscope, a JEOL 3000F transmission electron microscope
operated at 300 kV acceleration voltage for HRTEM and a FEI Titan ‘cubed’ microscope operated at 300 kV for
ADF-STEM-EELS. The convergence semi-angle α used was 22 mrad, the inner acceptance semi-angle β for
ADF-STEM imaging was 22 mrad, the EELS collection angle used was also 22 mrad. The TEM specimens of these
samples were prepared by Focused Ion Beam technique.h The FEE characteristics of these samples were measured with a tunable parallel plate setup, in which a microm-
eter was used to control the cathode-to-anode distance. www.nature.com/scientificreports/ hBN is an insulating layered material. To make a specific application of hBN in nanoelectronics, it is impor-
tant to modify the electronic properties of hBN by doping it with suitable dopant elements. Particularly, when
doped carbon, hBN exhibited semiconducting properties due to the appearance of dopants or defect states in the
bandgap72,73. In the present study, it has been observed from FTIR (cf. Fig. 2) and STEM-EELS (cf. Fig. 6) that
there is incorporation of carbon in the hBNNWs, resulting in the formation of C-N and B-C bonding in hBN
region which could improve the electrical conductivity of hBNNWs. From these observations, the field emis-
sion mechanism for the improvement in the FEE behavior of hBNNWs-nNCD heterostructures can be enlight-
ened: first, the sp2 graphite phase in the grain boundaries of nNCD films conducts the electrons efficiently to the
hBNNWs-nNCD interface. Second, the direct growth of hBN on the diamond surface lowers the resistivity of the
interfacial layer and therefore the electrons can be transferred readily from nNCD films across the interfacial layer
to the hBNNWs. Finally, the incorporation of C in the hBNNWs provides efficient electron transport paths for
the emitted electrons to reach the tip of the nanowalls from which they escape into vacuum without any difficulty
as the hBN surfaces are NEA in nature9,10 All these factors reduce the E0 value by lowering the barrier for the
emitting electrons and thus enhances the FEE Je. Moreover, the vertically aligned nanowall structure facing the
anode could be considered as an additional reason for improvement of the FEE properties of hBNNWs-nNCD
heterostructures. 8 Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 www.nature.com/scientificreports/ Conclusions A facile and reproducible way of fabricating vertically aligned hBNNWs-nNCD heterostructures with excel-
lent FEE and PI performances is demonstrated. The hBNNWs grown directly on the nNCD surfaces markedly
enhance the FEE properties (viz. low E0 (15.2 V/μm), high Je of 1.48 mA/cm2 at an applied field of 21.3 V/μm
and high life-time stability of 248 min). Such an improvement in the field emission behavior originates from
the unique material combination used. The cross-sectional TEM results show that, in the hBNNWs-diamond
heterostructures, hBN nucleated and grew directly on the diamond surfaces, eliminating the formation of an
aBN/tBN phase. There is also incorporation of C in the hBN region, which leads to improvement of the con-
ductivity for the hBNNWs. Moreover, the addition of N2 in the diamond growth plasma induces sp2-bonded
carbon phases in the grain boundaries of the NCD films that improve the electron transport from diamond to the
hBNNWs, resulting in the enhanced FEE properties of hBNNWs-nNCD heterostructures. The potential appli-
cation of these heterostructures is demonstrated by the PI measurements for a microplasma device, that is, the
lowering of the threshold voltage by 370 V and the increase in plasma current density to Jpl = 2.48 mA/cm2. These
observations confirmed the role of the diamond interlayer in the enhancement of the electron emission behav-
ior of hBNNWs-nNCD heterostructures. Consequently, the present approach for fabricating hBNNWs-nNCD
heterostructures is a simple and a direct process that opens new aspects in flat panel displays and high brightness
electron sources. Methods
Th
b An electrometer (Keithley 237) was used to measure the
current-voltage (I-V) characteristics at pressures below 10−6 Torr. The point of intersection of the straight lines
extrapolated from the low and high-field segments of the F-N plots, viz. log (Je/E2) versus 1/E, is labeled as the E0. The fabrication of a plasma microcavity using hBNNWs-NCD heterostructures as cathodes are given elsewhere22. 1. De Heer, W. A., Chatelain, A. & Ugarte, D. A Carbon nanotube field emission electron source. Science 270, 1179 1180 (1995)
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The authors like to thank the financial support of the Research Foundation Flanders (FWO) via Research Project
G.0456.12, G0044.13N and the Methusalem “NANO” network. Kamatchi Jothiramalingam Sankaran, Stuart
Turner, and Paulius Pobedinskas are Postdoctoral Fellows of the Research Foundations Flanders (FWO). The authors like to thank the financial support of the Research Foundation Flanders (FWO) via Research Project
G.0456.12, G0044.13N and the Methusalem “NANO” network. Kamatchi Jothiramalingam Sankaran, Stuart
Turner, and Paulius Pobedinskas are Postdoctoral Fellows of the Research Foundations Flanders (FWO). The authors like to thank the financial support of the Research Foundation Flanders (FWO) via Research Project
G.0456.12, G0044.13N and the Methusalem “NANO” network. Kamatchi Jothiramalingam Sankaran, Stuart
Turner and Paulius Pobedinskas are Postdoctoral Fellows of the Research Foundations Flanders (FWO) www.nature.com/scientificreports/ l
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195–200 (2009). Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 10 www.nature.com/scientificreports/ 64. Zhou, D. et al. Synthesis of nanocrystalline diamond thin films from an Ar-CH4 microwave plasma. J. Appl. Phys. 83, 540
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4737–4740 (1996). (
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66. Terrones, M. et al. Metal particle catalysed production of nanoscale BN structures. Author Contributions K.J.S. and K.H. contributed to design the study, grow NCD films and write the manuscript. D.Q.H. contributed to
synthesis hBNNWs and perform FTIR measurements. S.K., K.-C.L. and I.-N.L. contributed to perform FEE and
PI measurements. S.K., S.T. and J.V. contributed to perform TEM characterization. P.P. and M.K.V. contributed
to perform Raman measurements. S.D. and J.D’.H. contributed to perform SEM characterization. All authors
contributed to discussions of the results. All authors reviewed the manuscript. Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Sankaran, K. J. et al. Enhanced optoelectronic performances of vertically aligned
hexagonal boron nitride nanowalls-nanocrystalline diamond heterostructures. Sci. Rep. 6, 29444; doi: 10.1038/
srep29444 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:29444 | DOI: 10.1038/srep29444 11
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Negative pressure wound therapy with instillation (NPWTi): Current status, recommendations and perspectives in the context of modern wound therapy.
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Negative Pressure Wound Therapy Journal
| 2,016
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cc-by
| 10,479
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8
NEGATIVE PRESSURE WOUND THERAPY JOURNAL, VOL. 3, NO. 1, 2016
Negative pressure wound therapy with instillation
(NPWTi): Current status, recommendations and
perspectives in the context of modern wound
therapy.
Adam Bobkiewicz, Adam Studniarek, Michał Drews, Tomasz Banasiewicz
REVIEW
Abstract—Introduction of negative pressure wound therapy
(NPWT) revolutionized the conception of wound healing. Currently, increasing number of studies confirmed the high efficiency
of this therapy in many clinical scenarios. Moreover, some
innovations have been introduced in recent years to improve
the management of complex and chronic wounds. NPWT with
instillation (NPWTi) combines traditional NPWT with application of topical irrigation solutions within the bed of the wound.
Bioburden reduction, decreases time to wound closure, promotes
granulation and tissue formation. Fewer operative visits are
required when using NPWTi compared to standard NPWT.
However, there are still questioned aspects of the NPWTi and thus
its superiority over standard NPWT has not been fully indicated.
Moreover, based on current studies no firm conclusions have
been taken concerning the type of instilled solution preferably
used, range of dwell- time phase, range of negative pressure
and others. The main goal of the publication is to overview
and summarize the current state of art concerning NPWTi.
Moreover, mechanisms of action, review of the most commonly
used instilled solutions are discussed and clinical evidence of
NPWTi are described.
Keywords—NPWT, instillation,
I. I NTRODUCTION
HE introduction of negative pressure wound therapy
(NPWT) in the late 1980s simultaneously in the United
States and Germany revolutionized the management of complex and chronic wounds.1–3
Its initial clinical indication was designed for chronic
wounds of lower extremity such as leg ulcers, decubitus ulcers
and traumatic wounds after open fractures. Very quickly the
indication for NPWT was extended to other varying wounds
with different underlying pathologies.
Because of excellent results of NPWT and the progress in
both basic science research and clinical trials regarding NPWT,
T
Manuscript received 18.03.2016; revised 28.03.2016. This work did not
receive any financial support. Authors declare no conflict of interest.
Author affiliations: Department of General, Endocrinological Surgery and
Gastroenterological Oncology Poznan University of Medical Sciences, Poland
, (AB, AS, MD, TB)
*Correspondence to: Adam Bobkiewicz, MD, Department of General, Endocrinological Surgery and Gastroenterological Oncology Poznan University
of Medical Sciences, Przybyszewskiego 49, 60-355 Poznan, Poland. Phone:
+48 618 691 122; fax: +48 618 691 684; e-mail: bobofon007@gmail.com
FRACO Publishing c b
this therapy has become a widely accepted and common
therapeutic modality. Currently, there is a wide spectrum of
indications for NPWT including open fractures, amputation
wounds, sternal wound infections, enterocutaneous fistulas,
open abdomen management, abdominal wound dehiscence,
vascular bypass site infection and many others.4–6
In 2003, Kinetic Concept Inc. introduced a first commercialized system of NPWT instillation (NPWTi): V.A.C Instill
Wound Therapy. A new method of negative pressure wound
therapy combined the benefits of standard NPWT with an introduction of incorporated, controlled and periodic instillation
of topical solution to the wound bed. The mechanism of action
allows for the delivery of instilled fluid to the bed wound
facilitating a removal of cellular debris, exudate and inflammatory molecules that may impair the processes of proper wound
healing. Moreover, NPWTi decreases the bacterial bioburden
within the wound and influences the reduction of biofilm
formation.
Nowadays, NPWTi has been commonly applied to numerous clinical scenarios. In 2013, following an expert panelists’
discussion, the first International Consensus Guidelines was
created to establish the principles for NPWTi.7 Although some
recommendations and guidelines have been reported before,
there are still number of questions concerning NPWTi. Additionally, there is an increased list of publications regarding
NPWTi including different type of instilled solutions, setting
parameters such as instillation time, dwell time as well as time
of NPWT. Therefore, there is a constant debate in regards to
the most accurate recommendations.
The majority of publications concerning NPWTi are expert
opinions, case reports or case series therefore it is difficult to
establish evidence-based conclusions.
In this review, we focused on the current state of art
regarding NPWTi: present indications, types of administered
instilled solutions and potential problems with the utility of
NPWTi were highlighted. We analyzed other instilled solutions
used in NPWTi that were previously evaluated by other authors and are currently not firmly recommended. Additionally,
we described the mechanisms of action for the purpose of
highlighting the principles of NPWTi and thus showing the
DOI: 10.18487/npwtj.v3i1.23
BOBKIEWICZ et al. : NPWTI IN WOUND THERAPY
potential superiority to the standard NPWT.
We believe that the presented detailed review may help
in optimal selection for NPWTi settings for specific clinical
indications and scenarios. Moreover, we hope this work may
increase researchers’ knowledge and improve the quality of
designed experimental studies regarding ongoing research in
the field of NPWTi.
II. M ECHANISM OF ACTION
The mechanism of action for NPWTi remains the same in
regards to the standard NPWT and is the basis for successful
outcomes of NPWTi. The applied negative pressure within
the surgical wound indicates its multi-dimensional action that
positively influences the wound healing process.
The increase of local blood flow influences an enhanced
collagen synthesis and promotes mechanisms that stimulate
angiogenesis. NPWTi also leads to a decreased local tissue
edema, lowers the number of bacteria within the wound
and removes inhibitory agents. The use of NPWT positively
affects tissue granulation process and maintains a moist wound
environment. Additionally, NPWT reduces lateral tension of
the wound edge, positively influences the wound contraction
and supports a proper wound edge vascularity.8–10
III. C ELLULAR AND MOLECULAR CHANGES
Both in vitro and in vivo models showed, that the administration of reticulated open-cell foam (ROCF) generates
microstrain at the cellular level which has a direct influence
on the elevation of proliferating cells within the wound and
enhances vascularity.11, 12
At the cellular level, a mechanical strain generated with negative pressure stimulates sensory cells to molecular changes
within cells.13 Gravity and hemodynamic forces, as constituents of NPWT, significantly influence the improvement
in microenvironmental conditions of the wound bed resulting
in an enhanced wound healing.14 Although, some mechanisms
of action at the cellular level are still unknown, the conception
of cellular mechanotransduction seems to accurately describe
the mechanisms of alteration and enhancement of cellular
properties with the usage of NPWT through the direct use
of administered negative pressure.15
In vitro studies demonstrated that the administration of
negative pressure wound therapy positively influences the
function and activity of fibroblasts.11 The use of NPWT
stimulates proliferation, production and remodeling of fibroblasts, extracellular matrix, as well as increases growth
factors production.11, 16 The results of in vitro studies were
confirmed by in vivo results. Scherer et al. confirmed that cell
proliferation expressed as a percentage of Ki67-positive nuclei
was significantly greater in NPWT group compared to other
groups in mice model.11
IV. B IOFILM
It has been presented that NPWTi significantly influences
the reduction of biofilm composed of varying microorganisms
within the wound, which is considered one of the main reasons
for impaired wound healing.
9
Based on research studies with an animal model and clinical
practice, it was stated that the presence of microorganisms
within the wound, their influence on the wound bed, quick
replication and tendency to form colonies result in biofilm
formation. Bacteria is the most common reason for biofilm
formation, however usually within a wound there is a complex
biofilm formation by bacteria, fungi and protozoa embedded
in a self-produced extracellular matrix of polysaccharides or
other extracellular polymeric substance (EPS), cellular debris
and exudates.17
Consequently, providing a substance to the wound, should
by definition remove not only the exudates and cellular debris
but also influence the destruction and removal of biofilm
bacteria which is a basis for its success.
Mechanical debridement, reduction of biofilm and autolytic
mechanisms are considered the most important components
of NPWTi action and they are key elements emphasizing the
advantage of NPWTi over the standard NPWT.18
The above-mentioned concept and the need for modification
of systems and treatment therapy of chronic wounds are
associated with the current state of the art, indicating that
microbial biofilm is one of the crucial factors impairing
wound healing.19–21 Phillips et al. analyzed based on an
animal model various antimicrobial solutions on Pseudomonas
aeruginosa biofilm.22 Using seven solutions with periodic
NPWT instillation, they compare their efficacy versus NPWT
alone and NPWT with saline solution. Using NPWT alone
(no instillation used), there was no statistical significance
in comparison to the untreated control group even though
the reduction in total CFUs was observed. All experimental
groups using both antimicrobial solutions and saline showed
statistical significance in the reduction of CFUs compared
to the untreated group. However, in authors’ opinion, in the
saline group the reduction in CFUs was rather associated with
mechanical removal than other potential mechanisms acting on
the bacteria’s biofilm. Comparing the antimicrobial solution
groups with the saline groups, all of them except one (Ssolution) showed statistical significance in reduction of CFUs.
Conventionally used lavage, which was initially considered
an important element of wound cleansing, has an increased
risk of bacteria dispersion within the wound bed as well as
beyond the wound. Allen et al. based on the wound models
compared the standard lavage technique and NPWTi and its
influence on the degree of bacterial dispersion and crosscontamination using ex-vivo model with fluorescent bacterial
particles inoculation.23 Both low-pressure lavage and NPWTi
showed comparable effectiveness of wound cleansing (debris
reduction >90%). However comparing tissue damage, based
on three-dimensional photography, more severe tissue damage
was revealed in NPWTi treatment group (P <0.05). The most
important is the fact of no evidence for cross-contamination in
NPWTi. Gabriel et al. comparing bacterial bioburden reduction
in NPWTi group and the control group (standard wound-care
therapy) showed statistical significance in the time required to
decrease bacterial bioburden ( 6.0 ± 1.5 versus 25.9 ± 6.6 days
respectively).18
It is important to note that regardless of the type of the
negative pressure therapy, settings, type of instilled fluid, the
10
NEGATIVE PRESSURE WOUND THERAPY JOURNAL, VOL. 3, NO. 1, 2016
crucial aspect is initial debridement of the wound. Appropriate
debridement is necessary to eliminate poorly vitalized and
necrotic tissue, all foreign bodies within the wound bed as well
as any excessive cellular debris. Currently, there are numerous
methods for debridement including lavage, autolytic agents,
ultrasound, chemicals and enzymes or surgical procedures.24
Regular debridement also plays an important role in bacterial
biofilm reduction.25, 26
V. T ISSUE GRANULATION
Leung et al. comparing the standard NPWT versus NPWTi
analyzed the acceleration of wound granulation in porcine
model. Using the NPWT with instillation and normal saline in
4 cycles of instillation per day (dwell times: 5 or 60 minutes),
they demonstrated statistically significant wound filling and
collagen deposition within the granulated tissue within a bed
wound.27 Brinkert et al. in a prospective study evaluated the
effect of NPWTi used in different clinical scenarios.28 A total
of 131 patients were enrolled to either NPWTi group as the primary method of treatment (n=85; 64,9%) or received NPWTi
after a failed standard NPWT therapy (n=46; 35,1%). Granulation of the wound bed was increased in NPWTi group versus
standard NPWT. Moreover, significant reduction of wound
volume was also shown in NPWTi versus standard NPWT.
Dead space of the wound as well as any undermined cavities
were granulated more rapidly in NPWTi in comparison to
the standard NPWT. Lessing et al. investigated the influence
of different application of standard NPWT and NPWTi on
granulation of the wound in porcine model.29 NPWTi with
saline ( 5 minutes of dwell time every 2,5 hours, negative
pressure -125 mmHg) and various settings of standard NPWT
(intermittent, continuous, dynamic) were applied on dorsal
excisional wound for 7 days. At the time of end point, tissue
samples were taken for histological examination. In NPWTi
group granulation thickness (p<0,05), greater reduction of
wound volume (p<0,05) and higher filling rate of the wound
(p<0,05) were statistically significant compared to the standard
NPWT.
VI. W OUND D RESSINGS
The proper application and arrangement of negative pressure
within the wound dressing is guaranteed with the use of
reticulated open-cell foam (ROCF). The most commonly used
foams on the market are: ROCF- G (V.A.C. R GranuFoamTM
Dressing, KCI USA, Inc, San Antonio, TX) oraz ROCF-V
(V.A.C. VeraFlo CleanseTM Dressing System, KCI USA, Inc.).
Although pore sizes are comparable in both types of dressings,
the different chemical composition of dressings leads to the
fact that physical and chemical properties are different in both
cases of wound dressings. Therefore, the interactions between
the dressing and a wound are also different. ROCF-G is composed of polyether- based polyurethane foam, whereas ROCFV is made of polyester-based polyurethane foam. ROCF-V is
less hydrophobic than ROCF-G, which allows for an easier
adherence and distribution of instilled fluid within the wound.
It was confirmed that the less hydrophobic property of
ROCF dressing is, the more affinity of fluid adherence within
the dressing is observed.30 Moreover, it is easier to drain the instilled fluid with wound exudate and cellular debris outside the
wound. The better susceptibility of ROCF-V for preserving the
fluid within the wound dressing also allows for a lower risk of
pooling the fluid beneath the dressing and outside the wound,
which may contribute to an incidence of a leak and unsealing
of the wound dressing.31 Scanning electron microscopy images
showed similar pore size and structure of both ROCF-G and
ROCF-V.32 Comparable results were presented by Lessing et
al.30 The mean value of pore size was estimated to be 400 µm
to 600 µm in both ROCF-G and ROCF-V. Lessing et al. using
scanning electron microscopy compared mechanical properties
of ROCF-G and ROCF-V regarding tension, compression and
tearing properties.30 Moreover, the properties of individual
foams were analyzed in both wet and dry conditions. Wet
ROCF-V showed statistically better properties under tensile
and tear condition than ROCF-G, in both wet and dry circumstances. Comparing fluid distribution, ROCF-V showed better
capabilities of accumulating fluid versus ROCF-G. Based on
histological analysis, ROCF-V showed increased granulation
within the wound in comparison to ROCF-G after 7 days of
NPWTi or NPWT alone (P < 0.05).
It is important to note that both dressings ROCF-V and
ROCF-G present characteristic to all polymers, a type of
plasticizing effect and hydrolytic degradation due to the interaction with instilled fluid; however, ROCF-V has shown
weaker above-mentioned properties.30
VII. C ONTINUOUS - VERSUS PERIODIC - INSTILLATION
Although NPWTi has been used worldwide in many clinical
scenarios, there are no firm conclusions and recommendations
regarding the optimal type of instillation or a range of dwell
time and amount of instilled fluid.
Rycerz et al. described a wound model for the purpose of
assessment of optimal fluid distribution within a wound based
on two different methods of fluid administration (stained with
methylene blue).33 Using agar-based model, they studied the
impact of continuous- and periodic- instillation on two types
of designed wounds: 1) a simple wound and 2) a complex
tunneled wound. In the model using continuous instillation,
30 ml of fluid/ hour throughout 3,5h was administered using
the negative pressure of -125 mmHg. On the other hand,
in the model using periodic instillation, 75 ml of fluid in
a simple wound model and 120 ml of fluid in a complex
wound model were administered and held for 10 min and later
repeated (-125 mmHg was set up). Comparing the two types
of therapies, an isolated penetration of the fluid within the
wound bed in both simple and complex wound was observed in
continuous instillation, whereas a regular pattern of wound bed
staining was revealed in periodic instillation. Despite the fact
that in the above-mentioned study they demonstrated better
administration parameters of instillation in the case of periodic instillation, according to some studies the efficiency of
continuous instillation was also proven. Independently Lessing
et al. and Scimeca et al. demonstrated the effectiveness of
continuous instillation even though both used different instilled
fluids: saline solution and doxycycline, respectively.30, 34
BOBKIEWICZ et al. : NPWTI IN WOUND THERAPY
Argenta and Morykwas presented the benefits resulting from
the use of intermittent therapy in standard NPWT, emphasizing
the better perfusion of both the wound tissue and surrounding
tissues.3, 35 The element of intermittent therapy in NPWTi is
unequivocally part of the administered therapy composed of
instilled phase with the following dwell time phase, and then
with a formation and maintenance of negative pressure. However, it seems that both the benefits of instilled solution and
the intermittent pauses of active negative pressure positively
influence the effects of treatment with NPWTi.
VIII. I NSTILLATION SOLUTIONS
Based on recent International Consensus Guidelines from
2013, the following instillation solutions were approved as
efficient for the purpose of instillation: Lavasept R (polyhexanide 0.04%), Prontosan R (polyhexanide 0,1% with betaine)
and Microcyn/Dermacyn R (hypochlorous acid solution).7 The
final consensus was established when more than 80% of expert
panelists agreed on the appropriate efficiency of analyzed
instillation solution.
However, there are some limitations to these agreements.
Firstly, the recommendations are based on a personal opinion of twelve expert panelists’ agreement. Secondly, various
studies with different methodologies were included into the
consensus such as prospective, randomized, comparative and
controlled studies leading to a creation of a bias in the
recommendations.
Based on the same consensus other solutions were also
evaluated. Despite the fact that they did not meet the agreement
criteria, it was stated that other instillation solutions may
still be considered in particular clinical scenarios. However,
ongoing research and clinical trials are required to confirm its
efficiency.
Below, we present the most common instillation solutions
used in NPWTi. The mechanism of action of a particular
solution was briefly described. Additionally, we indicated their
potential disadvantages and described both preclinical and
clinical studies regarding the utility of instillation solutions.
The most common instilled solutions are summarized in Table
1.
A. Isotonic solutions
Leung et al. by comparing NPWT with normal saline
instillation versus standard NPWT in porcine model showed
a statistical significance in collagen deposition and tissue
granulation within the wound in the experimental group with
NPWTi.27 Kim et al. conducted a prospective randomized
study comparing NPWT instillation with normal saline and
antiseptic solution (0,1% polyhexanide plus 0,1% betaine).43
There was no statistical significance in any of the analyzed
parameters (a number of operative visits, a length of hospital
stay, a wound healing rate and a wound healing rate within
30-day follow-up) in comparison to two cohorts of patients.
However, the time to final surgical procedure was statistically
shorter in the normal saline group (p= 0,038). In authors’
opinion the effectiveness of normal saline utility in NPWTi
is comparable to this antiseptic solution.
11
Brinkert et al. showed a high rate of wound closure in
98% of patients using NPWTi with normal saline in case
series of 131 patients.28 However, this study has one important limitation. Almost half of the patients (48,8%) received
standard NPWT prior to implementation of NPWTi. Thus the
firm conclusion suggesting NPWTi as a more efficient therapy
is questionable. Fluieraru et al. in a retrospective case series
study proved the efficiency of NPWTi with saline instillation
in patients who previously did not recover under the standard
therapy as well as in patients with chronic complex wound
with no previous NPWT treatment.44 In general, in 23 out
of 24 patients tissue granulation was achieved and surgical
wound closure was possible using either flaps or skin grafts
treatment. Phillips et al. and Davis et al. independently proved
in porcine model that the reduction of P. aeruginosa bacterial
bioburden is significantly lower using saline when compared
to 0.1% polyhexanide and 0.1% betaine solution.22, 45
B. Hypochlorite-based solutions
1) Dakin’s solution: The historical root for the utility of
Dakin’s solution goes back to the times of World War I.46
At the time of its development it was considered one of the
most efficient bactericidal agents for combat injuries.47 The
main component of Dakin’s solution is sodium hypochlorite.
Dakin’s solution is a slightly alkaline solution with the concentration of sodium hypochlorite ranging from 0,125% - 0,5%.
0,5% Dakin’s solution is termed “full strength” and it is the
highest concentration of sodium hypochlorite which is well
tolerated by the skin without any side effects. Consequently,
0,25% Dakin’s solution is termed “half strength”, whereas
0,125% is known as “quarter strength”. Based on both in
vitro and in vivo studies the efficiency of 0,125% Dakin’s
solution was confirmed against S. aureus, P. aeruginosa, E. coli
and Enterococcus.48–51 The cost, availability and simplicity of
using Dakin’s solution makes it one of the most commonly
used bactericidal solutions.
One of the raised controversies about Dakin’s solution is
its potential cytotoxicity. Kozol et al. confirmed the decreased
viability and migration of neutrophils when Dakin’s solution
was used.52 Similar results were obtained independently by
Heggers et al. and Lineaweaver et al., who studied the effect
of Dakin’s solution on fibroblasts.51, 53 Both studies confirmed
the dose- dependent effect of cytotoxicity on fibroblasts.
Thus, the optimal concentration of Dakin’s solution should be
considered in respect to both: the bactericidal effect resulting
in reduction of bacterial bioburden and the risk of alteration
of the function of macrophages and leucocytes which are the
crucial molecules acting in the inflammatory phase of wound
healing.
2) Microcyn R / Dermacyn R : Microcyn is a solution composed of hypochlorous acid and sodium salt. Contained
hypochlorous acid is similar to the one naturally occurring
in humans. In in vitro model, Microcyn reduced the level of
P. aeruginosa, E. coli and S. aureus with a statistical significance.54, 55 Microcyn R / Dermacyn R creates a moist woundcare therapy with the property of rehydration of the necrotic
tissue and promoting autolysis. Goretti et al. compared Dermacyn and diluted povidone iodine in the management of
12
NEGATIVE PRESSURE WOUND THERAPY JOURNAL, VOL. 3, NO. 1, 2016
Table I
T HE MOST COMMON SOLUTIONS AND AGENTS USED IN NEGATIVE PRESSURE WOUND THERAPY WITH INSTILLATION
Solution class
Study
Solution/ Agent
Advantages
Disadvantages
Isotonic solutions
Brinkert et al.,
2013,28
Saline Solution, Lactated
Ringer’s solution,
1/ availability and low costs
1/ No antimicrobial property
2/ No sensitization observed
2/ In some studies lower efficiency
compared to antiseptic solution
Dakin’s solution,
1/ Availability, simplicity and low costs
1/ potential cytotoxicity
DermacynTM ,
1/ There are no known drug interactions
or contraindications
Fluieraru et al.,
2013
Hypochloritebased solutions
Goss
et
201436
al,
Wolvos, 201337
2/ Contained hypochlorous acid naturally occurring in human
Microcyn R
Biguanidines
Kim
et
2014,38
al.,
Polyhexamethylene,
1/ High efficiency in varying type of
wounds
Lehner
201139
al.
Polyhexanide
(Lavasept R ),
2/ No sensitization/ interaction observed
et
Polyhexanide plus Betaine
(Prontosan R )
Silver nitrates
Gabriel
200818
et
al.,
0,5% silver nitrate
1/ Broad- bactericidal spectrum
2/ Variety of commercial dressings
1/ Must be protected from light exposure
2/ Potentially cytotoxic
3/ P. aeruginosa, Enterobacteriaceae or
Salmonella resistance wasreported
Antibiotics
Fleischmann
al., 199840
et
Wolvos, 200441
Neomycin, Gentamicin,
Tobramycin,
Recommended strictly according to
bacterial culture results
Vancomycin,
Polymyxin B, Bacitracin
Catatonic
solutions
Matiasek et al.
2014?
Octenidine
Effective in
No firm conclusions based on prospective studies
contaminated wounds with
multiresistant bacteria
Insulin
Scimeca et al.,
201034
Insulin
1/ Recommended in wounds due to DM
2/ No influence on systemic glycemia
level
Anesthetic
Povidone-iodine
solution
Wolvos, 200441
Chang
200642
Lidocaine
Reduction of pain
No antimicrobial property
Povidone-iodine
1/Availability and low costs
1/ contraindicated in patients with hyperthyroidism, dermatitis herpetiformis
Cadexomer iodine
2/ Rapid antimicrobial action
2/ Cytotoxicity and sensitization
3/ Tissue staining
postsurgical infected ulcers of the diabetic foot.56 Patients
treated with Dermacyn presented significantly shorter healing
time and a higher wound healing rate at 6 months. Landsman
et al. evaluated the effect of Microcyn in the treatment of
mildly infected diabetic foot ulcers.57 In comparison to the
oral levofloxacin treatment group, Microcyn showed higher
clinical success rate of treatment (p= 0,033).
C. Biguanidines
1) Lavasept R /Prontosan R : Both Lavasept R /Prontosan R
belong to the group of biguanides composed of polyhexamethylene biguanide. Additionally, Prontosan contains 0,1%
betaine which has a comparable mechanism of action to
surfactant, reducing the surface tension of a water solution
allowing for a better penetration into the wound and to the
bacterial biofilm.
Minnich et al. conducted an in vitro study using a solution of 0.1% polyhexanide and 0.1% of betaine.58 The
reduction of 13 tested microorganisms was evaluated after
7, 14, and 28 days. Based on this study, the reduction of S.
epidermidis, P. aeruginosa, Serratia marcescens, C. albicans,
S. aureus, vancomycin-resistant E. faecalis, P. mirabilis, E.
coli, methicillin-resistant S. aureus, A. baumannii, E. cloacae,
and E. faecalis was observed confirming the efficiency of
0.1% polyhexanide and 0.1% of betaine for microorganisms
reduction. Romanelli et al. in a randomized controlled trial
investigated the effect of the utility of polihexanide and betaine
BOBKIEWICZ et al. : NPWTI IN WOUND THERAPY
solution in patients with venous leg ulcers.59 Using a portable
device measuring pH of the wound surface (which correlates
with the level of bacterial burden), the authors confirmed the
efficacy of polihexanide and betaine solution in reduction and
stability of wound pH level in comparison to the control group
(p< 0,05). Hübner et al. evaluated the efficacy of 0.02 and
0.04% polyhexanide (polyhexamethylene biguanide, PHMB)
against Pseudomonas aeruginosa SG81 biofilm in in vitro
studies.? Results achieved in the PHMB group was comparable
to 0.1% chlorhexidine digluconate (CHX) in regards to the
amount of biofilm and bacterial metabolism in biofilms formed
with Pseudomonas aeruginosa. Sibbald et al. conducted a
multicenter, prospective, randomized clinical trial comparing
the effectiveness of polyhexamethylene biguanide (PHMB)
foam dressing and non-antimicrobial foam in the treatment
of chronic wounds.60
Bacterial bioburden was significantly reduced in PHMB
foam group (P = 0,016). In addition, pain reduction was
revealed as a statistically significant result at 2 weeks and at
4 weeks of the therapy in the group of PHMB management.
Wound size in PHMB was also reduced in comparison to the
non- antimicrobial foam.
The efficiency of Lavasept R was independently confirmed
in clinical practice by a number of authors. Lehner et al.,
described the utility of Lavasept R in 23 patients with infected
hip endoprosthesis (19 defined as an early infection and 4 as a
late infection).39 The success rate was 84% in early infection,
whereas in late infection 50% of success was reported. Thus in
authors’ opinion, NPWTi with Lavasept may be considered as
a salvage management for infected endoprosthesis, especially
in the early course of an infected hip endoprosthesis.
Köster evaluated the effect of using NPWT with Lavasept
instillation in patients with an early periprosthetic infection
following a knee endoprosthesis placement.61 Only in one
patient the implant needed to be removed, whereas in the
majority of patients implant was preserved. NPWTi was continued from three to nine days. In all except one patient no
infection was present at the time of follow-up (between 12 and
34 months postoperatively) confirmed by clinical, radiological
and laboratory examination. In authors’ opinion NPWTi with
Lavasept reduces the number of surgical revisions, enhances
the wound healing and reduction of infection leaving the knee
implant in situ.
D. Silver nitrate
Silver nitrate possesses a potential property for a creation
of somehow impermeable barrier against microorganisms’
penetration in the bed wound.62 However, in in vitro models it
was observed that silver nitrate (but also nanocrystalline silver)
exhibited a cytotoxic effect to cells, therefore playing a key
role in the healing processes with the effect on leukocytes and
macrophages, as well as fibroblasts and keratinocytes.?, 53
In porcine model study presented by Wright et al., slightly
different results were observed.63 An increased apoptosis and
decreased level of matrix metalloproteinase may potentially
support the process of wound healing. The crucial element
making the difference between in vitro and in vivo studies
13
seems to have a potential to bind the ionic silver which
is different in organic and inorganic constituents. Gabriel et
al. did not observe any side effects of using silver nitrate
for wound healing in the fifteen patients that they analyzed.
Consequently, the instillation with silver nitrate was continued
until the time of confirmed clearance of bacterial bioburden
within the wound.18 Statistical significance was revealed comparing the association between bacterial bioburden and the rate
of wound closure in NPWTi group versus moist wound-care
therapy (p< 0,001).
E. Povidone-iodine solution
Povidone-iodine solution is a well- known disinfecting agent
commonly used in trauma and surgical wounds. Optimal
effectiveness of povidone-iodine solution was established with
1:100 dilution. However, solution with 1:10,000 dilution still
presents bactericidal activity.64, 65 Potential disadvantages of
povidone-iodine solution are tendency for an irritation to
the applied site, cytotoxicity and staining of the tissues.
Although in some in-vivo and animal studies the cytotoxicity
was observed, these results were not confirmed in humans.66
Povidone-iodine solution did not negatively influence bed
wound healing.67, 68 Additionally, in comparison to other antiinfective agents (e.g. neomycin), the sensitization rate of
povidone-iodine is relatively low.69 Chang et al. confirmed
the efficiency of povidone-iodine solution in spinal surgery
indicating a higher surgical site infection rate in the control
group (P<0,05).42
F. Insulin
The potential positive effect of topical application of insulin on wound healing has been reported in basic science
research. However, there are not many studies on insulin
instillation in humans, therefore firm conclusions should not
be taken. In both rat and rabbit studies, the application of
insulin (or combined insulin-zinc therapy) promoted wound
healing.?, 70 Wilson et al. recommended the use of insulin
solution for complex and chronic wounds resulting from
diabetes mellitus including pressure ulcers and amputation site
stumps.71 Rezvani et al., investigated the use of topical insulin on wound healing in randomized, double-blind, placebocontrolled trial.72 The mean rate of wound healing was 46.09
mm2/day in the insulin treatment group and 32.24 mm2/day in
the control group (P = 0.029). It is important to note that the
symptoms of hypoglycemia resulting from the insulin therapy
were not observed in any of the patients. Similar results were
supported by Greenway et al. indicating that topical insulin
is an accelerator of wound healing in humans.73 Scimeca et
al. using NPWTi with insulin in a case report confirmed the
efficacy for the treatment of chronic wounds due to emergency
amputation at the midfoot level.34
IX. C LINICAL INDICATIONS
The review of recent publications concerning NPWTi are
summarized in Table 2.
In 1988, W. Fleischmann who is considered as a pioneer in
NPWT instillation and negative pressure therapy, published
14
NEGATIVE PRESSURE WOUND THERAPY JOURNAL, VOL. 3, NO. 1, 2016
Table II
TABLE 2. R ECENT STUDIES CONCERNING NEGATIVE PRESSURE WOUND THERAPY WITH INSTILLATION
Author and year
Type of study
Number
of
NPWTi
patients
Type of instilled fluid
Instill
time
(sec)
Dwell
time
Instillation cycle
(hour)
Negative
pressure
(mmHg)
mean (range)
Fleischmann
al., 199840
Case series
27
Nebacetin (neomycin and
bacitracin) plus polyhexanidine
NA
30 min
NA
Od
do
33,5 (30-37)
Retrospective
15
Silver nitrate
30
1 sec
Every 2 hour
-125
9,8 (5-20)
Wolvos, 200441
Case series
5
1 or 2% Lidocaine + antibiotic1
15-60
5 min
Every 3 hour
-125
15 (5-24)
Bernstein
and Tam
200574
BH
H,
Case series
5
Polymyxin (500.000 IU)
plus Bacitracin (50.000
IU) in 2L of saline
90
5 min
Every 6 hour
-125
NA
Brinkert et al.,
201328
Prospective
131
Saline
20
10 min
Every 6 hour
(range:
every
4-12 hour)
-125
12,19
Kim et al., 2015
Prospective,
randomized
100
Saline versus 0.1% Polyhexanide + 0,1% Betadine
NA
20 min
Every 2 hour
NA
NA
al.,
Retrospective
48
Saline or Prontosan R
NA
1-60 sec
Every 2 hour
-125
NA
Kim
et
201438
al.,
Retrospective
68
Prontosan R
NA
6 or 20
min
Every 2 or 3,5
hour
-125
NA
Goss
et
201436
al.,
Prospective
7
0,125% Dakin’s solution
(“quarter strength”)
NA
10 min
Every 1 hour
-125
7
Fluieraru et al.,
201344
Retrospective
24
Saline
30
10 min
Every 4 hour
-125
10,1 (6-15)
Wolvos, 201337
Case series
6
Microcyn or Dakin’s solution (“quarter strength”)
NA
5 or 10
min
Every 2 or 4 hour
From
-100 to
-125
Jul 54
Lehner
201139
Prospective,
multicenter
32
Lavasept R (n=31), saline
(n=1)
<60
19 min
(5-30)
From 5 to 40 cycles/day
From
-125 to
-200
16,3 (9-46)
Koster, 200961
Case series
10
Lavasept R
Okt 20
10-15
min
NA
NA
03. Sep
Leffler
200976
Case series
6
Lavasept R
20
20 min
Every 4 hour
-125
NA
et
-50
Days
NPWTi
of
-600
Gabriel
200818
et
al.,
43
Gabriel
201475
et
et
et
al.
al.,
an initial study concerning the influence of instilled fluid
on the wound treatment.40 Using NPWTi with antiseptic or
antibiotic solutions in 27 patients with acute and chronic
infections of bone and soft tissue as well as chronic osteomyelitis, they confirmed the efficiency of NPWTi in 26
patients with one recurrence of infection during 3-14 months
of follow-up. Gabriel et al. published a retrospective study
comparing patients treated with NPWTi and standard moist
wound care.18 The majority of patients were treated with
NPWTi due to pressure ulcers, extremity trauma (including
bone exposure) and abdominal surgical wounds. In the NPWTi
group of treatment the time of required treatment, wound
closure, resolution of wound infection and hospital stay were
significantly shorter in comparison to the standard method of
treatment (p< 0.001). Wolvos in his initial report of clinical
practice with NPWTi described a routine use of combined
Lidocaine and antibiotic solution for the purpose of chronic
and complex wound treatment.41 Surprisingly, all patients
reported a reduction in pain following NPWT instillation
with 1-2% Lidocaine as a topical anesthetic. Wolvos also
observed that an appropriate, targeted antibiotic (based on the
microbiological results) decreased the bacterial burden in the
wound. Bernstein and Tam described a case series of patients
with post-surgical diabetic foot wounds treated with NPWTi.74
Based on their initial experience, the application of topical
antibiotics positively influences the progress of wound healing
in chronic and complex wounds after surgical management in
DM patients. In authors’ opinion, NPWTi positively affects
the wound fluid viscosity, decreasing inflammatory agents and
cellular debris and it also reduces bacterial burden. Brinkert et
al. in a prospective study compared the efficiency of NPWTi
and standard NPWT in a group of 131 patients treated in
three referral orthopedic or surgical centers in France.28 The
most common clinical indication for both NPWTi and standard
NPWT was an open fracture (n=46), pressure ulcer (n=27) and
non-healing postoperative dehiscence (n=25). Wound closure
BOBKIEWICZ et al. : NPWTI IN WOUND THERAPY
was possible to be achieved in 98% of patients treated with
NPWTi with the mean duration of the therapy 12,19 days. In
the recent prospective randomized study, Kim et al. analyzed
the effect of NPWTi on wound healing using two different
solutions: normal saline versus antiseptic solution (0.1% polyhexanide plus 0,1% betaine).43 In the majority, chronic or
complex wound was located within the lower extremity in
both analyzed groups of treatment. There was no statistical
difference between the compared groups of treatment for
the number of surgeries, the length of hospital stay, wounds
closed/ covered ratio and wounds ratio that remained closed
within 30 days of follow up. The only significance was the
time to final surgical procedure which was favorable in NPWTi
with saline solution group (p=0,038). Based on this study,
normal saline and antiseptic solution (0.1% polyhexanide plus
0,1% betaine) demonstrate a similar efficacy. Gabriel et al.
compared the standard NPWT and NPWTi with saline or
polyhexanide in patients with extremity or trunk wounds.75
A total of 48 patients treated with NPWTi showed a statistical
significance in comparison to the standard NPWT for the time
of hospital stay (8,1vs 27,4 days), duration of the therapy (4,1
vs 20,9 days), time of wound closure (4,1 vs 20,9 days) and
mean operating room debridement (2,0 vs 4,4). Goss et al. in a
prospective pilot study evaluated the efficacy of the reduction
of a wound bacterial bioburden comparing the standard NPWT
and NPWTi.36 Dakin’s solution was used as an instilled bactericidal agent. Chronic venous stasis and diabetic foot ulcer
were the most common underlying wound pathologies in both
treatment groups with the mean time of wound duration 30
months in the standard NPWT group and 23 months in NPWTi
group. At the time of end point (7th day of the therapy), there
was no statistically significant difference between these two
groups in reduction of bacterial bioburden (CFU/ gram of
tissue culture; p=0,43). However, the mean absolute reduction
of bacterial bioburden was statistically significant in NPWTi
group versus standard NPWT (p=0,016). Fluieraru et al. used
NPWTi as the primary method of treatment for extensive
undermining deep wounds (n=12) as well as in patients who
failed the standard NPWT (n=12).44 Isotonic saline was used
as an instilled fluid with a 10-minute dwell time and 30
seconds of instillation. There was no complication associated
with NPWTi. In 23 patients wound closure was achieved using
flaps or skin grafts following preconditioning of the wound bed
with the use of NPWTi. In all patients, good results of tissue
granulation and filling of the wound cavities were observed.
Recently, Wolvos published a small case series of patients treated with NPWTi in contaminated, chronic abdominal
wounds (n=3) or infected wounds within the lower extremity
(n=2) and chest wall (n=1).37 Wound healing and closure
were achieved in all patients using skin graft or surgical
closure (primary, secondary or delayed primary). There was no
difference in quality and the amount of tissue granulation in
patients treated with NPWTi (n=6) and standard NPWT (n=1),
even though both groups were small and inconsistent regarding
the types of the underlying pathologies of the wound and
the degree of the contamination, which is a limitation of the
study. In 2011 Lehner et al. published a multi-center prospective observational study concerning the utility of NPWTi in
15
patients following hip and knee replacements with surgical
site infection associated with orthopaedic implants.39 Clinical
indication for introduction of NPWTi included infected hip
implant (n=20), infected knee implant (n=10) and 2 patients
with infected osteosynthesis material. Routinely, polyhexanide
was used as an instilled fluid in all but one patient in whom
saline was used. Twenty-two patients had an acute infection
(<8 weeks after orthopedic implant placement), whereas ten
patients had a chronic infection (between 8 and 36 weeks
postoperatively). After NPWTi course, an eradication of the
wound infection was confirmed in 24 patients (75%), whereas
in 6 patients the recurrence of the wound infection was
revealed (18,8%) and in 2 patients ongoing wound infection
was reported. Koster presented ten patients with an implantassociated infection following a knee implant placement.61
After a required wound debridement, NPWTi was initialized
using Lavasept R . During the observational period of 13-34
months after a completed NPWTi therapy, only in one patient
there was a case of a reinfection. Similar results were achieved
by Schintler et al., who used NPWTi with Lavasept R in
patients with soft tissue infections and necrotizing fasciitis.77
Additionally, in eight patients bone exposure or septic arthritis
were observed. The time of administration of NPWTi ranged
from 4 to 18 days. In all patients wound closure was achieved
using skin graft, flaps or secondary closure. Leffler et al.
described a small case series of 6 patients with osteomyelitis
within the lower extremity (n=5) or the upper extremity (n=1)
treated with NPWTi.76 Lavasept R was used as an instilled
fluid with the following settings: 20 seconds of instillation
with 20-minute dwell time followed by NPWT at -125 mmHg.
After the NPWTi therapy, sterile bacterial cultures from the
site of an infection were confirmed in all patients. There
was no recurrence of a wound infection following a flap
reconstruction. Also, they did not observe a flap loss due to
the impairment of wound healing.
X. C ONTRAINDICATIONS AND WARNINGS
Similarly to the standard NPWT, the list of indications for
NPWT with instillation has been recently increased. There has
been a tremendous progress in in the field of NPWT as well
as NPWT with instillation leading to the fact that this therapy
is currently used in many clinical scenarios.
However, there are some clinical situations when more
attention should be taken. Some solutions should also be
avoided as an instillation. Contraindications for NPWTi are
exactly the same as for standard NPWT and include: exposed
blood vessels or nerves, exposed bowels (or qualified for
abdominal NPWT) or anastomotic sites. Solutions containing
octenidine, hydrogen peroxide and other alcohol-based products are contraindicated in NPWT with instillation because
of their interactions and potential destructive effect on foam
dressings.? Certain contraindications are specific to the applied
solution. For example, neomycin may be absorbed locally and
an increased serum concentration may result in nephrotoxic
and ototoxic reactions.78 Anaphylaxis caused by the local
administration of Bacitracin or Polymyxin B or when used
as an irrigation was also reported.79–81 Although some authors
16
NEGATIVE PRESSURE WOUND THERAPY JOURNAL, VOL. 3, NO. 1, 2016
reported shock, coma or even death related to povidone iodine
solution and hydrogen peroxide utility in surgical debridement,
generally both local antiseptics seem to be safe and generally
recommended for the purpose of wound cleansing.82, 83
Fluid instillation should not be delivered directly to the
abdominal or thoracic cavities.84 Firstly, a potential retention
within the human cavity may decrease the body temperature.
Secondly, instilled fluid may be retained within the body cavities and may not be properly suctioned. Thus, the cases with
a present bacterial inflammation may lead to intra-abdominal/
intrathoracic abscesses formation, despite the proper maintenance and application of negative pressure. However, D’Hondt
et al. recently described a case report of NPWTi use in a
patient treated with open abdomen management following
a pancreatic surgery.85 Due to a failure of previous therapy (included abdominal NPWT), NPWT with gentamicin
and metronidazole (based on antibiogram results) was implemented. Instillation time was set for 20 seconds with 10
minutes of dwell time and a negative pressure of -125 mmHg.
During the 12-day no local or systemic side effects of NPWTi
were observed and the eradication of B. fragilis, P. aeruginosa
and Lactobacillus was confirmed based on abdominal cultures.
It is important to note that described patient developed frozen
abdomen. Thus, possibly the retention of instilled fluid is minimized by the granulated tissue preventing it from pooling the
solution directly through the entire abdominal cavity. Based
on our experience, we agree with the authors that NPWTi
may serve as an important alternative when the standard open
abdomen therapy fails. However, further research and clinical
trials are required to evaluate the safety and efficiency of
NPWTi in open abdomen. Because of the above mentioned
reasons, NPWT with instillation should be avoided in clinical
situations with unexplored wound or a potential tunnel drained
into body cavities. NPWTi should not be placed over skin flaps
or grafts with a potential risk of failure and problems with
adapting and healing of the skin flaps or grafts.
XI. C ONCLUSIONS
Based on current knowledge supported by clinical trials,
NPWTi is found as an important alternative to standard NPWT
in many clinical scenarios. Moreover, in some publications the
superiority of NPWTi over standard NPWT was highlighted.
Further studies regarding both basic science as well as
clinical trials are needed to establish the firm conclusions
concerning the efficiency of NPWTi.
From practical point of view, in authors’ opinion it is
important to share experience and collect data from varying
institutions in different clinical indications to create firm
conclusions and form guidelines for NPWTi.
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https://econjournals.com/index.php/ijeep/article/download/9806/5254
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English
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ANALYSIS AND PROSPECTS FOR THE DEVELOPMENT OF REGIONAL ENERGY INTEGRATION OF THE EURASIAN ECONOMIC UNION COUNTRIES
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International journal of energy economics and policy
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cc-by
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Received: 02 April 2020 Received: 02 April 2020 ABSTRACT In the presented paper, it is proved that a new objective trend appears in the current conditions of Eurasian region development. It will strengthen mutual
cooperation between the countries of Eurasia and it will enrich regionalization process in the context of world energy markets restructuring, intensification
of competition, deglobalization tendencies and other challenges of the world economy. At the same time, fuel and energy complex (FEC) is becoming
the main driving force for the countries of Eurasian region and their further development. The concept of regional energy integration of the Eurasian
Economic Union (EAEU) countries is offered in the paper. It’s based on the strategic advantages of economic integration in relation to the fuel and
energy complex. Energy potential analysis of the EAEU member countries is performed. The issues of common energy markets formation in the region
are considered. The main problems and a number of uncoordinated tasks between the countries participating in integrational process in this area are
highlighted in terms of common gas, oil and oil products market formation, although it is noted that development and approval stage of the Programs
for such formation is mostly completed. It is expected that these markets will be fully formed by 2025. It was emphasized that energy integration is
one of the most important areas for the EAEU countries development, which is expected to bring significant economic benefits in the long-term period. Keywords: Regional Energy Integration, Common Energy Markets, Energy Potential, The Eurasian Economic Union
JEL Classifications: F02, F63, O13 International Journal of Energy Economics and
Policy International Journal of Energy Economics and Policy, 2020, 10(5), 13-20. Natalya Yuryevna Sopilko1*, Olga Yuryevna Myasnikova2, Nataliya Vital’evna Bondarchuk3,
Natalia Anatolyevna Navrotskaia4, Tatyana Evgenyevna Migaleva5 Natalya Yuryevna Sopilko1*, Olga Yuryevna Myasnikova2, Nataliya Vital’evna Bondarchuk3,
Natalia Anatolyevna Navrotskaia4, Tatyana Evgenyevna Migaleva5 1Russian State Social University, Moscow, Russia, 2Peoples’ Friendship University of Russia (RUDN University), Moscow,
Russia, 3Financial University under the Government of the Russian Federation; Russian Academy of National Economy and Public
Administration under the President of the Russian Federation, Moscow, Russia, 4Saint Petersburg University, Saint Petersburg,
Russia, 5Plekhanov Russian University of Economics, Moscow, Russia. *Email: sheremett73@gmail.com DOI: https://doi.org/10.32479/ijeep.9806 DOI: https://doi.org/10.32479/ijeep.9806 DOI: https://doi.org/10.32479/ijeep.9806 Accepted: 13 June 2020 This Journal is licensed under a Creative Commons Attribution 4.0 International License 2. LITERATURE REVIEW It should be highlighted that today different types of electricity
markets having been already operating in the EAEU countries. Kurbanaliyev and Drogovoz in their study (2016) offer a
conceptual approach to the development of common electricity
market in the EAEU, based on the formation of supranational
electricity market without significant reform of national markets. It is noted that globalization of energy markets, especially in Asia,
can have a significant impact on the trends in supply and demand of
fuel and energy resources. Moreover, the logistical factor becomes
more and more important in the world and regional trade (Telegina
and Khalova 2017; Gillessen et al., 2019). Looking at experience of many regional associations, it can be noted
that they are based on the process of international labor division
with the greatest benefit and realization of economic interests of
the participating countries. For example, the first step in European
economic integration was the signing of agreement for the European
Coal and Steel Association (EUSC) (Hay and Rosamond, 2002). The
main vector of this process development was possibility of favorable
environment creating in the European region for goods movement
of metallurgical and coal industries between the countries. Such
a union has become an incentive for the development of further
integration processes in the European region. It’s obvious that today different types of markets have been
already operating in the EAEU countries. It’s considered that these
aspects also directly effect on developmental character of energy
integration in the EAEU. At the end of the last century, integration processes were
developed in the countries of the Southern Cone Common
Market (MERCOSUR) in manufacturing and energy sectors. This
interaction has intensified further integration in the region (Mares
and Martin, 2012). A program was adopted for the economic
cooperation of the countries, which goal was intersectoral
specialization in strategically important economic areas, and
especially in energy sector. In recent years serious work to understand the basic principles,
mechanisms and conditions of energy integration has been
carried out by a number of major domestic scientists, mainly
by scientific teams with the guidance of Telegina et al. (2019). Energy integration experience and single energy space formation
in the European Union is studied in details, as well as conceptual
vision of the goals, objectives and forms of energy integration
in the EAEU is presented. 1. INTRODUCTION Energy cooperation has been declared as unconditional priority
for the EAEU development for the period until 2025, when the
formation of common markets for electricity, gas, oil and oil
products should be completed. Such cooperation should contribute
to the formation of unified energy space, transcontinental energy
corridors, should increase energy resources availability for the
citizens of the EAEU member countries and their economic
entities, as well as it could generally lead to more intensive
economic growth and increase of well-being level of the population
of the EAEU countries over long term perspective. inherited energy system, comparable common technological and
technical standards, it has a cross-bordered infrastructure and
capacities, etc. According to experts the formation of common
energy market will allow obtaining a large number of advantages
for countries, such as possibility of energy flows using, energy
capacities redistributing, balancing, and also phasing out obsolete
equipment from enterprises funds, competitiveness rising of the
EAEU as a whole (Sopilko et al., 2020). Some experts consider energy integration in the Union region in the
context of energy security (Cherp and Jewell, 2014; Myasnikova
et al., 2019), which means an ability to protect member countries
from lack of energy resources and energy shortages. The main
goal of this approach is the formation of a single energy space on
the principles of sustainability. Energy integration is the locomotive of integration process in the
region because of rich resource base, high role of fuel and energy
complex (FEC) in economies of the EAEU countries, historically
established community and inextricable economic ties in them. 1. INTRODUCTION In that context the priority is international cooperation between
the countries and common markets formation. As a rule,
partnership initially develops in specific economic spheres of
strategic significance for countries, and it is assumed that in case
of successful convergence some certain integration effects appear. And that give impetus to cooperation in related economy sectors,
and then such effects will be obtained in other spheres. The current stage in the development of international economic
relations is characterized by uncertainty and structural changes. The period of 2008-2019 and the beginning of 2020 was marked by
series of events of various scales: The global economic crisis, fall
and partial restoration of energy prices, the shale revolution, the
development of military and political conflicts, and the slowdown
in global economic growth. That leads to the world markets
restructuring, changes in the relationship between consumers and
sellers of goods, services, and, in particular, energy resources, and
contributes to integration development. In 2015, five countries (Russia, Belarus, Kazakhstan, Armenia
and Kyrgyzstan) created a world-wide international integration
association – the Eurasian Economic Union (the EAEU), which
gave new incentive to international economic integration processes
in Eurasia space. In 2015, five countries (Russia, Belarus, Kazakhstan, Armenia
and Kyrgyzstan) created a world-wide international integration
association – the Eurasian Economic Union (the EAEU), which
gave new incentive to international economic integration processes
in Eurasia space. International Journal of Energy Economics and Policy | Vol 10 • Issue 5 • 2020
13
This Journal is licensed under a Creative Commons Attribution 4.0 International License This Journal is licensed under a Creative Commons Attribution 4.0 International License Sopilko, et al.: Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic U Energy cooperation has been declared as unconditional priority
for the EAEU development for the period until 2025, when the
formation of common markets for electricity, gas, oil and oil
products should be completed. Such cooperation should contribute
to the formation of unified energy space, transcontinental energy
corridors, should increase energy resources availability for the
citizens of the EAEU member countries and their economic
entities, as well as it could generally lead to more intensive
economic growth and increase of well-being level of the population
of the EAEU countries over long term perspective. 2. LITERATURE REVIEW The following components of energy
integration are highlighted: Theoretical studies allow us to consider integration processes
development in energy sector of Eurasian region, taking into
account certain specifics. It is noted that large projects have
already been successfully implemented in the region, such as
Electrification Plan of Russia (GOELRO) was implemented at the
beginning of the last century (Dynkin et al., 2018). Special role
in this program was given to energy industry, which served as a
driver for the development of other industries and fields. Technical integration (rational integration of many technical
facilities into a single complex for production, transmission,
distribution and consumption of energy). Technological integration as combination of individual
technologies into a single technological chain (from energy
production to its consumption) and the formation of single
technological space on the basis of individual energy systems. In connection with the EAEU formation and its reliance on
fuel and energy complex, it acquires special scientific interest
and theoretical comprehension of regional energy integration
processes. Functional integration, which can ensure the unity of goals, criteria
and procedures harmonization, the implementation of all functions
and processes aimed at goal achieving. Some authors emphasize that “unique” conditions have developed
in the post-Soviet space, especially in terms of common electricity
market creating and developing in the EAEU countries (Zemskova,
2018; Perskaya, 2020; Pastukhova and Westphal, 2018). It is also
noted that the Union has great energy potential, it has common Organizational integration provides the interaction of participants
and their concerted actions to their goal achievement. Sopilko, et al.: Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic Unio Information integration, which supplies single integrated approach
to creation and maintenance of informational base of energy
metasystem. The main background of the concept is based on the fact that
energy integration provides participating countries with a number
of unique and strategic opportunities and advantages (Figure 2). Software integration allows using of coordinated and interconnected
complex of models, algorithms and programs to ensure the overall
functioning of all system components. The first advantage is increasing of energy security level and
sustainability of integrated energy systems, such as trunk electric
networks and gas piping systems, which rise reliability and security
level of electric power supply to consumers. 2. LITERATURE REVIEW For example, in the
framework of isolated energy systems, or in poorly integrated
electric power systems, emergency shutdown of some generating
or transmitting capacities, or change in mode of their operation, is
likely to lead to the damage of adjacent systems and subsequent
cascading blackouts of consumers. Some experts note that today there are objective backgrounds
for globalization in energy sector, two of them are distinguished. It’s market factor – constant increase in energy demand in such
developing countries as China, India and others (Meynkhard,
2020); technological factor – existing innovative technologies
in energy sector in developed countries (renewable energy
technologies, deep-sea mining, etc.) (Nazarova et al., 2019). On the contrary, in case of appearance of electric system with high
integration level, the availability of reserve capacities and high
coordination level between electric power zones allows avoiding
such negative consequences. Modern Unified Electric System
of Russia (UES of the Russian Federation) can be an example
of highly integrated system in which even breakdowns of some
elements will not lead to collapse of the entire system. However,
such efficient arrangement of power grids is customary only for
few regions of the world. It is advisable to make more detailed analysis of regional energy
integration in the EAEU space, taking into account all existing
development backgrounds. 3. METHODOLOGY OF THE RESEARCH Currently, Eurasian Economic Commission (2019) has identified
the main directions of energy sector development in terms of
common energy markets formation of the EAEU countries. Created markets should be opened for participation of energy
companies of the EAEU’s countries, have special architecture
involving direct interaction between business entities in terms
of extraction, processing and transportation of energy resources. And the trade in these markets should be realized on exchange
platforms or under direct contracts. The second advantage is higher production efficiency of electric
and thermal energy, more flexible possibilities for energy system
balancing, higher performance coefficient of generating plants
due to the rational use of maneuverable and low-maneuverable
generation capacities within a single energy space. It provides
significant economic benefits, since it allows reducing the cost of
resources per unit of generated electricity and to increasing the In accordance with the goals set by the EEC, common energy
markets formation in the Union will contribute to the sustainable
development of economies, citizen’s well-being increasing, as
well as energy security strengthen and competitiveness growth of
goods produced in the EAEU on the world market. All of these can
make a significant contribution to strengthening the integration
foundation of the Union and helps to become a powerful factor in
the development of the Eurasian region as a whole. Figure 1: The mechanism of energy resources redistribution in the
process of energy integration
Source: Compiled by the authors. Source: Compiled by the authors. Figure 2: Opportunities provided by energy integration Figure 1: The mechanism of energy resources redistribution in the
process of energy integration
Source: Compiled by the authors. Figure 1: The mechanism of energy resources redistribution in the
process of energy integration It should also be kept in mind that energy resources can be
classified in the global energy market as own and acquired. The
predominance of own or acquired resources in the country’s
energy balance determines its role in integration association as
net consumer or as net producer of energy resources. This, in turn,
determines the necessity, scale and forms of energy resources
redistribution within the framework of energy integration in
accordance with general distribution mechanism. In our view
the effective redistribution of energy resources enhances energy
security within entire integration association (Figure 1). Source: Compiled by the authors. Source: Compiled by the authors
Figure 2: Opportunities provided by energy integration Source: Compiled by the authors. 3. METHODOLOGY OF THE RESEARCH Figure 2: Opportunities provided by energy integration In our opinion, it is possible to suggest further development of
ideas connected with energy integration in the EAEU region in
the context of regional energy integration concept. Theoretical
justification for further expansion of energy integration process
in the EAEU countries and neighboring countries of Central Asia
and the Far East can be offered for consideration. 15 International Journal of Energy Economics and Policy | Vol 10 • Issue 5 • 2020 International Journal of Energy Economics and Policy | Vol 10 • Issue 5 • 2020 Sopilko, et al.: Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic Unio service life of both generating plants and network infrastructure
due to their more balanced loading during the periods of decline
and peak consumption. In 2017, Russia possessed 85.7% of total oil production in the
EAEU, and 14.0% were in Kazakhstan. Between the period of
2015-2017 oil production grew by 3.6% in Russia, by 6.3% in
Kazakhstan. In Kazakhstan there was a temporary decrease in oil
production in 2015-2016 due to delay of industrial development
starting of Kashagan oil field. Price decrease and temporary excess
of crude oil in the world market had also negatively impacted
on oil industry of Kazakhstan during this period. In Russia, a
slight decrease in oil production in 2017 was due to production
restrictions introduction as part of the “OPEC +.” The third advantage is increased ability to deploy various types of
generating capacities within electricity system. Different types of
capacities are defined by complementary characteristics. The most
stable and reliable is such energy system, in which both highly
maneuverable and low maneuverable capacities are represented
in sufficient quantities, as well as generating facilities of various
installed capacities. Today the most advanced electric power
systems allow to control the loading of various capacities types and
the distribution of generated electricity in large geographic areas. This, in fact, was implemented within the framework of unified
electric power system of the USSR and is partially preserved in
the UES of the Russian Federation. Main facilities for petroleum products production are located in
Russia, Kazakhstan and Belarus. In 2017, the EAEU countries
produced 314.3 million tons of oil products, 89.5% of them came
from Russia. 3. METHODOLOGY OF THE RESEARCH The production of petroleum products has decreased
since 2014 by 6.7 million tons of oil equivalent (2.3%) in Russia, by
3.7 million tons of oil equivalent (16.9%) in Belarus, by 1.0 million
tons of oil equivalent (6.5%) in Kazakhstan. The decrease in oil
refining in Russia is associated with the phased implementation
of “tax maneuver” (Federal Law of 03.08.2018 No. 301-FZ), in
Belarus it’s connected with restrictions introduction on the re-
export of oil products produced from Russian oil. In view of all the above, it is possible to formulate comprehensive
definition of regional energy integration. It is a process of mutual
technological and economic ties forming between fuel and
energy complexes of the countries of regional association, which
contributes to formation and development of spatial distributed
energy systems and improves economic and energy efficiency of
fuel and energy complex, provides higher availability of energy
and energy carriers for consumers and strengthens energy security
of the countries. The consumption of oil and oil products steadily increased during
the period under review in the EAEU and by 2017 the total
demand reached 217.7 million tons of oil equivalent (4.1% higher
than in 2014). In 2017 net oil exports from Russia reached 320.1
million tons of oil equivalent (14.3% growth compared to 2014),
it reached 86.1 million tons of oil equivalent (3.9% increase) from
Kazakhstan (Table 1). Such an approach requires regional energy integration, detailed
analysis of available resource potential of the EAEU countries and
the determination of main vectors for the development of these
processes in the context of integration. The EAEU countries import oil for refining and oil products for
their own needs mainly from Russia. In 2017, gasoline, diesel
fuel and fuel oil (1.8 and 1.2 million tons) were delivered from
Russia to Kazakhstan and Armenia, gasoline and diesel fuel (960
and 940 thousand tons) were delivered to Kyrgyzstan and Belarus
(Eurasian Economic Commission, 2019) (Table 2). 4. RESULTS AND ANALYSIS International Journal of Energy Economics and Policy | Vol 10 • Issue 5 • 2020 ysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic Union Countries Sopilko, et al.: Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic Unio from oil fields. There are only few gas fields in Kazakhstan: More
than 70% of explored reserves of free gas are concentrated in the
Karachaganak field. Russia and Kazakhstan export gas to markets of the EAEU
countries and to the third countries. Export orientation level of gas
industry (calculated as the ratio of net export to gas production)
was 32% for Russia and 25% for Kazakhstan. Net gas exports had
significantly increased in Russia (by 40.5 billion cubic meters,
or by 21.7%) and Kazakhstan (by 6.2 billion cubic meters, or by
93.5%) for 2015-2017. Overall gas consumption in the EAEU countries had decreased
by 5.8 billion cubic meters (or 1.1%) for the period 2015-2017. In
Russia gas consumption decreased due to the effects of economic
crisis in 2015, as well as due to warm conditions in winter months. It was restored to the level of 475.9 billion cubic meters only by
2017. In Kazakhstan gas consumption had increased for 4.0 billion
cubic meters m (12.4%) in 2015-2017. In Belarus gas consumption
had decreased (by 1.6 billion cubic meters, or by 7.6%) and in
Armenia (by 0.4 billion cubic meters, or by 18.1%). In Armenia and Belarus gas demand is almost completely covered
by supplies from Russia, Kyrgyzstan’s demand is recovered by
Kazakhstan. Mutual deliveries are made between Russia and
Kazakhstan: From Kazakhstan to Russia in order to provide raw
materials for the Orenburg gas processing plant, from Russia to
Kazakhstan for gas supply to the northern regions of the country
that do not have communication with the main gas producing
regions (Table 4). 4. RESULTS AND ANALYSIS Table 1: Key indicators of oil industry in the EAEU
countries during 2014-2017, million tons
Country/year
2014
2015
2016
2017
Oil production
Armenia
0,0
0,0
0,0
0,0
Belarus
1,7
1,7
1,6
1,6
Kazakhstan
84,3
82,7
81,3
89,6
Kyrgyzstan
0,1
0,1
0,1
0,1
Russia
528,7
536,3
548,6
547,9
The EAEU, total
614,7
620,8
631,6
639,2
Petroleum products
Armenia
0,0
0,0
0,0
0,0
Belarus
22,2
23,3
18,8
18,4
Kazakhstan
14,6
13,5
13,5
13,7
Kyrgyzstan
0,1
0,3
0,7
0,8
Russia
288,1
283,6
281,5
281,4
The EAEU, total
325,0
320,7
314,5
314,3
Oil and oil products consumption
Armenia
0,4
0,3
0,3
0,3
Belarus
8,0
6,8
6,3
6,3
Kazakhstan
12,5
14,9
15,9
15,8
Kyrgyzstan
1,5
1,7
1,8
1,9
Russia
186,2
185,9
191,8
193,4
The EAEU, total
208,7
209,6
216,1
217,7
Net exports of oil
Armenia
0,0
0,0
0,0
0,0
Belarus
−26,4
−27,5
−21,4
−21,3
Kazakhstan
82,9
81,7
78,0
86,1
Kyrgyzstan
−0,1
−0,3
−1,1
−1,1
Russia
280,0
306,3
322,7
320,1
The EAEU, total
336,5
360,3
378,3
383,9
Source: According to Eurasian Economic Commission, 2019 Table 1: Key indicators of oil industry in the EAEU
countries during 2014-2017, million tons Coal production had increased by 41.3 million tons (or 11.7%)
compared to 2014 in the EAEU countries mainly due to Russia’s
contribution. In Russia coal production had increased by 45.9
million tons (16.4%). At the same time, in Kazakhstan coal
production had decreased by 5.3 million tons (by 7.2%) (Table 5). In Russia coal consumption had increased by 16.0 million tons
(10.5%) for 2014-2017 compared with the results of 2014. In
Kazakhstan coal consumption was characterized by unstable
dynamics and amounted to 50.0 million tons in 2017, it was 4.6
million tons (8.4%) less than the level of 2014. Net coal export from the EAEU countries in the reporting period
had increased by 33.6 million tons (23.8%) according to the level
of 175.2 million tons in 2017. Russia is the largest exporter of coal
in the EAEU, providing all net export growth. Coal export from
Kazakhstan was stable at the level of 16-19 million tons. Gross electricity production in the EAEU had increased by 47.4
TW-h (3.9%) and had reached 1272 TWh for the period 2014-
2017. Russia accounted for 86.3% of all electricity generated in
the EAEU by the end of 2017 (1097 TWh), Kazakhstan – 119
TWh (9.4%), Belarus – 35 TWh (2.7%). 4. RESULTS AND ANALYSIS Energy sector of economy is one of key sectors of economic
development of the EAEU countries. Therefore, it is efficient
to maximize existing resource potential using of oil and gas,
coal, nuclear and transport industries as a locomotive for further
successful development of integration processes in the region. Energy should play a stimulating role in the development of other
economy sectors, especially knowledge-based and high-tech. Until the end of tax maneuver in 2019-2024 export of crude oil
and petroleum products from Russia is subject to export customs
duty calculated on the basis of world prices for crude oil. Crude
oil supplies from Russia to Belarus under the current regulatory
regime are exempted from customs duties. In Kazakhstan, payment
of export customs duty for crude oil export is not provided. Kazakhstan has repeatedly imposed a ban on petroleum products
imports from Russia in order to protect the interests of their own
oil companies in 2014-2018. For this purpose, it would be advisable to analyze the existing
potential in energy sector of the EAEU. The share of fuel and energy complex in GDP structure of the
EAEU countries is about 17%, and in total industrial production, the
share reaches one third – 33% (Eurasian Economic Commission,
2019). The main producers, net exporters and consumers of energy
resources in the EAEU are Russia and Kazakhstan. The rest of the
EAEU countries are net importers of energy resources. In general,
the Eurasian Economic Union has the most significant energy
potential in the world without any exaggeration. By the end of 2017, gas production in the EAEU countries reached
754.8 billion cubic meters, 93.1% of total production has been
produced in Russia (Table 3). In Russia gas production has increased by 36.5 billion cubic meters
(5.4%) since 2014 due to the development of new gas fields on
Yamal Peninsula. In Kazakhstan the volume of gas production in
2017 had increased by 11.4 billion cubic meters compared with
2014 (12.4%) and reached 50.6 billion cubic meters. It’s 6.7% of
all production in the EAEU. Associated gas is mostly produced About a quarter of all proved world mineral reserves are
concentrated on the territory of the EAEU countries, including
40% of world gas reserves, 25% of world coal reserves, 20% of
world oil reserves and more than 20% of world uranium reserves. 4. RESULTS AND ANALYSIS Electricity production
had increased in all EAEU countries, except Kyrgyzstan for the
period under review. Gas generation predominated in the EAEU International Journal of Energy Economics and Policy | Vol 10 • Issue 5 • 2020
17
Table 2: Mutual supplies of oil and oil products to the EAEU in 2017, thousand tons
Oil
In Armenia
In Belarus
In Kazakhstan
In Kyrgyzstan
In Russia
From Kazakhstan
0
0,065
0
0
0,8
From Russia
0
24
0
0,04
0
Petrol
In Armenia
In Belarus
In Kazakhstan
In Kyrgyzstan
In Russia
From Belarus
0
0
0
0
40
From Russia
130
140
1230
470
0
Diesel fuel
From Belarus
0
0
0
0
100
From Kazakhstan
0
0
0
5,9
18
From Russia
140
800
520
490
0
Fuel oil
From Russia
970
0
85
0
0
Source: According to Eurasian Economic Commission, 2019 Table 2: Mutual supplies of oil and oil products to the EAEU in 2017, thousand tons Table 2: Mutual supplies of oil and oil products to the EAEU in 2017, thousand tons
Oil supplies of oil and oil products to the EAEU in 2017, thousand tons Table 3: Key indicators of gas industry in the EAEU
countries in 2014–2017, billion cubic meters
Country
2014
2015
2016
2017
Gas production
Armenia
0,0
0,0
0,0
0,0
Belarus
0,2
0,2
0,4
0,4
Kazakhstan
39,2
41,9
42,8
50,6
Kyrgyzstan
0,0
0,0
0,0
0,1
Russia
666,8
658,1
682,4
703,1
The EAEU, total
706,4
700,2
725,7
754,2
Gas consumption
Armenia
2,4
2,2
2,1
1,9
Belarus
21,3
19,9
19,8
19,7
Kazakhstan
32,6
34,5
35,5
36,6
Kyrgyzstan
0,3
0,3
0,3
0,3
Russia
483,7
457,2
470,8
475,9
The EAEU, total
540,2
514,0
528,4
534,4
Net gas export
Armenia
−2,4
−2,2
−2,1
−1,9
Belarus
−20,9
−19,6
−19,4
−19,3
Kazakhstan
6,7
7,2
10,0
12,9
Kyrgyzstan
−0,2
−0,3
−0,3
−0,2
Russia
186,7
198,0
211,7
227,2
The EAEU, total
169,8
183,1
199,9
218,7
Source: According to Eurasian Economic Commission, 2019 Table 3: Key indicators of gas industry in the EAEU
countries in 2014–2017, billion cubic meters Significant amount of electricity production also fell on coal
generation (205 TWh, or 18.9%), as well as nuclear power plants
and hydroelectric power stations (206 TWh, or 16.2% each). In the EAEU countries, electricity production at nuclear power
plants had significantly increased (by 22.6 TWh, or 12.3%) due
to the commissioning of new power units in Russia. 4. RESULTS AND ANALYSIS Electricity
production at wind farms and solar power plants increased from
0.3 TWh in 2014 to 5.5 TWh in 2017. The consumption of biomass
and other types of fuel for electric power industry needs decreased
slightly, while other types of generation showed weak growth (not
more than 4%). In Armenia, its own energy production is based on the power
generation of nuclear power plants and hydroelectric power
plants, providing <1/3 of the country’s needs. Armenian nuclear
power plant is only one in the region. It was put into operation in
1980 and stopped in 1989 after the Spitak earthquake. In 1995, in
connection with the most acute energy crisis in Armenia, operation
of one of two power units of nuclear power plant with 440 MW
was resumed. Nowadays, it generates more than 70% of the whole
electricity produced in Armenia, and the period of its operation was
extended by 10 years until 2026 with the support of the Russian
company “Atomtehenergo JSC.” Table 4: Mutual gas supplies to the EAEU in 2017, billion Table 4: Mutual gas supplies to the EAEU in 2017, billion
cubic meters Supply direction
From Kazakhstan
From Russia
In Armenia
0,0
2,3
In Belarus
0,0
19,5
In Kazakhstan
–
10,0
In Kyrgyzstan
0,2
0,0
In Russia
13,3
–
Source: According to Eurasian Economic Commission, 2019 Lack of own energy resources and small volumes of electricity
production at the Armenia’s existing nuclear power plants and
hydroelectric power stations make it highly dependent on Russian
energy supplies (including uranium raw materials for nuclear
power plants). The basis of Armenia’s energy strategy is electricity generation
development at its own sources (construction of small and
medium-sized hydroelectric power stations, as well as new
nuclear power plant construction) and provision of additional
gas supplies from Iran. Potential risks for Armenia include
interruptions in energy supplies from Russia and Iran (for various
reasons), risks associated with the operation of obsolete nuclear
reactor in seismically dangerous zone, and the difficulties of
conducting market reforms in presence of exclusive energy
suppliers. 5. DISCUSSION 5. DISCUSSION There is no doubt that in energy integration development
significant role is played by the fact that each country participating
in integration association has its own unique energy potential. Energy potential of countries depends on large number of factors,
primarily it’s the availability of natural resources that can be used
to generate energy. Controversial issues are integral part in the integration processes
development. These issues must be resolved taking into account the
geopolitical and economic factors of the EAEU member countries,
which will allow them getting integration effects in future. The results of analysis indicate that the EAEU countries have
sufficient energy potential, developed infrastructure and other
opportunities for the further successful development of energy
integration processes. The integration processes have reached high
level of institutional development in the EAEU by 2020. Under
the EAEU Treaty, countries are developing long-term mutually
beneficial cooperation in energy sector, pursuing coordinated
energy policy and phasing the formation of common energy
markets. It is necessary to highlight the problems that are currently
presented in economy and energy system of all the EAEU member
countries. General problems of economy sectors should include:
high degree of depreciation of fixed assets (depreciation of
fixed assets reaches 70% and more in some sectors); significant
material and energy intensity of manufactured products; low
susceptibility to innovation; technological backwardness and
low labor productivity; lack of staffing with high qualifications;
lack of investment, etc. We can also note the obsolescence of
infrastructure and production assets in the context of growing
needs of domestic market, insufficiently high level of energy
efficiency, organizational and technical difficulties with the
creation and implementation of new equipment and technologies,
limited internal competition, insufficiently favorable investment
climate, etc. The internal energy markets of the EAEU member
countries remain narrow, low in terms of consumption and
fragmented. There is no single investment space. Rules and
principles of government regulation significantly vary in different
countries. Penetration scale of the EAEU members companies into
the global energy market does not correspond to infrastructure and
resource potential that they have. The consensus of the EAEU countries on the issue of electricity
market was reached on April 20, 2019, when the last disagreement
over the intentions to maintain tariff protection of government
monopolies was overcome. 5. DISCUSSION Free energy supplies from countries
with lower prices threaten Russian companies with regulated
tariffs (they grow by 4% per year in Russia) with loss of market
shares, and the necessity to perform transit supplies in the opposite
direction with decrease in profitability. As a result, the scheme to
enter common electricity market will be two-stage for companies. Firstly, energy companies will have to obtain the approval of national
regulators. And then, this requirement will be removed only after
signing agreement on common gas market, tariff and organizational
balancing of the market under new conditions. Development and
adoption of the EAEU documents in gas and oil sectors is planned
after the formation of regulatory framework for electricity industry. 4. RESULTS AND ANALYSIS Table 5: Key indicators of coal industry of the EAEU
countries in 2014-2017, million tons Table 5: Key indicators of coal industry of the EAEU Table 5: Key indicators of coal industry of the EAEU
countries in 2014-2017, million tons
Country/year
2014
2015
2016
2017
Coal mining
Armenia
0,0
0,0
0,0
0,0
Belarus
0,0
0,0
0,0
0,0
Kazakhstan
73,6
69,5
63,4
68,3
Kyrgyzstan
1,0
1,0
1,3
1,7
Russia
279,4
295,0
307,1
325,3
The EAEU, total
354,0
365,5
371,8
395,3
Coal consumption
Armenia
0,0
0,0
0,0
0,0
Belarus
1,2
1,1
1,0
1,1
Kazakhstan
54,6
50,5
46,9
50,0
Kyrgyzstan
1,7
1,7
2,3
3,0
Russia
153,3
171,7
165,3
169,3
The EAEU, total
210,9
224,9
215,4
223,5
Net coal export
Armenia
0,0
0,0
0,0
0,0
Belarus
−0,8
−0,6
−0,6
−0,6
Kazakhstan
18,8
19,3
16,5
18,4
Kyrgyzstan
−0,8
−0,8
−0,9
−0,7
Russia
124,2
126,0
140,1
158,1
The EAEU, total
141,5
143,8
155,1
175,2
Volumes of production, consumption and net export of coal are reduced to caloric value
of 6,000 kcal/kg. Source: According to Eurasian Economic Commission, 2019
electricity generation structure (597 TWh, or 47% of the total
generation in 2017). In mutual electricity trade between the EAEU countries, electricity
flows are of the greatest importance: Between the UES of Russia’s Center, where the main generating
facilities of PJSC Mosenergo are located, as well as Ryazan and
Cherepovets State District Power Plants of PJSC OGK-2, and
Belarus. Between the UES of Urals, where Surgutskaya State District Power
Plant –1, Troitskaya, Serovskaya State District Power Plants of
PJSC OGK-2 are located, and Kazakhstan. Volumes of production, consumption and net export of coal are reduced to caloric value
of 6,000 kcal/kg. Source: According to Eurasian Economic Commission, 2019 Between the UES of South, where Adler TPP, Stavropol and
Novocherkasskaya State District Power Plants of PJSC OGK-2
are located (the launch of Grozny State District Power Plant was
also expected in 2019), and Kazakhstan. electricity generation structure (597 TWh, or 47% of the total
generation in 2017). 18 Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic Union Countries Sopilko, et al.: Analysis and Prospects for the Development of Regional Energy Integration of the Eurasian Economic U PJSC “INTER RAO” is an electricity operator of export and import
from Russia’s UES. 4. RESULTS AND ANALYSIS At the same time, the average actual export price of Russian oil in
2019 (as of June) was $ 466.2 per 1 ton, and the average price of
Kazakh oil on the foreign market in 2019 was $ 460–470 US per
1 ton. It should be noted that the world market price for “Urals”
oil for the same period was 479.5 US dollars per 1 ton (data from
the RF Ministry of Finance). It can be emphasized that the average
price of oil imported to Belarus from Russia was 364 US dollars
per 1 ton (Belstat data), which was almost 20% lower than Russian
export oil price and 25% lower than the world one. At the same time, the average actual export price of Russian oil in
2019 (as of June) was $ 466.2 per 1 ton, and the average price of
Kazakh oil on the foreign market in 2019 was $ 460–470 US per
1 ton. It should be noted that the world market price for “Urals”
oil for the same period was 479.5 US dollars per 1 ton (data from
the RF Ministry of Finance). It can be emphasized that the average
price of oil imported to Belarus from Russia was 364 US dollars
per 1 ton (Belstat data), which was almost 20% lower than Russian
export oil price and 25% lower than the world one. 6. CONCLUSIONS Gillessen, B., Heinrichs, H., Hake, J., Allelein, H. (2019), Energy security
in context of transforming energy systems: A case study for natural
gas transport in Germany. Energy Procedia, 158, 3339-3345. Energy security ensuring of the EAEU region, based on reliable
access to energy resources, as well as guaranteed sales and transit
volumes. Hay, C., Rosamond, B. (2002), Globalization, European integration and
the discursive construction of economic imperatives. Journal of
European Public Policy, 9(2), 147-167. As practical recommendations, it is proposed to consider
the possibility of gas consumption growth as effective and
environmentally friendly source of energy for electric and heat
generation, districts gasification level increasing and pipeline
network developing. Using of oil as a fuel is offered in oil
production and oil refining fields. It should be combined with
highly efficient oil refining. In turn, the refining process having
high added value will contribute to high technologies development,
and the products will be more competitive both in the EAEU and
in the world market. It also requires joining forces in nuclear
energy field which contributes to the development of peaceful use
of nuclear energy by member countries, common energy policy
formation, coordination of decision-making in this industry,
its stability improvement, and also conducting research and
development for innovative technologies introduction. Khasbulatov, R. (2017), Russia between two subcontinents of Eurasia:
Advantages and new threats. Economy of Region, 4(4), 1005-1015. Kurbanaliyev, A., Drogovoz, P. (2016), Analysis of the Eurasian economic
union commitment applicability for the organization of the common
electricity market. Journal of Economy and Entrepreneurship, 67,
424-431. Mares, D., Martin, J. (2012), Regional energy integration in Latin
America: Lessons from Chile’s experience with natural gas. Third
World Quarterly, 33(1), 55-70. Meynkhard, А. (2020), Priorities of Russian energy policy in Russian-
Chinese relations. International Journal of Energy Economics and
Policy, 10(1), 65-71. Myasnikova, O., Lysytska, S., Shcherbakova, N., Shamsheev, S.,
Spitsyna, T., Kubasova, E. (2019), Ecological approach in managing
the technology of oil refineries. International Journal of Energy
Economics and Policy, 9(3), 165-171. Successful implementation of energy integration will lead to
powerful growth in energy sector production, activation of
investment and cash flows within the EAEU, and more available
access to energy for consumers. It should also be emphasized that
energy integration will positively affect the social sphere. First of
all, due to industry development, numerous jobs will be created
in the EAEU regions. 6. CONCLUSIONS But at the same time, there are some contradictions that arose
in the process of joint activities despite the emerging vector
of common energy market development in the EAEU region,
adopted documents and agreements reached between participating
countries, (Khasbulatov, 2017; Butorina, 2016). On the one
hand, this is geopolitical factor that includes various kinds of
controversial issues, for example, in the framework of so-called
triangle “Russia – Belarus – Armenia,” and on the other, economic
interests of the members. One of the reasons for the disputed
relations between the EAEU member countries is the pricing
policy in energy field. Despite the existing problems, there is awareness of common
geopolitical and economic interests of Russia, Belarus,
Kazakhstan, Kyrgyzstan and Armenia at governmental level of
the countries. Clear integration vector has been chosen and it’s
supported by the presence of close common cultural and economic
ties. The key areas of integration processes should include unified
transport and energy infrastructure development, unified legal
framework, and common energy markets (economies of scale,
neighborhood effects, lower costs for the production of material
resources, synergies), which, in turn, will strengthen production
ties and ensure sustainable energy development of participating
countries and provide safety of the whole region. As noted above, domestic needs for hydrocarbon energy carriers
of Armenia and Belarus are provided by supplies from Russia at
below market prices. For example, Belarus purchased Russian
gas at price $ 127 per 1000 m3 (Rosstat data), and Armenia within
$ 165 per 1000 m3 (for reference, the average gas price in Europe
in 2018 and 2019 was about 200-250 US dollars per 1000 m3). At the same time, the price of gas supplied to Kyrgyzstan from
Kazakhstan amounted to about 195 US dollars per 1000 m3. In this regard, the EAEU member countries will face the following
tasks: Priority development of energy sector, as of its strategical
importance for economies of the countries and because it is the
basis for other economy sectors growth. 19 Energy infrastructure development, as strategically important
issue in long-term development, which is the basis of Eurasian
energy integration aimed at sustainable growth achievement, both
at economic and social aspects. Eurasian Economic Commission. (2019), Eurasian Economic Union
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Secondary Metabolites Produced during the Germination of Streptomyces coelicolor
|
Frontiers in microbiology
| 2,017
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cc-by
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Edited by:
Dirk Tischler,
Freiberg University of Mining and
Technology, Germany
Reviewed by:
Dennis Claessen,
Leiden University, Netherlands
Yinhua Lu,
Shanghai Institutes for Biological
Sciences (CAS), China
*Correspondence:
Jan Bobek
jan.bobek@lf1.cuni.cz Edited by:
Dirk Tischler,
Freiberg University of Mining and
Technology, Germany Reviewed by:
Dennis Claessen,
Leiden University, Netherlands
Yinhua Lu,
Shanghai Institutes for Biological
Sciences (CAS), China *Correspondence:
Jan Bobek
jan.bobek@lf1.cuni.cz Specialty section:
This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology Specialty section:
This article was submitted to
Microbial Physiology and Metabolism,
a section of the journal
Frontiers in Microbiology Keywords: spore germination, Streptomyces, cell signaling, secondary metabolism, albaflavenone, germicidin,
chalcone Secondary Metabolites Produced
during the Germination of
Streptomyces coelicolor
Matouš ˇCihák 1, Zden ˇek Kameník 2, Klára Šmídová 1, 2, Natalie Bergman 3,
Oldˇrich Benada 2, 3, Olga Kofro ˇnová 2, Kateˇrina Petˇrí ˇcková 1 and Jan Bobek 1, 2, 3* Matouš ˇCihák 1, Zden ˇek Kameník 2, Klára Šmídová 1, 2, Natalie Bergman 3,
Oldˇrich Benada 2, 3, Olga Kofro ˇnová 2, Kateˇrina Petˇrí ˇcková 1 and Jan Bobek 1, 2, 3* 1 First Faculty of Medicine, Institute of Immunology and Microbiology, Charles University, Prague, Czechia, 2 Institute of
Microbiology, The Czech Academy of Sciences, Prague, Czechia, 3 Chemistry Department, Faculty of Science, J. E. Purkinje
University, Ústí nad Labem, Czechia Spore awakening is a series of actions that starts with purely physical processes and
continues via the launching of gene expression and metabolic activities, eventually
achieving a vegetative phase of growth. In spore-forming microorganisms, the
germination process is controlled by intra- and inter-species communication. However,
in the Streptomyces clade, which is capable of developing a plethora of valuable
compounds, the chemical signals produced during germination have not been
systematically studied before. Our previously published data revealed that several
secondary metabolite biosynthetic genes are expressed during germination. Therefore,
we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the
sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are
produced during germination of the model streptomycete, S. coelicolor. Interestingly,
the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may
coordinate the development of the producer (quorum sensing) and/or play a role in
competitive microflora repression (quorum quenching) in their nature environments. Keywords: spore germination, Streptomyces, cell signaling, secondary metabolism, albaflavenone, germicidin,
chalcone ORIGINAL RESEARCH
published: 13 December 2017
doi: 10.3389/fmicb.2017.02495 INTRODUCTION Received: 29 August 2017
Accepted: 30 November 2017
Published: 13 December 2017 A large variety of compounds is produced by various microorganisms by means of specialized
biosynthetic pathways. Although the special (or secondary) metabolites (Hopwood, 2007; Baltz,
2008; van Keulen and Dyson, 2014) are not essential for growth and reproduction, they often
provide the producing organism with a bioactive role (Keller et al., 2005). Reaching further than
a cell itself physically can, the small diffusible molecules may give an advantage to its producer
by effectively adapting to extracellular conditions to some degree. They may provide defense
(or attack), competition, signaling, or interspecies interactions, depending on the environmental
cues, thus increasing the likelihood of survival in an inhospitable environment (Brachmann et al.,
2013; Martinez et al., 2017). The bioactivity of the small molecules is mostly achieved by affecting
transcription in receiving cells (Camilli and Bassler, 2006). Citation: The chemical structure has so far been
elucidated in less than 30 percent of the compounds, belonging
to the following groups of natural substances: polyketides,
pyrones, peptides, siderophores, γ-butyrolactones, butenolides,
furans, terpenoids, fatty acids, oligopyrroles, and deoxysugars
(van Keulen and Dyson, 2014). The remaining 70 percent are
called “cryptic compounds” as they are not produced at standard
laboratory conditions (Bentley et al., 2002; Ikeda et al., 2003;
Ohnishi et al., 2008; Tanaka et al., 2013; van Keulen and
Dyson, 2014). To activate these cryptic pathways, streptomycetes
are cultivated under non-standard physical and nutritional
conditions or co-cultured with other microorganisms (Wakefield
et al., 2017). Genetic manipulations within the genes (Luo et al.,
2013) or the transfer of the whole biosynthetic gene cluster into a
heterologous producer (Kalan et al., 2013; Tanaka et al., 2013) are
also commonly used strategies. The successful activation of the
biosynthetic pathways often leads to biosynthesis of previously
unknown compounds (Ikeda et al., 2003; Ohnishi et al., 2008;
Gomez-Escribano et al., 2012; Tanaka et al., 2013). For example,
a polyketide alkaloid, coelimycin P1 (so-called yellow pigment),
is produced from the cpk cryptic gene cluster in S. coelicolor
(Gomez-Escribano et al., 2012). Further development requires the re-activation of the
transcriptional apparatus (Paleckova et al., 2006; Mikulik et al.,
2008, 2011) controlled by the activity of a set of sigma factors,
whose expression takes place from the very beginning of the
process (Bobek et al., 2014; Strakova et al., 2014). Genome-
wide expression data revealed that the activity of most metabolic
pathways is stabilized after the first DNA replication that occurs
between 120 and 150 min of germination of S. coelicolor (Bobek
et al., 2014). After this period, morphologically observable
changes, like the first germ tube emerging from the spore, occur
(Kelemen and Buttner, 1998; Claessen et al., 2006; Ohnishi et al.,
2008). In the case of non-activated spores, it was found that
about 10–20% of spores do not germinate even under optimal
incubation conditions (Yoshida and Kobayashi, 1994). Sole
spores of S. viridochromogenes have been shown to germinate
more slowly than in the dense population (Xu and Vetsigian,
2017). This indicates an existence of germination activator
produced into the medium. On the other hand, the extract
from the S. viridochromogenes supernatant has been shown
to inhibit the germination of unactivated spores when added
prior to incubation (Hirsch and Ensign, 1976a). Citation: ˇCihák M, Kameník Z, Šmídová K,
Bergman N, Benada O, Kofro ˇnová O,
Petˇrí ˇcková K and Bobek J (2017)
Secondary Metabolites Produced
during the Germination of
Streptomyces coelicolor. Front. Microbiol. 8:2495. doi: 10.3389/fmicb.2017.02495 December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. stage (Sello and Buttner, 2008; Seipke et al., 2012). The aerial
hyphae are dissected into chains of uninucleoid spores. Spores
are subjected to maturation which ensures their survival in
unfavorable conditions and allows them to spread into new
niches. Secondary metabolism is of special interest in Streptomyces,
a clade of multicellular bacteria that occupies a high position
in the developmental hierarchy of bacteria due to their
advanced morphology and physiology. Streptomycetes have
evolved a plethora of biosynthetic pathways to produce various
secondary metabolites, especially signal molecules (see below),
or antibiotics (van Keulen and Dyson, 2014). These compounds
provide the organism with a competitive advantage, protection
from unfavorable living conditions and/or facilitate interspecies
interactions (Maxwell et al., 1989). The dormant state of spores is characterized by limited
metabolic activity or its complete stagnation (McCormick and
Flardh, 2012). Subsequent germination is the spore’s transition
into a metabolically active vegetative phase. Reactivation of the
dormant exospore takes place in an aqueous environment. In
addition to energy sources (e.g., trehalose) and various nutrients
(Ranade and Vining, 1993), the dormant spores of streptomycetes
also contain transcriptome which is a remnant of sporulation
and spore maturation (Mikulik et al., 2002). The residual pool
of mRNA appears to be necessary for the initial germination
phase, serving as a template for the early synthesis of proteins,
such as chaperones and hydrolases. Whereas chaperones are
indispensable in the re-activation of present proteins upon their
release from the trehalose milieu (Bobek et al., 2017), hydrolases
reconstitute the thick hydrophobic spore cell wall (Bobek et al.,
2004; Haiser et al., 2009). The genes for the biosynthesis of streptomycete secondary
metabolites are mostly clustered and their expression is highly
regulated (Bentley et al., 2002; Tanaka et al., 2013). The model
S. coelicolor possesses the best annotated genome that encodes
biosynthetic pathways for more than 20 secondary metabolites
(Bentley et al., 2002). Frontiers in Microbiology | www.frontiersin.org LC-MS Analyses environmental conditions (Petersen et al., 1993; Aoki et al.,
2007, 2011; Ma et al., 2017). Since it is considered to be
non-productive (Seipke et al., 2012), the initial developmental
phase has hitherto not been given sufficient attention. It is
nevertheless apparent from the genome-wide expression analysis
of S. coelicolor’s germinating spores (germlings) performed by
Strakova (Strakova et al., 2013), that 163 genes involved in the
biosynthesis of secondary metabolites are transcribed during
germination (including those cryptic). It is for this reason that we
chose to focus on the biosynthetic activities of the germinating
spores of S. coelicolor in this article. Secondary metabolites
produced in this phase would possibly function as germinative
signals in the frame of intercellular communication (Rutherford
and Bassler, 2012; Brachmann et al., 2013) or may suppress
competing microflora. y
LC-MS analyses were performed on the Acquity UPLC system
with 2996 PDA detection system (194 - 600 nm) connected
to LCT premier XE time-of-flight mass spectrometer (Waters,
USA). Five µL of sample was loaded onto the Acquity UPLC
BEH C18 LC column (50 mm × 2.1 mm I.D., particle size 1.7 µm,
Waters) kept at 40◦C and eluted with a two-component mobile
phase, A and B, consisting of 0.1% formic acid and acetonitrile,
respectively, at the flow rate of 0.4 mL min−1. The analyses were
performed under a linear gradient program (min/%B) 0/5; 1.5/5;
15/70; 18/99 followed by a 1.0-min column clean-up (99% B) and
1.5-min equilibration (5% B). The mass spectrometer operated in
the positive “W” mode with capillary voltage set at +2,800 V, cone
voltage +40 V, desolvation gas temperature, 350◦C; ion source
block temperature, 120◦C; cone gas flow, 50 L h−1; desolvation
gas flow, 800 L h−1; scan time of 0.15 s; inter-scan delay of 0.01
s. The mass accuracy was kept below 6 ppm using lock spray
technology with leucine enkephalin as the reference compound
(2 ng µL−1, 5 µL min−1). MS chromatograms were extracted for
[M+H]+ ions with the tolerance window of 0.03 Da, smoothed
with mean smoothing method (window size; 4 scans, number
of smooths, 2). The data were processed by MassLynx V4.1
(Waters). Citation: The inhibitor
present was later isolated (along with other congeners) and
described as germicidin A (Petersen et al., 1993; Aoki et al.,
2011; Ma et al., 2017). The launch of germination within a spore
population is stochastic, as was shown not only in streptomycetes
(Xu and Vetsigian, 2017) but also in other spore-forming
bacteria (van Vliet, 2015). The probability of germination within
a population differs between different streptomycete strains;
S. viridochromogenes and S. granaticolor exhibit fast and robust
germination whereas S. coelicolor and S. venezuelae show more
complex behavior with a fraction of germlings that stop growing
soon after germination (Mikulik et al., 1977; Bobek et al., 2004;
Xu and Vetsigian, 2017). Activity of early released compounds,
germination activators and inhibitors, may affect the stochasticity
of germination in order to adapt the germination strategy to The complicated development of streptomycetes requires
highly sophisticated control mechanisms mediated by multiple
molecules linked to signaling cascades (Kelemen and Buttner,
1998; Claessen et al., 2006; Gao et al., 2012). A widely studied
signaling system is quorum sensing, in which stimuli are spread
within a population and induce appropriate responses (Phelan
et al., 2011). Based on the signal assessment, the organism can
adapt to its environment and coordinate further development
in response to local population densities (Waters and Bassler,
2005).One of the assumptions made in this work is that cellular
signaling is also employed in spore germination (see below). However, the nature of the signaling in this developmental
phase remains unclear, as do the chemical characteristics and the
possible regulatory effect of the produced substances. Streptomyces undergo a cellular differentiation that resembles
the fungal life cycle (Seipke et al., 2012). Their growth starts
with germinating spores that develop into a vegetative mycelium
of branching hyphae. Subsequent development of aerial hyphae
is considered to be a cell response to nutrient depletion; most
of the secondary metabolites are formed at this developmental December 2017 | Volume 8 | Article 2495 2 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. Germination and Microbial Growth Spores were washed twice in 10 mL sterile distilled water and
resuspended in 50 mL NMMP (Kieser et al., 2000), R3 (Shima
et al., 1996), or AM (Bobek et al., 2004) liquid medium to a final
spore concentration 108 ml−1. Glucose, glycerol, or mannitol
was used as a carbon source. For boosting synchronicity of the
population, spores were incubated for 10 min at 50◦C, followed
by 6-h germination at 37◦C (Hirsch and Ensign, 1976a; Kieser
et al., 2000) before screening for produced secondary metabolites. To prepare samples from the stationary phase of growth,
S. coelicolor was further cultivated 48 h at 29◦C in the same
medium. The grown mycelium or supernatant was then used in
the screening for produced secondary metabolites. Spores were washed twice in 10 mL sterile distilled water and
resuspended in 50 mL NMMP (Kieser et al., 2000), R3 (Shima
et al., 1996), or AM (Bobek et al., 2004) liquid medium to a final
spore concentration 108 ml−1. Glucose, glycerol, or mannitol
was used as a carbon source. For boosting synchronicity of the
population, spores were incubated for 10 min at 50◦C, followed
by 6-h germination at 37◦C (Hirsch and Ensign, 1976a; Kieser
et al., 2000) before screening for produced secondary metabolites. Germicidin A standard was purchased from Cayman Pharma
(the Czech Republic), chalcone’s standard was obtained from
Sigma-Aldrich (Merck, Germany). Dimethylsulfoxid (DMSO)
was purchased from Lach-Ner (Czech Republic). On a six-
sector culture microtiter plate, ONA medium (1.4% Oxoid
nutrient agar, pH 7.2; Kieser et al., 2000) with a linear
concentration gradient of germicidin A 0–8 µg mL−1 (standard
dissolved in sterile distilled water) or chalcone 0–8 µg mL−1
(standard dissolved in DMSO), or the albaflavenone-hexane
extract (concentration unknown) was poured into three sectors
as follows. Pure ONA medium was poured into an inclined plate
and allowed to solidify to form a wedge; the plate was then placed
horizontally and ONA medium containing selected compound in
concentration 8 µg mL−1 was poured to form a complementary
wedge. The remaining three sectors were filled with either pure
ONA medium for control cultivation respective to germicidin A,
or ONA medium with DMSO for control cultivation respective
to chalcone, or ONA medium with hexane for control cultivation
respective to albaflavenone. 105 spores were spread on each sector
and incubated 48 h at 29◦C. Number of colony-forming units
(CFU) was assessed and compared. Bioactivity Assays y
y
Although albaflavenone is not commercially available, we have
verified its presence using LC-MS in the hexane extract from the
supernatant after 48 h of S. coelicolor’s growth in R3 medium
with glycerol as the carbon source. Cells were centrifuged for
5 min (7,000 × g). The supernatant was extracted three times
with 300 mL of n-hexane (Lach-Ner, Prague, Czech Republic). The cells were washed in distilled water and mycelial products
were extracted with 200 mL of n-hexane. Cells were removed
by filtration. After separation of the phases, the n-hexane
layers were pooled (below called as albaflavenone-hexane extract,
Figure 1B). Preparation of Streptomyces Spores Streptomyces coelicolor M145 was cultivated on cellophane discs
on solid agar plates (0.4% yeast extract, 1% malt extract, 0.4%
glucose, 2.5% bacterial agar, pH 7.2) at 28◦C for 14 days. Harvested dormant spores were filtered through cotton wool
and used to screen for associated secondary metabolites. Spores
mixed with 20% glycerol were stored frozen at −20◦C. MATERIALS AND METHODS Preparation of Streptomyces Spores Frontiers in Microbiology | www.frontiersin.org Scanning Electron Microscopy Scanning Electron Microscopy
Streptomyces spores were fixed with 3% glutaraldehyde overnight
at 4◦C. The fixed spores were extensively washed and then
allowed to sediment at 4◦C overnight onto circular coverslips
treated with poly-L-lysine. The coverslips with attached spores
were dehydrated through an alcohol series followed by absolute
acetone and critical point dried from liquid CO2 in a K850
Critical Point Dryer (Quorum Technologies Ltd, Ringmer, UK). The dried samples were sputter-coated with 3 nm of platinum in
a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies
Ltd, Ringmer, UK). The final samples were examined in a
FEI Nova NanoSEM scanning electron microscope (FEI, Brno,
Czech Republic) at 5 kV using CBS and TLD detectors. Beam
deceleration mode of scanning electron microscope was used in
some cases for minimization of charging artifacts. Solid Phase Extraction of Secondary
Metabolites Secondary metabolites were taken from the culture supernatants
extracted using ethyl acetate (Rajan and Kannabiran, 2014),
QuEChERS (Schenck and Hobbs, 2004), or solid phase extraction
(Kamenik et al., 2010), which was found to be the most suitable
and was carried out as follows. An Oasis HLB 3cc 60 mg
cartridge (hydrophilic-lipophilic balanced sorbent, Waters, USA)
was conditioned with 3 mL methanol (LC-MS grade, Biosolve,
Netherlands), equilibrated with 3 mL water (prepared using
Milli-Q water purifier, Millipore, USA) and then 3 mL culture
supernatant (pH adjusted to 3 with formic acid, 98–100%, Merck,
Germany) was loaded. Subsequently, the cartridge was washed
with 3 mL water and absorbed substances were eluted with 1.5 mL
methanol. The eluent was evaporated to dryness (Concentrator
Plus, 2013 model, Eppendorf), reconstituted in 200 µL 50%
methanol and centrifuged at 12,000 × g for 5 min. December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 3 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. FIGURE 1 | Albaflavenone. (A) LC-MS analysis of culture broth extract of S. coelicolor’s spores after 6 h germination in AM medium with glycerol. MS chromatogram
recorded for m/z 219.175. (B) Mass spectrum of compound with tR = 9.01 min. Acquired m/z 219.1741 corresponds to [M+H]+ of albaflavenone with theoretical
m/z 219.1749. (C) LC-MS analysis of hexane extract from supernatant of S. coelicolor after 48 h of culture in R3 medium with glycerol (further referred to as
albaflavenone-hexane extract). MS chromatogram recorded for m/z 219.175. (D) Mass spectrum of compound with tR = 9.00 min. Acquired m/z 219.1736
corresponds to [M+H]+ of albaflavenone ion with the theoretical m/z 219.1749. FIGURE 1 | Albaflavenone. (A) LC-MS analysis of culture broth extract of S. coelicolor’s spores after 6 h germination in AM medium with glycerol. MS chromatogram
recorded for m/z 219.175. (B) Mass spectrum of compound with tR = 9.01 min. Acquired m/z 219.1741 corresponds to [M+H]+ of albaflavenone with theoretical
m/z 219.1749. (C) LC-MS analysis of hexane extract from supernatant of S. coelicolor after 48 h of culture in R3 medium with glycerol (further referred to as
albaflavenone-hexane extract). MS chromatogram recorded for m/z 219.175. (D) Mass spectrum of compound with tR = 9.00 min. Acquired m/z 219.1736
corresponds to [M+H]+ of albaflavenone ion with the theoretical m/z 219.1749. A Dehydrogenase Activity Test A Dehydrogenase Activity Test RESULTS Two milliliter of the albaflavenone-hexane extract was mixed
with 5 mL R3 medium with glycerol and used for 6 h germination
of 5 × 106 spores. A negative control culture was performed
in 5 mL R3 medium with glycerol with 2 mL pure hexane. The dehydrogenase activity test (as described in Burdock et al.,
2011) was used to measure metabolic activity of germinating
spores by means of triphenyl tetrazolium chloride (TTC). After
the germination course, cells were incubated in the presence of
TTC and an electron-donating substrate for 1 h. Rising triphenyl
formazan (TF) was extracted using ethanol and its concentration
was determined colorimetrically by measuring the optical density
at wavelength of 484 nm. The absorbances were compared
between the tested and negative control samples. Frontiers in Microbiology | www.frontiersin.org In Silico Analysis of the Expressed
Secondary Metabolite Biosynthetic Genes
during Germination of S. coelicolor Genes that are expressed in the consecutive time points
of germination have been reported by Strakova (Strakova
et al., 2013). From their dataset we selected genes whose
products
are
involved
in
the
biosynthesis
of
secondary
metabolites by S. coelicolor, according to the StrepDB database
(http://strepdb.streptomyces.org.uk), where the annotated S. coelicolor genes are categorized into metabolic groups. We
updated the list of secondary metabolites with regard to newly
published findings (Zhao et al., 2009; van Keulen and Dyson,
2014). The resulting list of genes is summarized in Table 1. Predicted secondary metabolites, whose respective genes are
expressed during germination, include polyketides, pyrones,
peptides, siderophores, terpenoids, oligopyrroles, and fatty acids. Secondary Metabolites Produced by
S. coelicolor during Germination and/or in
the Stationary Phase The compounds detected
Actinorhodin Production
Actinorhodin
compounds
(actinorhodinic
acid
and
γ-
actinorhodin) were detected in both dormant and germinating
spores, as well as in samples from the stationary phase of TABLE 1 | An overview of biosynthetic genes expressed during germination, according to Strakova et al. (2013). Secondary metabolites
Biosynthetic (SCO) genes expressed during germination
Actinorhodin and related congeners
0331; 3978; 4280; 5072-5079; 5081-5086; 5088-5090
Albaflavenone
5223
Calcium-dependent antibiotic (CDA)
3210; 3212; 3214; 3220-3221; 3223; 3225; 3227; 3228; 3230-3232; 3234-3237; 3239; 3241-3242; 3246; 3249
Coelibactin
7682-7684; 7686-7687; 7689-7691
Coelichelin
0490-0498
Coelimycin P1
6273-6275; 6277-6287
Desferrioxamines B, E, G1 a D1
2783-2784
Eicosapentaenoic acid (EPA)
0124-0125
Flaviolin, THN
1206
Geosmin
6073
Hopanoids, ATBH
6759-6763; 6765; 6767; 6769; 6771
Isorenieratene, β-carotene
0187-0191
Streptorubin B, undecylprodigiosin
0126-0127; 5877-5878; 5881; 5891-5894; 5898
Triketid pyrones
7670-7671
Tw95a
5314; 5318; 5320 TABLE 2 | Secondary metabolites detected in the stationary phase of growth. Secondary metabolite
Medium (carbon source)
Molecular
formula
Theoretical
mass (m/z)
Acquired
mass (m/z)
Mass error
(ppm)
γ- actinorhodin
R3, AM, NMMP (glycerol, mannitol, glucose)
C32H22O14
631.1090
631.1081
1.40
Actinorhodinic acid
R3, AM, NMMP (glycerol, mannitol, glucose)
C32H26O16
667.1300
667.1289
1.60
Albaflavenone
R3 (glycerol)
C15H22O
219.1736
219.1749
5.90
CDA
R3 (glycerol)
C67H78N14O26
1495.5290
1495.5292
0.10
Coelimycin P1
R3 (glycerol)
C17H20N2O4S
349.1222
349.1229
2.00
Chalcone
R3 (glycerol/mannitol)
C15H12O
209.0960
209.0966
2.90
Desferrioxamine B
R3 (glycerol/mannitol) AM (glycerol/mannitol)
C25H48N6O8
561.3625
561.3612
2.30
Desferrioxamine D1
AM (glucose/mannitol)
C27H50N6O9
603.3711
603.3718
1.20
Desferrioxamine E
R3 (glycerol/mannitol) AM (glucose/glycerol) NMMP
(glucose/mannitol)
C27H48N6O9
601.3561
601.3561
0.00
Desferrioxamine G1
AM (glucose/glycerol)
C27H50N6O10
619.3677
619.3667
1.60
Germicidin A
R3 (glycerol/mannitol) AM (glucose/mannitol)
C11H16O3
197.1170
197.1178
4.10
Germicidin B
R3 (glycerol/mannitol) NMMP (glucose/mannitol)
C10H14O3
183.1016
183.1021
2.73
Kalafungin
R3 (mannitol)
C16H12O6
301.0709
301.0714
1.00
Streptorubin B
R3 (glycerol/mannitol) AM (glucose/glycerol)
C25H33N3O
392.2696
392.2702
1.50
Undecylprodigiosin
R3 (glycerol/mannitol) AM (glucose/glycerol)
C25H35N3O
394.2866
394.2858
2.00 TABLE 2 | Secondary metabolites detected in the stationary phase of growth. Frontiers in Microbiology | www.frontiersin.org Secondary Metabolites Produced by
S. coelicolor during Germination and/or in
the Stationary Phase The fact that genes whose products are involved in the
secondary metabolite biosynthesis were transcribed during
germination encouraged us to investigate whether germinating
spores produce respective compounds up to the 6th hour of
their development. For this purpose, we performed an LC-
MS analysis of the culture supernatants. In order to ensure
that any detected compound is synthesized de novo during Frontiers in Microbiology | www.frontiersin.org December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 4 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. TABLE 1 | An overview of biosynthetic genes expressed during germination, according to Strakova et al. (2013). Secondary metabolites
Biosynthetic (SCO) genes expressed during germination
Actinorhodin and related congeners
0331; 3978; 4280; 5072-5079; 5081-5086; 5088-5090
Albaflavenone
5223
Calcium-dependent antibiotic (CDA)
3210; 3212; 3214; 3220-3221; 3223; 3225; 3227; 3228; 3230-3232; 3234-3237; 3239; 3241-3242; 3246; 3249
Coelibactin
7682-7684; 7686-7687; 7689-7691
Coelichelin
0490-0498
Coelimycin P1
6273-6275; 6277-6287
Desferrioxamines B, E, G1 a D1
2783-2784
Eicosapentaenoic acid (EPA)
0124-0125
Flaviolin, THN
1206
Geosmin
6073
Hopanoids, ATBH
6759-6763; 6765; 6767; 6769; 6771
Isorenieratene, β-carotene
0187-0191
Streptorubin B, undecylprodigiosin
0126-0127; 5877-5878; 5881; 5891-5894; 5898
Triketid pyrones
7670-7671
Tw95a
5314; 5318; 5320
TABLE 2 | Secondary metabolites detected in the stationary phase of growth. Secondary metabolite
Medium (carbon source)
Molecular
formula
Theoretical
mass (m/z)
Acquired
mass (m/z)
Mass error
(ppm)
γ- actinorhodin
R3, AM, NMMP (glycerol, mannitol, glucose)
C32H22O14
631.1090
631.1081
1.40
Actinorhodinic acid
R3, AM, NMMP (glycerol, mannitol, glucose)
C32H26O16
667.1300
667.1289
1.60
Albaflavenone
R3 (glycerol)
C15H22O
219.1736
219.1749
5.90
CDA
R3 (glycerol)
C67H78N14O26
1495.5290
1495.5292
0.10
Coelimycin P1
R3 (glycerol)
C17H20N2O4S
349.1222
349.1229
2.00
Chalcone
R3 (glycerol/mannitol)
C15H12O
209.0960
209.0966
2.90
Desferrioxamine B
R3 (glycerol/mannitol) AM (glycerol/mannitol)
C25H48N6O8
561.3625
561.3612
2.30
Desferrioxamine D1
AM (glucose/mannitol)
C27H50N6O9
603.3711
603.3718
1.20
Desferrioxamine E
R3 (glycerol/mannitol) AM (glucose/glycerol) NMMP
(glucose/mannitol)
C27H48N6O9
601.3561
601.3561
0.00
Desferrioxamine G1
AM (glucose/glycerol)
C27H50N6O10
619.3677
619.3667
1.60
Germicidin A
R3 (glycerol/mannitol) AM (glucose/mannitol)
C11H16O3
197.1170
197.1178
4.10
Germicidin B
R3 (glycerol/mannitol) NMMP (glucose/mannitol)
C10H14O3
183.1016
183.1021
2.73
Kalafungin
R3 (mannitol)
C16H12O6
301.0709
301.0714
1.00
Streptorubin B
R3 (glycerol/mannitol) AM (glucose/glycerol)
C25H33N3O
392.2696
392.2702
1.50
Undecylprodigiosin
R3 (glycerol/mannitol) AM (glucose/glycerol)
C25H35N3O
394.2866
394.2858
2.00
germination, we included dormant spores (non-activated) as
negative control samples in the analysis. As a positive control
that tests effectiveness of our detection method, samples from the
sporulation phase were included too. Actinorhodin Production coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
ded for m/z 209.097. (B) Mass spectrum of compound with tR = 3.41 min. Acquired m/z 209.0965 corresponds to [M+H]+ of chalcone with the theoretical m/z
9.0966. TABLE 3 | Secondary metabolites produced during germination. Secondary metabolite
Medium (carbon source)
Molecular formula
Theoretical mass
(m/z)
Acquired mass
(m/z)
Mass error
(ppm)
Albaflavenone
AM (glycerol)
C15H22O
219.1741
219.1749
3.70
Germicidin A
R3 (glycerol/mannitol) AM (glucose/mannitol)
C11H16O3
197.1188
197.1178
5.10
Chalcone
R3 (glycerol/mannitol)
C15H12O
209.0965
209.0966
0.50
FIGURE 2 | Germicidin A. (A) LC-MS analysis culture broth extract of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
recorded for m/z 197.118. (B) Mass spectrum of compound with tR = 6.48 min. Acquired m/z 197.1188 corresponds to [M + H]+ of germicidin A with the theoretical
m/z 197.1178. (C) LC-MS analysis of germicidin A authentic standard. MS chromatogram recorded for m/z 197.118. (D) Mass spectrum of germicidin A. Acquired
m/z = 197.1170 corresponds to [M+H]+ of germicidin A with the theoretical m/z = 197.1170. TABLE 3 | Secondary metabolites produced during germination. FIGURE 2 | Germicidin A. (A) LC-MS analysis culture broth extract of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
recorded for m/z 197.118. (B) Mass spectrum of compound with tR = 6.48 min. Acquired m/z 197.1188 corresponds to [M + H]+ of germicidin A with the theoretical
m/z 197.1178. (C) LC-MS analysis of germicidin A authentic standard. MS chromatogram recorded for m/z 197.118. (D) Mass spectrum of germicidin A. Acquired
m/z = 197.1170 corresponds to [M+H]+ of germicidin A with the theoretical m/z = 197.1170. FIGURE 3 | Chalcone. (A) LC-MS analysis culture broth extract of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
recorded for m/z 209.097. (B) Mass spectrum of compound with tR = 3.41 min. Acquired m/z 209.0965 corresponds to [M+H]+ of chalcone with the theoretical m/z
= 209.0966. FIGURE 3 | Chalcone. (A) LC-MS analysis culture broth extract of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
recorded for m/z 209.097. (B) Mass spectrum of compound with tR = 3.41 min. Acquired m/z 209.0965 corresponds to [M+H]+ of chalcone with the theoretical m/z
= 209.0966. Actinorhodin Production germination, we included dormant spores (non-activated) as
negative control samples in the analysis. As a positive control
that tests effectiveness of our detection method, samples from the
sporulation phase were included too. The compounds detected
only in the sporulation phase (after 48 h of cultivation) but absent
from the germination are listed in Table 2. Actinorhodin
compounds
(actinorhodinic
acid
and
γ-
actinorhodin) were detected in both dormant and germinating
spores, as well as in samples from the stationary phase of
growth (Table 2). Streptomycetes produced these compounds
after 48 h of cultivation in R3, AM, and NMMP medium,
regardless of carbon source (glucose, glycerol, or mannitol). Although the suspensions of dormant spores were washed in
distilled water, we found that dormant spores also contained
these blue pigments. We also found actinorhodinic acid
and γ-actinorhodin in the cell-free supernatant after spore
germination in AM medium. All supernatants containing
the blue pigments also exhibited a pH-dependent color
change. Our LC-MS measurements revealed, however, that the
S. coelicolor’s germlings produce three different compounds
with masses corresponding to the sesquiterpenoid antibiotic
albaflavenone, the polyketide germicidin A, and chalcone
(Table 3; Figures 1–3, respectively). Furthermore, the identity of
germicidin A was confirmed by comparing the actual retention
time with the original standard obtained from Cayman Pharma
(Figure 2). December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 5 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. et al. Secondary Metabolism of Germinating Streptomycete Spores
E 3 | Secondary metabolites produced during germination. dary metabolite
Medium (carbon source)
Molecular formula
Theoretical mass
(m/z)
Acquired mass
(m/z)
Mass error
(ppm)
venone
AM (glycerol)
C15H22O
219.1741
219.1749
3.70
cidin A
R3 (glycerol/mannitol) AM (glucose/mannitol)
C11H16O3
197.1188
197.1178
5.10
ne
R3 (glycerol/mannitol)
C15H12O
209.0965
209.0966
0.50
URE 2 | Germicidin A. (A) LC-MS analysis culture broth extract of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol. MS chromatogram
ded for m/z 197.118. (B) Mass spectrum of compound with tR = 6.48 min. Acquired m/z 197.1188 corresponds to [M + H]+ of germicidin A with the theoretical
97.1178. (C) LC-MS analysis of germicidin A authentic standard. MS chromatogram recorded for m/z 197.118. (D) Mass spectrum of germicidin A. Acquired
= 197.1170 corresponds to [M+H]+ of germicidin A with the theoretical m/z = 197.1170. URE 3 | Chalcone. (A) LC-MS analysis culture broth extract of S. Biological Effects of Albaflavenone,
Germicidin A, and Chalcone
Albaflavenone Since albaflavenone was not commercially available, its possible
effect on germination was tested using the albaflavenone-
hexane extract (see Methods). A negative control, which
contained pure hexane, was included in the experiment. No quantitative or phenotypic changes were observed in
the
tested
conditions
(Figure 5C). To
verify
this
result,
a dehydrogenase activity test was performed (Figure 5D). Metabolic activity of living cells that are present in the medium
during germination was determined as a function of their
dehydrogenase activity, proportional to the concentration of
rising TF measured by the optical density at 484 nm (see
Methods for details). The optical density increased in time
during germination at the same rate in both tested and control
samples; the albaflavenone-hexane extract created no observable
effect. Chalcone Our initial experiments showed that chalcone suppressed
germination of S. coelicolor in concentrations down to 8 µg mL−1
on the solid medium (Figure 5B). The average number of CFU
was 20 on a medium with a chalcone gradient (0–8 µg mL−1),
contrary to 70 colonies on the chalcone-free medium. The
size of the colonies was inversely proportional to the chalcone
concentrations; the colonies were significantly smaller compared
to the negative control, suggesting a slower germination rate
and/or vegetative growth. Moreover, in the presence of chalcone,
actinorhodin was not produced throughout the whole cell cycle. The effect of chalcone was additionally examined in R3 liquid
medium. Whereas the concentration 8 µg mL−1 revealed to
be subinhibitory, the chalcone concentration of 80 µg mL−1
completely suppressed the development as could be seen in
electronmicroscopic images taken from the germination course
in the 4th and 6th hour of cultivation Figure 6. As could be seen
in the image, the developing germ tubes disrupt in the presence
of chalcone, leaving empty cell envelopes. The biological effect of the other two secondary metabolites
on germination was examined using standards of gemicidin A
(Cayman Pharma) and chalcone (Sigma-Aldrich). Experiments
were performed on six-section cultivation titration plates (Gama
Group). Three fields in one column contained ONA medium
with a gradient of tested compound (0–8 µg mL−1) and pure
medium without the tested substance was poured into the fields
in the other column as negative controls. Results were evaluated
in terms of the number of colony forming units (CFU) and
phenotypic changes. pH-Dependent Biosynthetic Activity of
Germlings albaflavenone. To see whether β-farnesene is produced in more
acidic conditions, we performed a germination experiment in
R3 medium with glycerol at pH 6.0. Secondary metabolites were
extracted by the solid phase extraction, followed by the LC-MS
analysis. To compare, we used an extract after 6 h of cultivation
at pH 7.2 without changing the other conditions. Although the
direct production of β-farnesene was not found, it is clear from
the total ionic and base peak chromatograms that under different Biosynthesis of albaflavenone involves the activity of cytochrome
P450 (CYP170A1) which, depending on pH, can either act as
a monooxygenase (pH 7.0-8.2) or as a farnesene synthase (5.5–
6.5) (Zhao et al., 2009). Whereas β-farnesene was not synthesized
in cultures in R3, NMMP, and AM medium at pH 7.0–7.2, we
showed that the germlings produce a substance corresponding to December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 6 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. Germicidin A
Germicidin A clearly inhibited the germination of S. coelicolor
from the concentration of 4 µg mL−1 (Figure 5A). The average
number of colony-forming units (germinating spores) was 20 on
an ONA medium with the linear gradient of germicidin A at 4 µg
mL−1 and lower; 60 colonies were grown without germicidin A as
a negative control. The tested colonies were of the same shape and
size as their controls. Actinorhodin production was quantitatively
the same. pH conditions S. coelicolor produces a different spectrum of
substances (Figure 4). Further analysis was beyond the scope of
this manuscript. DISCUSSION Although many secondary metabolites of streptomycetes have
been discovered, they were most often isolated from the
stationary phase of growth, i.e., in the context of the formation
of aerial mycelium (Janecek et al., 1997; Kieser et al., 2000;
van Keulen and Dyson, 2014). However, our in silico search
within the gene expression data (Strakova et al., 2013; Bobek
et al., 2014) revealed a number of genes (including those cryptic)
responsible for the biosynthesis of secondary metabolites to
be expressed during the course of S. coelicolor’s germination
(see Table 1); especially genes responsible for the synthesis of
desferrioxamines (sco2783-2784), gray spore pigment (sco5314,
sco5318, sco5320), or yellow coelimycin P1 (sco6273-6276,
sco6277-6287, so called cpk cryptic gene cluster) (Lakey et al.,
1983; Gomez-Escribano et al., 2012). We therefore focused our
work on whether germinating spores are capable of activating the
respective biosynthetic pathways and producing any compound
within the 6 h of germination. FIGURE 4 | pH-dependent biosynthetic activity of germlings. Comparison of
base peak MS chromatograms acquired for culture broth extracts of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol differing at
pH; (A) pH 6.0; (B) pH 7.2. Metabolites that could be bound to the spore surface or
present in germination medium were identified by means of
the LC-MS analysis. Simultaneously, the secondary metabolites
produced during the sporulation phase and those associated
with the dormant spores were also included in the analysis in
order to state whether the compounds found in the samples
from germlings were synthesized de novo. The cultivations were
carried out in three different liquid media (R3, NMMP, and AM, FIGURE 4 | pH-dependent biosynthetic activity of germlings. Comparison of
base peak MS chromatograms acquired for culture broth extracts of S. coelicolor’s spores after 6 h germination in R3 medium with glycerol differing at
pH; (A) pH 6.0; (B) pH 7.2. December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 7 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. FIGURE 5 | Biological activities of germicidin A, chalcone, and albaflavenone-hexane extract. Bioassays (A–C) were performed on the six-sector cultivation titration
plates in two sets with negative control triplets (see Methods for more details), representative pictures are shown. (A) (Neg.) negative control culture on ONA medium
without germicidin A. DISCUSSION (Grad.) culture on ONA medium with a linear concentration gradient of germicidin A (0–8 µg mL−1 respectively to the yellow wedge). (B) (Neg.)
negative control culture on ONA medium with a DMSO gradient without chalcone. (Grad.) culture on ONA medium with a linear concentration gradient of chalcone
(0–8 µg mL−1 respectively to the yellow wedge). (C) (Neg.) negative control culture on ONA medium with a pure hexane gradient. (Grad.) culture on ONA medium
with a linear concentration gradient of the albaflavenone-hexane extract respective to the yellow wedge. (D) A dehydrogenase activity test. Spores germinated in the
presence of the albaflavenone-hexane extract (red) or in the presence of pure hexane (black). Metabolic activity was measured at denoted time points by optical
density at 484 nm. FIGURE 5 | Biological activities of germicidin A, chalcone, and albaflavenone-hexane extract. Bioassays (A–C) were performed on the six-sector cultivation titration
plates in two sets with negative control triplets (see Methods for more details), representative pictures are shown. (A) (Neg.) negative control culture on ONA medium
without germicidin A. (Grad.) culture on ONA medium with a linear concentration gradient of germicidin A (0–8 µg mL−1 respectively to the yellow wedge). (B) (Neg.)
negative control culture on ONA medium with a DMSO gradient without chalcone. (Grad.) culture on ONA medium with a linear concentration gradient of chalcone
(0–8 µg mL−1 respectively to the yellow wedge). (C) (Neg.) negative control culture on ONA medium with a pure hexane gradient. (Grad.) culture on ONA medium
with a linear concentration gradient of the albaflavenone-hexane extract respective to the yellow wedge. (D) A dehydrogenase activity test. Spores germinated in the
presence of the albaflavenone-hexane extract (red) or in the presence of pure hexane (black). Metabolic activity was measured at denoted time points by optical
density at 484 nm. FIGURE 6 | An inhibitory effect of chalcone on spore germination. Electron microscopic images of dormant (left) and germinating spores after two (middle) and four
(right) hours of cultivation in the liquid R3 medium. Control cultivation was performed in the absence of chalcone. In the presence of chalcone (in a concentration of
80 µg.mL−1), spores or germ tubes are disrupted (indicated by black arrows). The white bars indicate 2.5 µm. FIGURE 6 | An inhibitory effect of chalcone on spore germination. DISCUSSION Electron microscopic images of dormant (left) and germinating spores after two (middle) and four
(right) hours of cultivation in the liquid R3 medium. Control cultivation was performed in the absence of chalcone. In the presence of chalcone (in a concentration of
80 µg.mL−1), spores or germ tubes are disrupted (indicated by black arrows). The white bars indicate 2.5 µm. In contrast to the R3 medium, the minimal liquid medium
NMMP, a poorer medium in which streptomycetes produce
fewer secondary metabolites (Hodgson, 1982), was also used. The reason is that NMMP enables the testing of the effects see Methods). The nutritionally rich medium R3 was chosen for
the capacity of S. coelicolor to produce a number of structurally
distinct secondary metabolites in it, such as actinorhodin (Shima
et al., 1996) or coelimycin P1 (Gomez-Escribano et al., 2012). December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 8 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. of various ions and nutrients on the production of secondary
metabolites. The AM medium containing 20 amino acids was
also implemented into our experiments as it had been specifically
designed for germination experiments and was used throughout
the whole genome expression analyses (Bobek et al., 2004;
Strakova et al., 2013). It is known that the presence of different
carbon and energy sources in the medium qualitatively affects
secondary metabolism (Janecek et al., 1997). That is why the
presence of various sugars—glucose, glycerol, and mannitol—in
all three media types was tested. In accordance with previously
published data (Kieser et al., 2000), both glycerol and mannitol
were revealed to be a more suitable source of carbon for
secondary metabolites production in the R3 medium in our
experiments. Mannitol and glucose exhibited a higher capacity
for secondary metabolism when NMMP medium was used and
glycerol was shown to have a higher capacity in cases where AM
medium was used. Our results also showed that both nutritionally
richer media R3 and AM are more suited to germination and the
production of secondary metabolites than the minimal NMMP
medium, probably due to the presence of Ca2+ ions (Eaton and
Ensign, 1980; Lakey et al., 1983), L-amino acids (Hirsch and
Ensign, 1976b), or various carbon sources (Romero-Rodriguez
et al., 2016) in the richer media. et al., 2013). Secondary Metabolites of S. coelicolor
Produced during Germination g
The latest study on the topic (Xu and Vetsigian, 2017)
concludes that the germination of S. coelicolor M145 may
be positively or negatively affected by unknown substances
produced by the germlings themselves or by other streptomyces
species (e.g., S. venezuelae). The results presented here unveiled
that S. coelicolor produces three secondary metabolites during
germination, belonging to the terpenoids (albaflavenone) and
polyketides (germicidin A, chalcone). As these compounds have
not been detected in dormant spore extracts, we assume that they
are produced de novo during germination. They show a variety
of biological effects and thus perhaps help S. coelicolor suppress
competitive microflora or coordinate its own development at
the early stage of development. The biosynthetic pathways of
the detected substances encompass only a few simple reaction
steps that do not require complicated precursors and whose
biosynthetic genes are expressed during germination, as can
be found in gene expression data (Strakova et al., 2013). In
contrast, structurally complex metabolites (like the CDA) were
not detected in germination (see above). The
elution
methods
with
ethylacetate
(Rajan
and
Kannabiran, 2014) or the QuEChERS (Schenck and Hobbs,
2004) were not the most appropriate for isolation of secondary
metabolites. Therefore, solid phase extraction (Kamenik et al.,
2010) was applied with optimization for streptomycete secondary
metabolites. These extracts from supernatants of S. coelicolor’s
cultures were used for LC-MS analyses. DISCUSSION Despite the detected expression, we cannot exclude
the possibility that the presence of actinorhodin originates from
the stationary phase rather than from de novo synthesis in
germination (therefore the compounds are not listed in Table 3). The reason is that actinorhodin (as well as other aromatic
pigments derived from the type II and type III polyketide
synthases) is known to be bound on the spore envelopes
throughout dormancy (Davis and Chater, 1990; Bystrykh et al.,
1996; Funa et al., 1999; Tahlan et al., 2007). This was also
confirmed by the actinorhodin detection in our samples of
dormant spores even after several washings. Frontiers in Microbiology | www.frontiersin.org Albaflavenone g
Most of the secondary metabolites, whose biosynthetic genes
had previously been shown to be expressed during germination
(Strakova et al., 2013), were not detected in samples from
germination. These include 21 genes (including sco3230-3232
that encode CDA peptide-synthetase I-III) from the cda gene
cluster [encoding synthesis of the calcium-dependent-antibiotic
(CDA)], the cryptic gene cluster cpk (genes sco6273-6288,
encoding synthesis of a polyketide antibiotic coelimycin P1),
genes sco5314, sco5318, and sco5320 (encoding synthesis of
the gray spore pigment), and genes sco5877-5878, sco5881,
sco5891-5894, and sco5898 from the so-called red gene cluster
(encoding synthesis of undecylprodigiosin), and genes sco2783-
2784 from the desABCD cluster (sco2782-2785, controlling
the synthesis of desferrioxamines). Despite the respective gene
expression, biosynthesis of more complex secondary metabolites
may not occur, since gene expression is only a requirement for
biosynthesis and not evidence of it actually taking place. Tricyclic sesquiterpenoid albaflavenone has an aroma similar to
geosmin (Gerber and Lechevalier, 1965; Gurtler et al., 1994). Its biosynthesis requires only two genes: sco5222-5223 (Moody
et al., 2012). The expression of these genes can be suppressed
by cAMP-receptor protein, Crp, which also occurs in other
bacteria (e.g., in Escherichia coli). The cAMP-Crp control system
is a key regulator of germination, secondary metabolism, and
further development of S. coelicolor (Derouaux et al., 2004; Gao
et al., 2012; Bobek et al., 2017). The system also influences
the expression of biosynthetic gene clusters in S. coelicolor that
extend beyond albaflavenone actinorhodin, prodigiosin, CDA,
and coelimycin (Gao et al., 2012). So far, the production of albaflavone in streptomycetes has
been described only in the stationary growth in S. albidoflavus
(Gurtler et al., 1994), S. coelicolor, S. viridochromogenes, S. avermitilis, S. griseoflavus, S. Ghanaensis, and S. albus (Moody
et al., 2012). However, the expression data analysis shows that
the gene sco5223 is activated in germination (Strakova et al.,
2013), indicating the possible formation of this metabolite during
the initial 6 h of cultivation. We actually found a substance
corresponding to albaflavenone in the germination sample in the
AM medium with glycerol. The compound was also detected On the other hand, we found two actinorhodin congeners
- actinorhodinic acid and γ-actinorhodin (Bystrykh et al.,
1996; Okamoto et al., 2009) in all tested developmental
phases, i.e., stationary phase, dormant and germinating spores. Albaflavenone If we reason that the thickness
of the hydrophobic spore envelope is not unified (Lee and
Rho, 1993), then the water influx comes into different spores
in a different intensity, and therefore, naturally, germination
is a non-synchronous process (Hirsch and Ensign, 1976a;
Xu and Vetsigian, 2017). The spores already germinated
would produce albaflavenone as a signal that environmental
conditions are appropriate for the growth of the whole
population. Germicidins belong to a richly represented group of α-pyrone
natural substances found in bacteria, fungi, plants, and animals
(Schaberle, 2016). Pyrones have many biological effects and
signal molecules for quorum sensing can be found among them
(Brachmann et al., 2013). Germicidin A is a known reversible
inhibitor of spore germination; it prevents the germination at
very low concentrations of 40 pg mL−1; i.e., only 2,400 molecules
per spore (Petersen et al., 1993). We verified this biological effect
in S. coelicolor, where germicidin A had a marked adverse effect
on germination already at as low a concentration in medium as
4 µg mL−1. It is known that germicidin A affects the respiration
of spores and mycelia by interacting with the membrane Ca2+-
ATPase, inactivating the enzyme. By this mechanism, germicidin
not only prevents spores from generating sufficient energy for
germination but also inhibits hyphal growth (Eaton and Ensign,
1980; Grund and Ensign, 1985; Aoki et al., 2011). Germicidin
A also exhibits antibacterial activity against gram-positive
bacteria such as Bacillus subtilis, Arthrobacter crystallopoietes, or
Mycobacterium smegmatis (Grund and Ensign, 1985; Aoki et al.,
2011). β-farnesene is a sesquiterpene relative to albaflavenone with a
wide range of bioactivities (Gibson and Pickett, 1983; Avé et al.,
1987) that serves as a precursor of a number of biosynthetic
pathways, including the geosmin synthesis. The synthesis of
both albaflavenone and β-farnesene is dependent on the type of
activity carried out by cytochrome P450 (CYP170A1). It may
function either as P450 monooxygenase or as P420 farnesene-
synthase. It has been shown that the farnesene-synthase activity
predominates at pH 5.5–6.5 and in the presence of bivalent
cations (Mg2+, Mn2+, Ca2+), while at pH 7.0–8.2, it functions as
the monooxygenase, oxidizing epi-isozizaen first to albaflavenol,
and then to albaflavenone (Moody et al., 2012). Conformation
and final enzymatic activity of CYP170A1 is thus affected by
the pH of the environment. We presume that S. coelicolor
may exploit the dual pH-dependent activity of the enzyme
in order to detect optimal external conditions. Albaflavenone In germinating spores, expression of several involved genes:
sco5072-5086, sco5088, and sco5090 had been found (Strakova December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 9 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. in samples from the stationary phase (positive control in R3
medium with glycerol), but not samples from dormant spores,
indicating its germination-associated de novo synthesis. in samples from the stationary phase (positive control in R3
medium with glycerol), but not samples from dormant spores,
indicating its germination-associated de novo synthesis. Germicidin A production has previously been described
in germination spores and in the stationary growth stage of
S. viridochromogenes (Hirsch and Ensign, 1978; Petersen et al.,
1993). It was also isolated after more than 24 h of submerged
cultivation of S. coelicolor and other streptomyces (Petersen
et al., 1993; Aoki et al., 2011; Ma et al., 2017). The results
of our work, however, show for the first time that germicidin
A is produced by germlings of S. coelicolor (in R3 medium
with glycerol or mannitol, and in AM medium with glucose or
glycerol). In contrast, germicidin B in germinating S. coelicolor
was not produced, which is consistent with the results reported
in S. viridochromogenes (Petersen et al., 1993). On the other hand,
both polyketides, germicidin A and germicidin B, were detected
here in samples from the stationary phase (in both, R3 medium
with glycerol, or mannitol and NMMP medium with glucose or
mannitol). g
g
y
Albaflavenone is not commercially available, which is why
we used hexane extracts, where the compound was detected
by LC-MS, for testing its biological activities. We did not see
any effect, however. On the other hand, it can be assumed that
the albaflavenone produced during the germination provides an
advantage in a highly competitive soil environment because it
has a demonstrable antibacterial effect on Bacillus subtilis at the
concentration of 8 µg mL−1 (Gurtler et al., 1994). Moreover, if
albaflavenone was incorporated into the hydrophobic envelope
of spores, as other terpenoids do incorporate into the lipophilic
membrane layers, it would affect the permeability of the
envelopes leading to an intense water influx into spores, thereby
accelerating their germination. Albaflavenone Therefore, we
tested whether the biosynthetic activity is dependent on the
pH of the medium during germination. For the experiment
we performed the same R3 medium with a pH of either 7.2
or 6.0. Although we were not able to directly demonstrate the
production of β-farnesene, we proved that the spectrum of
detected substances significantly differed. Further experiments
are required to confirm the expected pH-dependent signaling
activity of the albaflavenon/β-farnesene systems. Because of its inhibitory effect, the production of germicidin
during germination in optimal conditions might, at first glance,
seem surprising. Its production by germinating spores might
help to co-ordinate germination within the population. Its
self-regulating function could maintain a portion of spores in
their dormant state for a prolonged period as a reserve if the
environment proves to be unfavorable or when germination
occurs in higher spore densities. Conversely, the ungerminated
spores can be further propagated in the environment and,
after overcoming the reversible inhibition, can spread in new
niches. Frontiers in Microbiology | www.frontiersin.org Germicidin A The products of these
genes, Nod factors, are involved in the symbiosis where
rhizobacteria produce nitrogen for plants (Maxwell et al., 1989). Interestingly, other flavonoids - quercetin, kaempferol, and
myricetin—are known to stimulate pollen germination in
Nicotiana tabacum L. (Ylstra et al., 1992). Therefore, we
expected that chalcones produced by germinating spores
would stimulate germination under favorable environmental
conditions. Experimentally, however, we verified the opposite as
the chalcone of S. coelicolor remarkably inhibited germination. At
a concentration of 300 µg mL−1, it completely suppressed growth
and at 8 µg mL−1 and lower the compound visibly inhibited
spore germination, colony differentiation, and actinorhodin
production on solid medium. Electron microscopic images taken
from liquid cultivation revealed disrupted germ tubes. This
finding correlates with a described activity of chalcone which was
shown to interfere with cell membrane of Staphylococcus aureus
(Sivakumar et al., 2009). Chalcone
is
apparently
an
instrument
of
interspecies
interaction, as there are referred its antifungal, phytotoxic and
insecticidal effects are outlined (Diaz-Tielas et al., 2012). Chalcone
could
also
function
as
a
signaling
molecule
in
a
symbiotic
relationship,
such
as
4,4′-dihydroxy-2′-
methoxychalcone produced by legumes that induce transcription
of nod genes in symbiotic rhizobacteria. The products of these
genes, Nod factors, are involved in the symbiosis where
rhizobacteria produce nitrogen for plants (Maxwell et al., 1989). rhizobacteria produce nitrogen for plants (Maxwell et al., 1989). Interestingly, other flavonoids - quercetin, kaempferol, and
myricetin—are known to stimulate pollen germination in
Nicotiana tabacum L. (Ylstra et al., 1992). Therefore, we
expected that chalcones produced by germinating spores
would stimulate germination under favorable environmental
conditions. Experimentally, however, we verified the opposite as
the chalcone of S. coelicolor remarkably inhibited germination. At
a concentration of 300 µg mL−1, it completely suppressed growth
and at 8 µg mL−1 and lower the compound visibly inhibited
spore germination, colony differentiation, and actinorhodin
production on solid medium. Electron microscopic images taken
from liquid cultivation revealed disrupted germ tubes. This
finding correlates with a described activity of chalcone which was
shown to interfere with cell membrane of Staphylococcus aureus
(Sivakumar et al., 2009). The widespread autoregulator of germination, germicidin
A, is known to be produced by germinating spores of S. viridochromogenes. Here it was shown that the germlings of S. coelicolor are also capable of its production. Chalcone is probably
one of the precursors of biosynthesis of a not yet described
flavonoid in S. coelicolor. Germicidin A During germination it functions as a
germination inhibitor that may serve as a means of interspecies
communication. ACKNOWLEDGMENTS Access to the electron microscopy facility, supported by the
project LO1509 of the Ministry of Education, Youth and Sports
of the Czech Republic and by the Operational Program Prague–
Competitiveness project (CZ.2.16/3.1.00/24023) supported by
the European Union, is gratefully acknowledged. This work
was supported by a project by Charles University in Prague:
Progres Q26/LF1 to JB, SVV260369 to KŠ, a grant of the
Grant Agency of the Charles University (www.cuni.cz/UK-33. html) under contract no. 160214 to KŠ, a project of J. E. Purkinje University: UJEP-SGS-173-07-01 to JB, and by the
Czech research infrastructure for systems biology C4SYS (project
no. LM2015055) to JB. CONCLUSION In this work the production of secondary metabolites in
germinating streptomycetes is systematically analyzed for the
first time. Our investigation was based on the hypothesis
that germinating spores exploit intercellular communications
(quorum sensing) to support a coordinated development in
its early stage as well as interspecies communication (quorum
quenching) to suppress metabolic activities of competing
microflora (Chen et al., 2000). This work succeeds the previous
transcriptomic analysis of germination in streptomyces (Strakova
et al., 2013), which has shown the expression of genes from
different antibiotic clusters. Here using LC-MS, we detected three
potentially important secondary metabolites—sesquiterpenoid
albaflavenone, and polyketides germicidin A, and chalcone—
that are synthesized during spore germination of S. coelicolor. All three detected compound possess capacities to suppress AUTHOR CONTRIBUTIONS MˇC managed all experiments and evaluated data; MˇC and
KP performed analytical samples; MˇC and ZK performed
LC-MS measurements; KŠ and NB performed cultures and
experimental design for germination; OB and OK performed
the electronmicroscopy; JB concieved the project and wrote the
manuscript. Streptomyces coelicolor A3(2). J. Antibiot. 64, 607–611. doi: 10.1038/ja.
2011.59
Aoki, Y., Yoshida, M., Kawaide, H., Abe, H., and Natsume, M. (2007). Isolation and
characterization of a spore germination inhibitor from Streptomyces sp. CB-1-
1, a phytopathogen causing root tumor of melon. Biosci. Biotechnol. Biochem.
71, 986–992. doi: 10.1271/bbb.60649
Avé, D. A., Gregory, P., and Tingey, W. M. (1987). Aphid repellent sesquiterpenes
in
glandular
trichomes
of
Solanum
berthaultii
and
S.
tuberosum. Streptomyces coelicolor A3(2). J. Antibiot. 64, 607–611. doi: 10.1038/ja.
2011.59
Aoki, Y., Yoshida, M., Kawaide, H., Abe, H., and Natsume, M. (2007). Isolation and
characterization of a spore germination inhibitor from Streptomyces sp. CB-1-
1, a phytopathogen causing root tumor of melon. Biosci. Biotechnol. Biochem.
71, 986–992. doi: 10.1271/bbb.60649
Avé, D. A., Gregory, P., and Tingey, W. M. (1987). Aphid repellent sesquiterpenes
in
glandular
trichomes
of
Solanum
berthaultii
and
S.
tuberosum. Germicidin A The detection of chalcone in Streptomyces has not yet been
proven to our knowledge. Its tentative identification presented
here is based on the accurate mass of analyzed supernatants
from the stationary phase and germination in R3 medium
with glycerol or mannitol. Chalcones are intermediates of
flavonoid biosynthesis where the key role in their synthesis plays
1,3,6,8-tetrahydroxynaphthalene-synthase. One of its precursors
is the naringenin-chalcone, whose involvement in the flavonoid Gcs protein (a polyketide synthase type III, PKS III) is involved in
the germicidin biosynthesis in S. coelicolor (Chemler et al., 2012). However, expression of its gene sco7221 during germination was
below the detection limit (Strakova et al., 2013). The germicidin
biosynthesis could also be related to the activity of other
genes sco7670-7671, whose expression is active in germination
(Strakova et al., 2013). December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 10 Secondary Metabolism of Germinating Streptomycete Spores ˇCihák et al. naringenin biosynthesis was described in S. clavuligerus (Alvarez-
Alvarez et al., 2015). The enzyme, which belongs to PKS III,
is closely related to the plant chalcone synthase (Izumikawa
et al., 2003). Its sco1206 gene in S. coelicolor is expressed during
germination (Strakova et al., 2013). competitive microflora at the early stage of development. Their
biosynthetic pathways are simple, having only a few reaction
steps that do not require complex precursors. Albaflavenone had been previously detected only in the
stationary phase of growth of certain streptomycetes (Gurtler
et al., 1994; Moody et al., 2012). It exhibits an antibacterial
effect and could serve as a germination signal for coordinated
development or as a factor of interspecific communication (these
suggestions are not proven here). In contrary, the two other
compounds revealed inhibitory effects on germination that may
explain slower germination rate and less synchronicity in the
model S. coelicolor, in comparison with other Streptomyces
species, such as S. viridochromogenes. Our data are consistent
with the known inhibitory effect of the supernatant of S. coelicolor
on its own germination (Xu and Vetsigian, 2017). g
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is
apparently
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instrument
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interspecies
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also
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as
a
signaling
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quinone formation encoded in the act gene cluster. Chem. Biol. 16, 226–236. doi: 10.1016/j.chembiol.2009.01.015 van Vliet, S. REFERENCES (2015). Bacterial dormancy: how to decide when to wake up. Curr. Biol. 25, R753–R755. doi: 10.1016/j.cub.2015.07.039 Paleckova, P., Bobek, J., Felsberg, J., and Mikulik, K. (2006). Activity of
translation system and abundance of tmRNA during development of
Streptomyces aureofaciens producing tetracycline. Folia Microbiol. 51, 517–524. doi: 10.1007/BF02931615 Wakefield, J., Hassan, H. M., Jaspars, M., Ebel, R., and Rateb, M. E. (2017). Dual induction of new microbial secondary metabolites by fungal bacterial
co-cultivation. Front. Microbiol. 8:1284. doi: 10.3389/fmicb.2017.01284 Waters, C. M., and Bassler, B. L. (2005). Quorum sensing: cell-to-cell
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2158-9 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. REFERENCES Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Rutherford, S. T., and Bassler, B. L. (2012). Bacterial quorum sensing: its role
in virulence and possibilities for its control. Cold Spring Harb. Perspect. Med. 2:a012427. doi: 10.1101/cshperspect.a012427 Copyright © 2017 ˇCihák, Kameník, Šmídová, Bergman, Benada, Kofroˇnová,
Petˇríˇcková and Bobek. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Schaberle, T. F. (2016). Biosynthesis of alpha-pyrones. Beilstein J. Org. Chem. 12,
571–588. doi: 10.3762/bjoc.12.56 Schenck, F. J., and Hobbs, J. E. (2004). Evaluation of the quick, easy, cheap,
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0388-y December 2017 | Volume 8 | Article 2495 Frontiers in Microbiology | www.frontiersin.org 13
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Reshaping mental health practice with evidence: the Mental Health Research Network
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The scale and quality of psychiatric research The deficit in the quantity and quality of mental
health research is related to a deficit in the funding of
mental health research. For example, in 1999-2000 out
of a total research budget of »318.4 million, the Medical
Research Council spent »17.6 million (5.5%) on mental
health research, a proportion considerably less than
would be expected from the predicted burden of disease. A similar gap is obvious in the USA. A recent report
suggested that only 28.5% of National Institute of Mental
Health (NIMH) awards are in the area of serious mental
illness, despite the fact that 58% of all direct care costs
are to this group (Torrey et al, 2003). In our view, this
funding deficit does not reflect unfairness in the grant
awarding process so much as a quality gap between
mental health grant applications and those received from
competing disciplines. Even at a superficial level, research in mental health
appears to fall behind what might be expected from the
disease burden. For example, the World Health Organi-
zation estimates that mental disorders account for 12.3%
of the global burden of disease (approximately 20% in
developed countries) (Thornicroft et al, 2002), but less
than 5% (17 558 of 375143) of trials in the Cochrane
Controlled Trials Register are indexed under ‘mental
disorder’. Deeper examination reveals the type and
geographical distribution of research. When the Cochrane
Schizophrenia Group carried out a survey of 2000
controlled trials relating to schizophrenia research they
found the literature dominated by hospital-based drug
trials undertaken in the United States (Thornley et al,
1998). Although schizophrenia is not a short-term
disorder, over half of the trials (N=1082) lasted for no
more than 6 weeks and only one-fifth lasted longer than
6 months (N=382). Only 3% of trials had sufficient
power to detect a clinically important improvement in
mental state and over half had fewer than 50 partici-
pants, with the trial size not increasing over time. Recent Government reports (Office of Science and
Technology, 2003) and the Academy of Clinical Medicine
(2003) have suggested that one solution to small-scale
research, and perhaps to the timeliness of research, is to
develop infrastructures or networks to support larger-
scale studies. editorial editorial Psychiatric Bulletin (2004), 28,153^155 T I L W Y K E S A ND M A X M A R S H A L L
Reshaping mental health practice with evidence:
the Mental Health Research Network interventions, it would be astonishing if such differences
did not exist. With an evidence base of this quality, it is
perhaps not surprising that 75 of 109 recommendations
in the NICE Schizophrenia Guidelines (National Institute
for Clinical Excellence, 2002) were based on the clinical
experience of the Guidelines Development Group or on
expert committee reports (the two lowest grades of
evidence as defined by the Guidelines Development
Group). Many psychiatrists will have searched for the solution to
a clinical problem only to find that the evidence
uncovered was grossly inadequate or thoroughly
confusing. Some will have felt that such uncertainty is
part of the science of psychiatry, but we would argue
that it arises from deficits in the quantity and quality of
the underpinning research. Wykes & Marshall Mental Health Research Network Wykes & Marshall Mental Health Research Network Research creativity High-quality proposals in specific Department of Health
priority areas will be fostered through a limited number of
research groups. Each research group will have the task
of developing a programme of proposals to run on the
Network and to feed back issues for the Department of
Health to consider in the future. They will consist of
leading UK researchers, including service user
researchers, who have a strong research track record in
the relevant priority area. One research group in early
intervention has already been established and another in
self-help has just started. The Medical Research Council has been responsive in this
area and has already allocated new money in its Brain
Sciences Initiative (trial platforms and pathfinder grants:
http://www.mrc.ac.uk/index/funding.htm). We are
hopeful that other funders will follow this lead and also
develop partnerships to support larger, and perhaps more
meaningful, studies in mental health. The scale and quality of psychiatric research The National Institute of Mental Health for
England (NIMHE) had the forethought, together with the
Department of Health Research and Development, to
draw up the blueprint for such a network, which now
exists for supporting trials and projects in mental health. The network is called the Mental Health Research
Network (MHRN) and this organisation aims to close the
quality and the funding gap between mental health and
other branches of medical research. The small-scale and short-term nature of most
psychiatric trials makes it difficult to address questions of
clinical relevance. Although service users tend to base
their treatment preferences on an evaluation of risks and
benefits, few psychiatric trials have sufficient power to
evaluate such risks adequately. Hence, the mental health
research community was slow to realise the importance
of medication side-effects such as sexual impairment and
weight gain. Similarly, few psychiatric trials have much to
say about the benefits or risks to particular sub-groups
(women or ethnic minorities) or sub-populations (urban
versus rural). Yet, given the complexity of mental health The MHRN is run by a managing partnership of the
Institute of Psychiatry (King’s College London), and the
University of Manchester under the Directorship of
Professor Til Wykes (Institute of Psychiatry) and Deputy 153 https://doi.org/10.1192/pb.28.5.153 Published online by Cambridge University Press Wykes & Marshall Mental Health Research Network Directorship of Professor Max Marshall (University of
Manchester). It represents a unique opportunity to widen
participation within research and help reshape mental
health practice with evidence. By fulfilling a coordinating
role, we hope to raise the standard of mental health and
social care research throughout England. (SURGE). It is responsible for encouraging collaborative
efforts between service users and clinical academic and
supporting both partners in large research studies. Conclusion We have no doubt that establishing the Network will be a
complex and at times controversial process. The Network
facilitates but does not fund research, so in the end
success will depend on the desire of researchers, Structure of the MHRN The MHRN is a managed network and its structure is
shown in Figure 1. It brings together researchers and
providers of mental health and social care services. The
MHRN hosts, but does not fund research. Its strategy is
supported by an advisory group that has representatives
from Department of Health Funding agencies as well as
academics and service users. Getting research onto the MHRN The MHRN is an open network so it is not necessary to
be a member of a hub or a research group in order to
take advantage of the infrastructure. Investigators can
apply to run a project on the Network by a brief submis-
sion to the Adoptions Committee. The Adoptions
Committee considers the suitability of outline proposals
according to the principles and criteria detailed in Box 1. What will the MHRN add? The power to run larger studies is dependent on the
MHRN research hubs, which are based around England
including the Midlands, Bristol, Manchester, London and
Cambridgeshire. Hubs are chosen through a competitive
process on the basis of academic quality, NHS partner-
ships, and the ability to represent different and comple-
mentary geographical and cultural constituencies to
support both large randomised controlled clinical trials
and projects involving rare conditions. We anticipate that a coordinated managed network will
make large-scale mental health research easier in three
key ways: . Through research groups to create a culture of large-
scale studies relevant to key problems in mental health. . Through the Hubs to provide access to a stable infra-
structure ofclinical/academic centres withatrack record
of large-scale recruitment across a large and diverse
patient population. . If successful, the Network will encourage fundingbodies
to view mental health research as capable of competing
inqualityand quantity withresearchinother branches of
medicine. Involving service users Support for service user involvement in research will be
provided by the Service User Research Group in England Figure 1. Structure of the Mental Health Research Network. 154
192/pb.28.5.153 Published online by Cambridge University Press Figure 1. Structure of the Mental Health Research Network. Figure 1. Structure of the Mental Health Research Network. 154 https://doi.org/10.1192/pb.28.5.153 Published online by Cambridge University Press https://doi.org/10.1192/pb.28.5.153 Published online by Cambridge University Press Wykes & Marshall Mental Health Research Network Box 1
Principles
(i) thereis evidenceofserviceuserinputintodevelopmentof
the proposal;
(ii) the proposal is in line with national mental healthpolicy;
(iii) theproposalmustbe free ofethicalor majordesignflaws. Criteria
The proposal:
(i) requires multiple centres because ofa need for: a large
sample size, recruitment of participants with a rare
condition, or from a particular group or lifestyle, OR
(ii) is a cluster-randomised trial requiringmultiple centres or
units of randomisation, OR
(iii) requires multiple centres in order to establish whether a
finding or an intervention is a valid generalisation across
different settings or is applicable in specific settings (for
example, rural areas). Declaration of interest Box 1 The Mental Health Research Network (MHRN) is a
standing programme of the National Institute of Mental
Health in England (NIMHE) and is funded by the Depart-
ment of Health. For more information, see
www.mhrn.info Criteria ACADEMY OF MEDICAL SCIENCES
(2003) Strengthening clinical research. London: Academy of Medical
Sciences. Primary and Secondary Care. London:
Gaskell. Primary and Secondary Care. London:
Gaskell. The proposal: THORNICROFT, G. & MAINGAY, S. (2002) The globalresponse to mental
illness. BMJ, 325, 608-609. OFFICE OF SCIENCE ANDTECHNOLOGY,
DEPARTMENT OF TRADE AND
INDUSTRY (2003) Bioscience 2015. Improvingnational health, increasing
national wealth; a report to
Government by the Bioscience
Innovation and GrowthTeam. London:
HMSO. OFFICE OF SCIENCE ANDTECHNOLOGY,
DEPARTMENT OF TRADE AND
INDUSTRY (2003) Bioscience 2015. Improvingnational health, increasing
national wealth; a report to
Government by the Bioscience
Innovation and GrowthTeam. London:
HMSO. THORNLEY, B. & ADAMS, C. (1998)
Content and quality of 2000 controlled
trials in schizophrenia over 50 years. BMJ, 317,1181-1184. TORREY, F., ZDANOWICZ, M.,WOLFE,
S., et al (2003) A Federal Failure in
Psychiatric Research: Continued NIMH
negligence in funding sufficient
research on serious mentalillnesses. Arlington,VA:Treatment Advocacy
Center: www.psychlaws.org. NATIONAL INSTITUTE FORCLINICAL
EXCELLENCE (2002) Schizophrenia:
Care Interventions intheTreatment and
Management of Schizophreniain NATIONAL INSTITUTE FORCLINICAL
EXCELLENCE (2002) Schizophrenia:
Care Interventions intheTreatment and
Management of Schizophreniain clinicians and service users to work together. It is our
belief that the mental health research community will be
sufficiently far-sighted to make the most of this unique
opportunity and realise its full potential. *TilWykes
Department of Psychology, Institute of Psychiatry, De Crespigny
Park, PO Box 77, London SE5 8AF. E-mail: mhrn@iop.kcl.ac.uk,
Max Marshall
University of Manchester *TilWykes
Department of Psychology, Institute of Psychiatry, De Crespigny
Park, PO Box 77, London SE5 8AF. E-mail: mhrn@iop.kcl.ac.uk,
Max Marshall
University of Manchester 155 https://doi.org/10.1192/pb.28.5.153 Published online by Cambridge University Press https://doi.org/10.1192/pb.28.5.153 Published online by Cambridge University Press
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Peer Review #2 of "A comparison of the mineral element content of 70 different varieties of pear fruit (Pyrus ussuriensis) in China (v0.1)"
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Manuscript to be reviewed
A Comparison of the Mineral Element Content of 70 different
varieties of Pear Fruit (Pyrus ussuriensis) in China
Chang Liu Equal first author, 1, 2 , Honglian Li Equal first author, 3 , Aihua Ren 4 , Guoyou Chen 5 , Wanjun Ye 6 , Yuxia Wu 1 , Ping Ma 1, 7 ,
Wenquan Yu 2 , Tianming He Corresp. 1
1
2
College of Horticulture, Xinjiang Agricultural University, Urumqi, China
Mudanjiang Branch/Key Laboratory of Fruit Breeding and Cultivation in Cold areas of Heilongjiang Province, Heilongjiang Academy of Agricultural
Sciences, Mudanjiang, China
3
Fruit Research Institute, Jilin Academy of Agricultural Sciences, Gongzhuling, China
4
Horticulture Branch, Heilongjiang Academy of Agricultural Sciences, Harbin, China
5
Quality and Safety Institute of of Agricultural Products/Inspection and Testing Center for Quality of Cereals and Their Products (Harbin), Ministry of
Agriculture and Rural Affairs, Heilongjiang Academy of Agricultural Sciences, Harbin, China
6
Heilongjiang Academy of Agricultural Sciences, Harbin, China
7
Bayin Guoleng Vocational and Technical College, Korla, China
Corresponding Author: Tianming He
Email address: 1554272245@qq.com
Background. Pyrus ussuriensis (Maxim.) is a unique pear tree that grows in northern
China. The tree has strong cold resistance and can withstand low temperatures from -30
°C to -35 °C. Due to its unique growth environment, its fruit is rich in minerals and has
much higher levels of minerals such as K, Ca and Mg than the fruit of Pyrus pyrifolia
(Nakai.) and Pyrus bretschneideri (Rehd.) on the market, and many say the ripe fruit tastes
better than other varieties. A comprehensive analysis of the characteristics of mineral
elements in the fruits of different varieties of P. ussuriensis will provide a valuable
scientific basis for the selection, breeding and production of consumer varieties of P.
ussuriensis, and provide a more complete understanding of nutritional differences between
fruit varieties. Methods. In this study, 70 varieties of wild, domesticated and cultivated
species of P. ussuriensis from different geographical locations were compared. Targeting
four main mineral elements and eight trace mineral elements contained in the fruit, the
differences in mineral content in the peel and pulp of different varieties of P. ussuriensis
were analyzed, compared and classified using modern microwave digestion ICP-
MS.Results. The mineral elements in the fruit of P. ussuriensis generally followed the
following content pattern: K > P > Ca > Mg > Na > Al > Fe > Zn > Cu > Cr > Pb > Cd. The
mineral element compositions in the peel and pulp of different fruits were also significantly
different. The four main mineral elements in the peel were K > Ca > P > Mg, and K > P >
Mg > Ca in the pulp. The mineral element content of wild fruit varieties was higher than
that of cultivated and domesticated varieties. Correlation analysis results showed that
PeerJ reviewing PDF | (2022:11:79482:1:2:NEW 24 Mar 2023)
Manuscript to be reviewed
there was a significant positive correlation between K, P and Cu in both the peel and pulp
of P. ussuriensis fruit (P＜0. 01). Cluster analysis results showed that the 70 varieties of P.
ussuriensis could be divided into three slightly different categories according to the
content of the peel or pulp. According to the contents of the fruit peel, these varieties were
divided into: (1) varieties with high Na, Mg, P, K, Fe and Zn content, (2) varieties with high
Ca content and (3) varieties with medium levels of mineral elements. According to the fruit
pulp content, these varieties were divided into: (1) varieties with high Mg, P and K content,
(2) varieties with low mineral element content, and (3) varieties with high Na and Ca
content. The comprehensive analysis of relevant mineral element content factors showed
that ‘SSHMSL,’ ‘QYL,’ ‘SWSL’ and ‘ZLTSL-3’ were the best varieties, and could be used as
the focus varieties of future breeding programs for large-scale pear production.
PeerJ reviewing PDF | (2022:11:79482:1:2:NEW 24 Mar 2023)
Manuscript to be reviewed
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A Comparison of the Mineral Element Content of 70
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Chang Liu1,2, Honglian Li3, Aihua Ren4, Guoyou Chen5, Wanjun Ye6, Yuxia Wu1, Ping Ma1,7,
Wenquan Yu2, Tianming He1
1 College
of Horticulture, Xinjiang Agricultural University, Urumqi, Xinjiang, China
Mudanjiang Branch, Heilongjiang Academy of Agricultural Sciences/Key Laboratory of Fruit Breeding
and Cultivation in Cold areas of Heilongjiang Province, Mudanjiang, Heilongjiang, China
3 Fruit Research Institute, Jilin Academy of Agricultural Sciences, Gongzhuling, Jilin, China
4 Horticulture Branch, Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China
5 Institute of Quality and Safety of Agricultural Products/Quality Supervision, Inspection and Testing
Center of Grain and Products, Ministry of Agriculture and Rural Affairs, Heilongjiang, China
6 Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China
7 Bayin Guoleng Vocational and Technical College, Korla, Xinjiang, China
2
Corresponding Author:
Tianming He
311 Nongda east road, Urumqi, Xinjiang, 830052, China
Email address: 1554272245@qq.com
These authors contributed equally to this work:
Chang Liu, Honglian Li
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Background. Pyrus ussuriensis (Maxim.) is a unique pear tree that grows in northern China. The
tree has strong cold resistance and can withstand low temperatures from -30 °C to -35 °C. Due to
its unique growth environment, its fruit is rich in minerals and has much higher levels of
minerals such as K, Ca and Mg than the fruit of Pyrus pyrifolia (Nakai.) and Pyrus
bretschneideri (Rehd.) on the market, and many say the ripe fruit tastes better than other
varieties.
A comprehensive analysis of the characteristics of mineral elements in the fruits of different
varieties of P.ussuriensis will provide a valuable scientific basis for the selection, breeding and
production of consumer varieties of P. ussuriensis, and provide a more complete understanding
of nutritional differences between fruit varieties.
Methods. In this study, 70 varieties of wild, domesticated and cultivated species of P.
ussuriensis from different geographical locations were compared. Targeting four main mineral
elements and eight trace mineral elements contained in the fruit the differences in mineral
content in the peel and pulp of different varieties of P.ussuriensis were analyzed, compared and
classified using modern microwave digestion ICP-MS.
Results. The mineral elements in the fruit of P. ussuriensis generally followed the following
content pattern: K > P > Ca > Mg > Na > Al > Fe > Zn > Cu > Cr > Pb > Cd. The mineral
element compositions in the peel and pulp of different fruits were also significantly different.
The four main mineral elements in the peel were K > Ca > P > Mg, and K > P > Mg > Ca in the
pulp. The mineral element content of wild fruit varieties was higher than that of cultivated and
domesticated varieties. Correlation analysis results showed that there was a significant positive
correlation between K, P and Cu in both the peel and pulp of P. ussuriensis fruit (P < 0.01).
Cluster analysis results showed that the 70 varieties of P. ussuriensis could be divided into three
slightly different categories according to the content of the peel or pulp. According to the
contents of the fruit peel, these varieties were divided into:(1) varieties with high Na, Mg, P, K,
Fe and Zn content (2) varieties with high Ca content and (3)varieties with medium levels of
medium elements. According to the fruit pulp content, these varieties were divided into: (1)
varieties with high Mg, P and K content, (2)varieties with low mineral element content, and(3)
varieties with high Na and Ca content. The comprehensive analysis of relevant mineral element
content factors showed that 'SSHMSL', 'QYL', 'SWSL' and 'ZLTSL-3' were the best varieties,
and could be used as the focus varieties of future breeding programs for large-scale pear
production.
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Introduction
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The pear belongs to the Rosaceae family and is one of the most important fruit trees in the world.
Based on origin, pear varieties are generally divided into two categories: Asian pears and
western pears (Chen et al., 2018). In China, the Asian pear is the main category of pear, with a
cultivation history of more than 3,000 years, and is the third largest fruit grown, by volume,after
citrus and apple. Pears are widely planted in China, mainly distributed in North China, Northeast
China, Northwest China and the Yangtze River basin, with rich germplasm resources (Dong et
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al., 2018; Li et al., 2022). Previous research has been conducted on pear germplasm resources
using agronomic and quality traits (Zheng et al., 2022; Wu et al., 2018; Niu et al., 2019; Gong et
al., 2020), Relevant fruit research has found that there are many ascorbic acids, flavonoids,
sugars, organic acids and minerals (K, P, Mg, Ca) and other bioactive substances that are
beneficial to human health (Hou et al.2022; Lia et al. 2018a; Adhikary et al., 2020; Sun et al.,
2021; Che et al., 2018). Both Chinese traditional medicine and international modern medicine
believe that pears have an antioxidant effect and are capable of lowering blood pressure,
moistening the lungs, resolving phlegm clearing heat and detoxification the body, Eating pears
regular can help supplement the nutrition needed by the human body, and may even have
medicinal value (Wang et al., 2015; Li et al., 2014; Peng et al., 2023; Ogita et al., 2020; Bajwa et
al., 2015). With the increased enthusiasm for healthy, sustainable food and the acceleration of
farming technology the importance of pear cultivation as an industry has increased in many
places(Zhang et al.,2018; Li et al., 2018b).
This increase in pear cultivation has led to higher fruit quality standards in the global market,
especially the high-end market and recent research has focused on the development of more
nutritional and better tasing fruit varieties.Wild apples in Xinjiang have been used to cultivate
new varieties of (red pulp) apples with high-flavonoid content, and a new variety of pear
'Shannongsu' with good antioxidant quality,has been developed by crossing the unique ‘Korla
fragrant Pear’ with the ‘Dangshansu Pear.’ These examples show the potential for innovative
alongside the optimization of fruit resources (Chen et al., 2022).
Mineral element content is one way to measure fruit quality traits, as mineral elements are
closely related to fruit size, pulp hardness, and soluble solids ( Martin et al., 2021; Maity et al.,
2022; Sete et al., 2019), and play important roles in fruit disease resistance, storage resistance,
and maintaining good quality and flavor (Cui et al., 2020; Wei et al., 2017). Mineral elements are
also very important nutrients that are essential for healthy human growth and development
(Goracy et al., 2021). If the human body lacks mineral Elements, it suffers from 'hidden hunger'
(Gödecke et al., 2018).Previous studies have shown that mineral elements play a crucial role in
cell metabolism, biosynthesis and immune function of the human body by combining with
proteins and other organic molecular functional groups(Aranaz et al., 2020; Njoku et al., 2018).
For example, iron(Fe) is an essential component of hemoglobin, myoglobin and some functional
enzymes (McDevitt et al., 2020); Zinc(Zn) is a component of many important enzymes in the
human body, and a lack of Zn lead to declines in immune function and can cause a variety of
diseases (Nadeem et al., 2020). Many of the mineral elements needed by the human body cannot
be synthesize on their own and need to be supplemented by the intake of fruits such as apples,
pears, or grapes. Therefore, it is important to study the content of mineral elements in pears and
other fruits for the selection of better functional varieties for fruit tree cultivation
.
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Pyrus ussuriensis (Maxim.) is one of the main pear varieties cultivated in China (Qiu et al.,
2018), and includes popular varieties such as: ‘Nanguo Pear’, ‘Jinxiangshui pear’, and ‘Huagai
pear’. It is mainly distributed in Heilongjiang, Jilin, Liaoning and other eastern regions of China.
These areas are hot and rainy in the summer, which is conducive to vegetation growth.Winter in
these areas is long, cold and dry, with large temperature difference between day and night
causing the surface vegetation to form humus after long-term corrosion, which can evolve into
rich soil with high organic content. Pears planted under these conditions are favored because of
their good flavor and high nutritional value, and play a very important role in the cultivation of
related fruit trees and fruit production.
There are many previous studies on the content of mineral elements in pear fruits, but these
studies have mainly been carried out on P. pyrifolia , P. bretschneideri and P. communis L.
(Shen et al., 2019; Su et al., 2017; Kong et al., 2018; Saquet et al., 2019), The research reports
that have been done on P. ussuriensis have only analyzed the mineral content of the ‘Pingguo
Pear’ and ‘Nanguo Pear.’ These studies have found that K, N and Ca content in the fruits of
different orchards can vary greatly (Yan et al., 2022; Piao et al., 2018), and that Mg and Ca
content are much higher in P. ussuriensis than in P. pyrifolia. and P. bretschneideri (Liu et al.,
2022).
Although previous work has provided valuable contributions to the understanding of the
nutritional content of pears, because the sample sizes of varieties were too small in these
previous studies, the mineral element content findings are not universal.This study selected 70
mature P.ussuriensis varieties from wild, domesticated and cultivated species from different
geographical locations to better understand the distribution characteristics of materialelement
content and the differences between the different varieties. Targeting four main mineral elements
and eight trace mineral elements contained in P. ussuriensis, the differences in the mineral
contents of the peel and pulp of different varieties of P. ussuriensis were analyzed, compared,
classified using modern microwave digestion ICP-MS. The purpose of this study was to
determine the content characteristics and regional distribution of mineral elements in different
varieties of P. ussuriensis and screen out the resources with high K, Ca and P content. These
research results provide insights into maximizing the nutritional value of P. ussuriensis and
contribute to the further development and utilization of P. ussuriensis resources in different
regions. The results also provide valuable reference materials for the in-depth study of the
molecular construction mechanism of mineral elements in plant cell structure, and the
interactions and functions of various mineral elements.
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Materials & Methods
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Sample collection and preparation
This study was carried out in the Fruit Tree Institute of Jilin Academy ofAgricultural Sciences in
2021. There were 70 pear varieties tested, including 39 domesticated varieties, 19 cultivated
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varieties and 12 wild varieties(see Table 1). The sample fruit was collected from the China Cold
Region Fruit Germplasm Resource Nursery (Gongzhuling City, Jilin Province) . at the mature
stage (based on the blackening of the seed color). The trees were grown in a flat plot under
standardized management, and the fruit was not bagged. A total of 20 pears were randomly
picked from the same height around the crown of five 10-year-old trees of each variety, and 10
pears of similar same shape size and color were selected for analysis from the 20 sample fruits
from each variety. These fruits were then immediately sent to the laboratory under fresh-keeping
conditions where they were cleaned and dried.The peel and pulp of the pears were made into
uniform paddles by the uniform paddling machine. The plastic sealed bags were separately
packed and stored in cold storage for testing. Three replicates were made from each sample.
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Zn, Cd and Pb (100 mg·L-1) was provided by the National Institute of Nonferrous Metals and
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Electronic Materials, and was prepared to the required concentration when used. The lithium
(Li), cobalt (Co), indium (In), uranium (U) mixed mass spectrometry tuning solution (10mg·L-
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1), rhodium (Rh) element internal standard solution (1000 mg·L-1), nitric acid and hydrogen
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peroxide were MOS level.
Inductively coupled plasma mass spectrometry was performed using the method outlined by
Yang Minghua et al. (2022). This method included: weighing 1.0-1.5g of the samples into the
microwave digestion inner tank, adding 5mL of nitric acid and 2mL of hydrogen peroxide,
covering and tightening the nut, and placing for 30min. After a protective sleeve was installed,
the samples were then pre-digested and placed symmetrically on the turntable, which was loaded
with the Classic method. The star/pause key was then pushed to start the digestion procedure (the
digestion reference conditions are shown in Table 2. After the digestion procedure was
completed, the instrument automatically entered the cooling process. When the indicated
temperature in the instrument chamber was less than 50°C, the digestive tube ware removed, and
the nut in the fume hood was slowly unscrewed to relieve pressure. The digestive solution was
then transferred with deionized water to a constant volume of 50ml, mixed well for testing, and
then the sample blank test was conducted
.
Instruments and reagents
The instruments used for samples preparation and analysis include: microwave digestion system
(MARS 6, USA), ICP-MS inductively coupled plasma mass spectrometer (ICAP Q, USA),
ultrapure water manufacturing system (Milli-Q, USA), and 6875D fully-automatic frozen grinder
(SPEX Sample Prep, USA). The glassware and polytetrafluoroethylene digestion tank used in the
experiment were soaked in 20% nitric acid for more than 24h, and washed with ultra-pure water
repeatedly, and then dried before use. The mixed standard of Na, Mg, Al, P, K, Ca, Cr, Fe, Cu,
Preparation of the standard curve
ICP-MS has a wide dynamic linear range, high sensitivity and fast analysis speed. First, the
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appropriate concentration range of the standard working curve was selected based on the
characteristic response value of 12 elements and the content conditions of the sample. Then,the
standard curve equation and linear correlation coefficient were obtained.ICP-MS was then used
to determine the content of the 12 elements in the samples (no less than 11 sample blank parallel)
processed simultaneously with an unknown sample. The method detection limit test was then
carried out, and the standard deviation of the response value of each element was calculated, The
instrument detection limit was calculated by dividing three times the standard deviation by the
slope of the standard curve. The detection limit of the method was calculated using the mass of
the sample and constant volume (results shown in Table 3).
Statistical analysis
The mean value (MEAN), standard deviation (SD) and coefficient of variation (CV) of the traits
were calculated using Microsoft Excel 2017 and were statistically analyzed using IBM SPSS
Statistics 22.0 software. The average amount of each element in different varieties of P.
ussuriensis was analyzed using single factor analysis of variance and a Duncan multiple
comparison at 5% and 1% significant levels. The correlation analysis used an equidistant Pearson
similarity analysis, and Origin 2021b was used to draw a heat map. A principal component
analysis was used for the factor analysis, defining the factors with a cumulative contribution rate
greater than 80% as principal components, and then using these principal components to evaluate
different P. ussuriensis resources. The clustering analysis was performed using the "analysisclassification-system clustering" process in the SPSS software, and the hierarchical clustering
diagram was drawn using the square Euclidean distance in the Origin 2021b software.
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Results
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Difference analysis of mineral element content
The content range, average values, standard deviations and coefficients of variation of the 12
mineral elements in the 70 P. ussuriensis varieties are listed in Table 4.The results showed that
the average content of different mineral elements in fruit followed the following order from
highest to lowest: K >P > Ca > Mg > Na > Al > Fe > Zn > Cu > Cr > Pb > Cd.K content
accounted for 79.10% of the total mineral element content in the fruit, which is about 10, 14 and
12 times of the content of three other major elements P, Mg and Ca,respectively. K, P, Mg and
Ca content in the fruit peel and pulp were much larger than the content of the eight trace
elements, which had a relative range between 0.47 and 4132.51.The correlation analysis of
variance showed that there were significant differences in the content order of K, P, Mg and Ca
in the peel and pulp, with the mineral content in the peel following a K > Ca > P > Mg order,
and the mineral content in the pulp following a K > P > Mg > Ca order. The content of trace
elements were Na, Al, Fe, Zn, Cu, Cr, Pb, Cd (in this order) and was not significantly different
between the fruit peel and the fruit pulp. The largest coefficient of variation in the peel was Na,
followed by Al and Cr, and the largest coefficient of variation in the pulp was Ca, followed by
Cd and Al. These results showed that the Na content in peel and the Ca content in the pulp of
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different varieties of P. ussuriensis varied greatly.
The mineral element content in the peel of different varieties of P. ussuriensis varied greatly, as
shown in Figure 1, with K content being the highest, and Cd content being the lowest of the
measured elements. Ca content in the different varieties of P. ussuriensis had the largest range,
from 43.01 to 833.40 mg/kg, The K content range was the smallest, ranging from 885.26 to
3209.00 mg/kg in different varieties. Of the 70 cultivated, domesticated and wild varieties
included in the study, the P. ussuriensis varieties with the highest Ca content were 'YY351L'
(638.50 mg/kg), ‘LHHXSL’ (833.40 mg/kg) and 'ZLTSL-3' (318.54 mg/kg). The P. ussuriensis
varieties with the lowest Ca content were ‘DXS’ (43.01 mg/kg), ‘MTHL’ (64.05 mg/kg) and
'SSHMSL' (92.60 mg/kg). The varieties of P. ussuriensis with the highest K content were
‘HJQL’ (3,099.00 mg/kg), ‘HLL’ (2,643.00 mg/kg) and ‘SSDL’ (3,209.00 mg/kg), and the
varieties with the lowest K content were ‘YY1L’ (886.23 mg/kg), ‘XCZL’ (885.26 mg/kg) and
‘SSTESL’ (1,892.00 mg/kg).
Levels of the different mineral elements in the pulp of P. ussuriensis varieties are shown in
Figure 2, with K having the highest content and Cd having the lowest content. Al had the largest
content range between varieties, ranging from 0.62-15.13 mg/kg. The Mg content range was the
smallest, from 41.40-126.95 mg/kg. Of the 70 tested P. ussuriensis varieties, the varieties with
the highest Al content were 'QYL' (15.13 mg/kg), ‘QHL’ (7.60 mg/kg) and 'ZLTSL-2' (5.53
mg/kg), and the varieties with the lowest Al content were ‘FX’ (0.78 mg/kg),‘XPXL’ (0.62
mg/kg) and 'DNSL' (1.44 mg/kg). The P. ussuriensis varieties with the highest Mg content were
'NGL' (124.39 mg/kg), ‘YBDJBL’ (126.95 mg/kg) and ‘SWSL’ (125.70 mg/kg), and the
varieties of P. ussuriensis with the lowest Mg content were 'CXL' (45.00 mg/kg). ‘HXL’ (41.40
mg/kg) and 'ZLTSL-2' (54.60 mg/kg).
The 70 varieties of P. ussuriensis tested in this study were then classified into wild, cultivated or
domesticated varieties. The results showed that although the Na and Cu content in the wild
varieties were not significantly different from the Na and Cu content in the cultivated varieties,
Mg, P, K, Fe, Cr and Zn content in the wild varieties were significantly higher than in the
cultivated and domesticated varieties. Ca content in the cultivated varieties was significantly
higher than in the wild and domesticated varieties. These results show that the domesticated
varieties have no clear advantages in mineral element content. The comprehensive analysis of the
mineral element content in the peel and pulp of these P. ussuriensis varieties found that 'ZLTSL3' had the highest mineral element content, followed by ‘SSKWSL’ and ‘SSHMSL,’ and that
overall, the levels of mineral elements in fruits of wild varieties was higher than in the fruit of
the cultivated and domesticated varieties.
Correlation analysis of mineral element content
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Heat maps have been widely used to analyze, the nutritional quality of fruit in recent years
because this method intuitively shows experimental results with a gradual blue-red band. The
analysis results of this study show that there were significant or extremely significant
correlations between mineral elements in the tested fruits. This indicates that there is a complex
interaction between the cations of these mineral elements and the amino acid residues of plant
cell protease, the phosphate and carboxylate anions both inside and outside the cell membrane,
and the electrolytes of these mineral elements. These interactions are likely also impacted by
factors such as growth environment and growth stage.
Figure 3 shows that among the 12 mineral elements contained in the fruit peels of the 70 P.
ussuriensis varieties, there were 33 pairs with an extremely significant positive correlation
(P<0.01). The Na contained in the peel had a very significant positive correlation with P and Cr,
and their correlation coefficients were 0.541 and 0.514, respectively. Mg, P, Cr, Fe, Cu and
other, elements contained in the fruit peel samples were significantly positively correlated with
each
other, with correlation coefficients above 0.5. The correlation between P and Cr was relatively
high, with a correlation coefficient of 0.729, indicating that the varieties with high P content in
the peel were also likely to have high Cr content in the peel. Al was positively correlated with Fe
and Zn, with correlation coefficients of 0.689 and 0.722, respectively. There was also a very
significant positive correlation between P and K, P and Zn, K and Cu, K and Zn and Cu and Zn,
with correlation coefficients of 0.702, 0.625, 0.517, 0.544, 0.563, respectively. Na and Fe, Na
and K, Mg and Al, Mg and Cd, Al and Pb, Zn and Cd were all significantly positively
correlated. Although there was a very significant positive correlation between Na and Mg, Na
and K, Cu and Zn, Mg and Al, Ca and Zn, Al and P, Cr and Cu, K and Cr, and K and Fe, their
correlation coefficients were relatively low. There was a significant negative correlation between
K and Pb, with a correlation coefficient of -0.301, This abnormal result shows that in P.
ussuriensis varieties with high levels of K, the Pb content was relatively low.
Figure 4 shows that among the 12 mineral elements studied in the fruit pulp of P. ussuriensis
varieties, there were 30 pairs (P<0.01) with extremely significant positive correlations, Na and
Al, Na and Cr, Mg and K, Mg and Zn, Zn and Pb, had extremely significant positive
correlations, with correlation coefficients of 0.662, 0.573, 0.517, 0.593 and 0.620, respectively.
In addition, P had significant positive correlations with K, Fe, Cu and Zn. . The correlation
between P and K was high, with a correlation coefficient of 0.760, indicating that varieties with a
high P content in fruit pulp also had high K content. Although there was a significant positive
correlation between Al and Ca, P and Cr, and Cu and Pb, their correlation coefficients were
relatively low. Differing from the fruit peel results, there was no significant negative correlation
between any mineral elements in the fruit pulp.
Factor analysis of mineral element content in P. ussuriensis
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After standardization, this study conducted a factor analysis of the 12 mineral elements in 70
varieties of P. ussuriensis, mainly extracting common factors through a principal component
analysis. The results found in Table 5, show that the cumulative variance contribution rate of the
first five common factors selected in this study was greater than 80% of the total detected
elements, indicating that these 12 mineral Elements accounted for the majority of the variance
between these varieties.
Among the relevant factors of the fruit peel shown in Table 5, F1 included six mineral elements:
Na(factor load: 0.843), P (factor load: 0.768), Cu (factor load: 0.744), Cr (factor load: 0.671), K
(factor load: 0.620), and Mg (factor load: 0.565). F2 mainly contained three mineral elements: Al
(factor load: 0.889), Fe (factor load: 0.796) and Zn (factor load: 0.773). F3, F4 and F5 were
respectively related to Ca (factor load: 0.934), Pb (factor load: 0.953) and Cd (factor load:
0.969), Because the factor load was positive and in positive distribution, the higher the factor
score, the higher the corresponding mineral element content.Since the contribution of more than
50% of the total variance came from the sum of the two main factors, F1 and F2, the nine
elements, Na, P, Cu, Cr, K, Mg, Al, Fe and Zn, can be defined as the characteristic mineral
elements of the P. ussuriensis fruit peel.
Among the relevant factor of the fruit pulp shown in Table 5, F1 included four mineral elements:
Cu (factor load: 0.872), P (factor load: 0.836), K (factor load: 0.787) and Zn (factor load: 0.669).
F2 mainly contained three mineral elements: Na (factor load 0.836), Cr (factor load 0.817) and
Al (factor load 0.794). F3, F4 and F5 are respectively related to Ca (factor load 0.922), Mg
(factor load 0.679), Cd (factor load 0.920), Fe (factor load 0.787) and Pb (factor load -0.587),
respectively. The factor load of Pb was negative, meaning the higher the Pb factor score, the
lower the corresponding Pb content. Because more than 50% of the total variance come from the
sum of the two main factors, F1 and F2, the seven elements, Cu, P, K, Zn, Na, Cr and Al can be
defined as the characteristic mineral elements of P. ussuriensis fruit pulp.
Using the ratio of variance contribution rate of each principal component to cumulative variance
contribution rate as the weight, this study established a comprehensive score modeld as follows:
Peel: Zi = 0.4317Z (i, 1) + 0.1344Z (i, 2) + 0.1201Z (i, 3) + 0.0813Z (i, 4) + 0.0526Z (i, 5);
Pulp: Zi = 0.3685Z (i, 1) + 0.1724Z (i, 2) + 0.1154Z (i, 3) + 0.0877Z (i, 4) + 0.0714Z (i, 5)
Where, Zi is the comprehensive score; Z (i, 1), Z (i, 2), Z (i, 3), Z (i, 4), Z (i, 5) are the score of
the five principal components; and i is the identifying number of the P.ussuriensis variety (170)..
This formula can be used to calculate the sequence rule of the comprehensive component and
content of mineral elements in P. ussuriensis fruit,which is helpful in identifying the most
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nutritious varieties. The higher the comprehensive component scores, the better the
comprehensive mineral nutrition of the selected P. ussuriensis fruit variety (.see Table 6 for more
detailed results of different varieties). Among the 70 varieties of pears studied, the 15 varieties
with the highest mineral element content in the peel were: 'SPL', 'FX', 'PDX-1', 'SSDL',
'HJQL' , 'LXL' , 'SSHMSL' , 'PDX-2' , 'QYL' , 'SWSL' , 'ZLTSL-2' , 'JXL' , 'ZLTSL-1' ,
'ZLTSL-3' and 'HEBSL-1' . The 15 fruit varieties with the highest mineral element content in
the pulp were: 'HTL', 'SWSL', 'DXS', 'DML', 'JTL', 'ZLTSL-2', 'SSHMSL', 'CXL', 'LDL',
'YBDJBL', 'ZLTSL-3' ,'HEBSL-2', 'QHL', 'JBL' and 'QYL' . According to the comprehensive
data of the mineral element content in both the fruit peel and pulp, the best P. ussuriensis
varieties were 'SSHMSL', 'QYL', 'SWSL' and 'ZLTSL-3'.
Cluster analysis of mineral elements
In this experiment, the mineral element content of the fruit peel and pulp of 70 P. ussuriensis
varieties were analyzed by cluster analysis (results are shown in Figure 5). According to the
content characteristics of the main mineral elements in the fruit peel, these 70 varieties of P.
ussuriensis were divided into the following three categories:
Class I: Varieties with high Na, Mg, Al, P, K, Fe and Zn content in the peel. Nine
varieties fell into this category, including ‘HLL,’ ‘HJQL’ and ‘SSDL.’ Na, Mg, Al, P, K, Fe and
Zn content in these varieties were very high, with average values of 15.99 mg/kg, 193.58 mg/kg,
13.44 mg/kg, 293.96 mg/kg, 2882.81 mg/kg, 3.00 mg/kg and 2.16 mg/kg, respectively.
Class II: Varieties with high Ca content in the peel. A total of 26 varieties fell into this category,
including ‘DXL,’ ‘PDXL’ and ‘QPCL.’ The Ca content in these varieties was very high, with an
average value of 257.35 mg/kg.
Class III: Varieties with medium element content in the peel. Half (35) of the varieties such fit
into this category including ' SPL ', 'XPXL' and 'WXL', The Na, Mg, Al, P, K, Fe and Zn content
in these varietieswere higher than in the varieties in Class II,.with average values of 9.59 mg/kg,
165.97 mg/kg, 10.43 mg/kg, 193.49 mg/kg, 1992.96 mg/kg, 8.95 mg/kg and 1.50 mg/kg,
respectively. The Ca content was also significantly lower, with an average of 196.97 mg/kg.
There were no significant differences in Cr, Cu, Cd and Pb content in the fruit peel between the
three groups. Average Cr levels in the three groups were 0.40 mg/kg, 0.16 mg/kg and 0.21
mg/kg,
respectively, average Cu levels were 1.97 mg/kg, 1.06 mg/kg and 1.28 mg/kg,respectively.
average Cd levels were 0.0073 mg/kg, 0.0050 mg/kg and 0.0052 mg/kg, respectively, and the
average Pb levels were 0.0323 mg/kg, 0.0484 mg/kg and 0.0435 mg/kg, respectively.
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Similarly, according to the content characteristics of the main mineral elements of the fruit pulp
these 70 varieties of P. ussuriensis were divided into three categories (results are shown in
Figure 6):.
Class I: Varieties with high Mg, P and K content in the pulp. Nine varieties fell into this
category, including ‘HHGL,’ ‘JBL’ and ‘SWSL.’ Mg, P and K content in these varieties were
very high, with average values of 97.51 mg/kg, 221.84 mg/kg and 2634.31 respectively.
Class II: Varieties with low levels of all mineral content in the pulp. This category consisted of
24 varieties, including ' DXL', 'HXL' and 'FX'.
Class III: Varieties with high Na, Al and Ca content in the pulp.A total of 37 varieties fell into
this category, including 'SPL', 'ML' and 'BLL'. The average Na, Al and Ca content in these
varieties were 6.12 mg/kg, 4.67 mg/kg and 72.72 mg/kg, respectively.
There were no significant differences in Cr, Fe, Cu, Zn, Cd and Pb levels in the fruit pulp
between the three groups. The average Cr content in the three groups was 0.11 mg/kg, 0.10
mg/kg and 0.10 mg/kg, respectively; the average Fe content was 4.20 mg/kg, 3.91 mg/kg and
5.51 mg/kg, respectively; the average Cu content was 0.60 mg/kg, 0.43 mg/kg and 0.83 mg/kg,
respectively; the average Zn content was 0.71 mg/kg, 0.56 mg/kg and 0.96 mg/kg, respectively;
the average Cd content was 0.0038 mg/kg, 0.0031 mg/kg and 0.0058 mg/kg,respectively; and
the average Pb content was 0.0223 mg/kg, 0.0210 mg/kg and 0.0280 mg/kg, respectively.
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Discussion
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Mineral element content of P. ussuriensis fruit
Mineral element content plays an important role in judging fruit quality and in maintaining the
normal physiological activities of the human body (Bai et al., 2021; Huang et al., 2022; Cao et
al., 2018; Choi et al., 2013; Renna et al., 2018). The coverage and sample size of previous P.
ussuriensis research samples are relatively small (Zhang et al., 2022a).
The results of this study showed that the main element in all 70 P. ussuriensis ruits tested was K
accounting for 79.10% of the total amount of 12 mineral elements studied. The average K
content was about 10, 14 and 12 times that of the other three major elements P, Mg and Ca,
respectively. Because its mobility is less bound by neighboring anions, the mobility of
monovalent K cations in plants is significantly higher than that of divalent Ca and Mg cations.
Moreover, in fruit cell tissue, it is easier to concentrate on the most active and necessary
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biochemical activities (Dróądą et al., 2018). Previously, Wei et al. (2019) found that the K ion
content of the ‘Korla fragrant pear’ and ‘Xinli 6’ were 1190 mg/kg and 960 mg/kg respectively,
and Shi et al. (2022) found that the K ion content of the ‘Huangguan pear’ was 1239.06 mg/kg.
The experimental results of this study showed for the first time that the average content of K ions
in the peel and pulp of P. ussuriensis was 1838.06 mg/kg and 1615.81 mg/kg, respectively,
indicating that P. ussuriensis is a very valuable, high-K fruit, as the K content values were far
higher than in the pear varieties previously studied.
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Calcium ion content was the second largest, followed by phosphorus and magnesium ion. In the
fruit pulp, the P content was second only to K content, followed by Mg and Ca content, which is
consistent with the research results and conclusions of Aizezi et al. (2018) on the ‘Korla fragrant
pear.’ Ca content was significantly higher in the peel than in the pulp. The ionic radius of Ca is
larger than that of magnesium, which leads to greater flow resistance in the phloem, so it has
lower fluidity than magnesium (Karley et al., 2009). Bivalent Ca ions with larger ionic radii are
more conducive to the formation of dense and stable calcium phosphate, calcium carboxylate
epidermal plant fiber and phloem protein bone structure with the amino acid residues in the cell
structure of the peel and the dense cell membrane carboxylate, which also causes the Ca content
to be significantly higher than P and Mg content (Zhang XN et al., 2022a, 2022b). Eating pears
without peeling the fruit first can help supplement the Ca content required by the human body
and help maintain the health of both bones and muscles.
Although the contents of trace elements in P. ussuriensis were far lower than the main elements,
they play important roles in the quality of the pear fruit, a conclusion that has also been reached
in the studies of other fruits and vegetables (Kuang et al., 2022; Liu et al., 2010; Liu et al., 2017;
Sardar et al., 2022). Although Wang et al. (2021) and Zhang et al. (2022b) found that the trace
elements in the ‘Laiyang Pear’ and ‘Hongzaosu Pear’ followed a Fe > Cu > Zn content pattern,
the results of this study showed that the trace elements in P. ussuriensis fruit followed a Fe >
Zn > Cu content pattern. This difference in the sequence of trace element content may be related
to factors such as variety, tree age, cultivation conditions, and also to the impact of the natural
environment such as the soil conditions and climate where the fruit trees grow (Fan et al., 2012).
P. ussuriensis varieties should be selected based on market demand and the conditions of the
growing area. The application of calcium and trace element fertilizers should, which can improve
the nutritional quality pears, should also be considered.
The results showed that the content of mineral elements in the peel and pulp of P. ussuriensis
changed in different degrees during their growth, showing rich genetic diversity. In the peel , the
largest coefficient of variation was Na, and the smallest coefficient of variation was K . The
relevant coefficient of variation ranged from 29.81% to 77.13%. The highest coefficient of
variation in pulp was Ca , and the smallest coefficient of variation was Mg. The relevant
coefficient of variation ranged from 28.03% to 67.66%. These results showed that the
germplasm resources of P. ussuriensis had great potential for improvement in mineral element
content, and the rich genetic background could provide sufficient parent materials for the
breeding of improved varieties.
Correlation of mineral elements in different parts of P. ussuriensis fruit
There is a correlation between the content of mineral elements in peel and pulp. Factor
analysis is an effective grouping method based on the correlation between variables that can
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show the high correlation between the same group of varieties. Although most mineral elements
in fruits have positive correlations and synergistic effects, a few of them also have negative
correlations and antagonistic effects (Reddy et al., 2020; Yao et al., 2017). This study also found
this phenomenon. For example, Duan et al. (2013) analyzed the mineral quality of 30 pear
resource fruits and found that K and Ca were significantly positively correlated in fruits, while
this study unexpectedly found that K and Ca were negatively correlated. This abnormal finding
may be due to the types of fruits tested, the content of mineral elements in the soil of the
sampling area, or the different sampling periods between the two. We also found that there was a
significant negative correlation between K and Pb content in the fruit of P. ussuriensis, with a
correlation coefficient of -0.301. This finding is also contrary to previous research results and
conclusions (conventional positive correlation). This abnormal result shows that the Pb content
in high-K P. ussuriensis varieties is relatively low, indicating that potassium fertilizer
application on fruit trees may help reduce harmful lead content in fruit.
There was a significant positive correlation between P and K, P and Cu, and P and Zn in the peel
and pulp of the P. ussuriensis varieties tested, with correlation coefficients above 0.5. This is
consistent with the research results of Kuang et al. (2017) on apples and Guo et al. (2019) on
pears. This phenomenon may be related to the synergistic absorption of mineral elements in
fruits, as most mineral elements form plant cell structures through chemical or ionic bonds, and
promote the biochemical reaction of plant growth through mutual balance and mutual restriction,
giving them specific physiological functions.
A factor analysis showed that the key factor affecting the evaluation of mineral elements in the
fruit of P. ussuriensis was the content of Cu, P, K, Zn, Na and Cr. These research results should
provide a valuable reference for the screening and identification of mineral elements in different
varieties of P. ussuriensis. Previous research results show that the peel is rich in Ca and P,
which are essential nutrient for the human body (Yoshimizu et al., 2015; Ayache et al., 2015),
and should be more widely used in food production. These mineral elements can be used as
nutritional fortifiers in food additives to supplement and strengthen certain foods (Liu et al.,
2020; Sun et al., 2016), and as tools to regulate and improve food quality. For example,
phosphorus can be used as an acidifier in soft drinks (Li et al., 2015), and calcium can be added
to improve the hardness of canned vegetables (She et al., 2014). P. ussuriensis varieties with
high calcium and phosphorus content play very important roles in improving the nutritional
value of food and protecting human health, and are a great source for research in balancing
nutrients in fruits. Although the content of mineral elements in fruit is small, these mineral
elements actively participate in many important functions in the human body. Future orchard
management plans can include fertilization adjustments based on local conditions and the
absorptio characteristics
of mineral elements in different autumn pear germplasms.
Cluster analysis of P. ussuriensis fruit
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In recent years, more researchers have used a cluster analysis to analyze the quality of fruit with
multiple samples and indicators (Qin et al., 2012; Yazdanpour et al., 2018). This method has
been widely used in the comprehensive evaluation of the quality of plum, peach, grape, apple
orange, as well as other fruits (Nie et al., 2000). In the cluster analysis of P. ussuriensis, the peel
and pulp of 70 P. ussuriensis varieties were divided into three categories according to mineral
element content. For example, in the correlation analysis of 'ZLTSL-1' , the high K and Fe
content in the peel placed this variety in Class I of peel groupings, and the high P, Cu and Zn
content in the pulp placed this variety into. Class II of pulp groupings. In the correlation analysis
of 'HJQ' the high Na, Mg, P and K content in the peel placed this variety into class I of peel
groupings, and the high Cr content in the pulp placed this variety into Class III of pulp
groupings.
Among the three peel groupings, Class I and Class III included wild cultivated and domesticated
varieties, but Class II included only domesticated varieties. Among the three pulp groupings,
Class I included both Iwild and domesticated varieties, Class II included both cultivated and
domesticated varieties, and Class III included wild, cultivated and domesticated varieties.These
results show that while domesticated varieties improve upon some traits of cultivated varieties,
such as disease resistance, they do not have significant advantages in mineral elements content
levels. Our comprehensive analysis of the mineral elements of P. ussuriensis fruits found that
there were significant differences in Mg, P, K, Na and Ca content between the peel and the pulp.
Future research can use molecular biology methods to carry out gene mapping and gene mining
of P. ussuriensis fruit for germplasm resources, aiming for polymorphism of these mineral
elements in the fruit, to provide a more in-depth basis for the future genetic improvement of the
mineral elements in P.Ussuriensis resources.
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Conclusions
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Our research results of P. ussuriensis show that content levels of the 12 mineral elements
measured were K > P > Ca > Mg > Na > Al > Fe > Zn > Cu > Cr > Pb > Cd, from high to low, in
which K and P were the most abundant mineral elements. Different varieties of P. ussuriensis
can be divided into three categories according to the mineral content of in peel: (1) varieties
with high Na, Mg, P, K, Fe and Zn content, (2) varieties with high Ca content, and (3) varieties
with medium element content. Different varieties of P. ussuriensis can also be divided into three
categories according to the mineral content of the pulp: (1) varieties with high Mg, P and K
content, (2) varieties with low element content, and (3) varieties with high Na, Ca content.
The comprehensive analysis results found that 'SSHMSL', 'QYL', 'SWSL' and 'ZLTSL-3' are
the P. ussuriensis varieties with the highest content of mineral elements. The study also found
that the application of potassium fertilizer can help reduce harmful lead content in fruit. In
addition, ingesting the peel when eating pears is important for supplementing the calcium needed
by the human body. These research results are important for the future selection, production and
breeding of P. ussuriensis fruits.
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Acknowledgements
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We would like to thank the National Cold Area Fruit Germplasm Resource Nursery (Gong
Zhuling City) for providing the fruit tested in this study.
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Figure 1
Content of mineral elements in the peel of Pyrus ussuriensis (Maxim.)
The vertical axis is the 70 P. ussuriensis varieties, the horizontal axis is the total content of
mineral elements in the peel of each variety, and the different colors of the columnar
superposition graph are the total content of each mineral element.
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Figure 2
Content of mineral elements in the pulp of Pyrus ussuriensis Maxim.
The vertical axis is 70 P.ussuriensis resources, the horizontal axis is the total content of
mineral elements in the pulp of each resource, and the different colors of the columnar
superposition graph are the content of mineral elements of each element.
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Figure 3
Correlation of mineral elements in the peel of Pyrus ussuriensis (Maxim.)
Heat map of the content of 12 mineral elements in the peel, analyzed by correlation, and
drawn using Origin 2021b software. The red and blue in the heat map indicate the degree of
association between mineral elements, with red representing a positive correlation and blue
representing a negative correlation. There are 33 pairs with extremely significant positive
correlations (P<0.01), and 8 pairs with significant positive correlations (P<0.05).
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Figure 4
Correlation of mineral elements in the pulp of Pyrus ussuriensis (Maxim.)
Heat map of the content of 12 mineral elements in the pulp, analyzed by correlation, and
drawn using Origin 2021b software. The red and blue in the heat map indicate the degree of
association between mineral elements, with red representing a positive correlation and blue
representing a negative correlation. There are 30 pairs with extremely significant positive
correlations (P<0.01), and 4 pairs with significant positive correlations (P<0.05).
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Figure 5
Cluster analysis of the fruit peel of 70 Pyrus ussuriensis (Maxim.) varieties
The hierarchical clustering diagram, drawn using the Origin 2021b software. According to the
content of mineral elements in the fruit peel, the 70 P. ussuriensis varieties were divided into
3 groups. Different colors are used to represent different groups. The first group includes 9
varieties and is marked in green, the second group includes 26 varieties and is marked in
blue, and the third group includes 35 varieties and is marked in red.
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Figure 6
Cluster analysis of the fruit pulp of 70 Pyrus ussuriensis (Maxim.) varieties
The hierarchical clustering diagram, drawn using the Origin 2021b software. According to the
content of mineral elements in the fruit pulp, the 70 P. ussuriensis varieties were divided into
3 groups. Different colors are used to represent different groups. The first group includes 9
varieties and is marked in green, the second group includes 24 varieties and is marked in
blue, and the third group includes 37 varieties and is marked in red.
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Table 1(on next page)
Basic information of Pyrus ussuriensis (Maxim.) varieties
The 70 samples we studied were selected according to the main production areas of Pyrus
ussuriensis (Maxim.) in northeast China. The geographical locations of the selected samples
include 5 from Inner Mongolia, 28 from Heilongjiang, 34 from Jilin and 3 from Liaoning.
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1
Table 1 Basic information of Pyrus ussuriensis Maxim.
Code
Name
Type
Code
Name
P1
SPL
Domesticated varieties
P25
FXL
P2
DXL
Domesticated varieties
P26
P3
XPXL
Domesticated varieties
P4
WXL
P5
Type
Code
Name
Domesticated varieties
P49
CXL
Cultivated varieties
QXSL
Domesticated varieties
P50
WX
Cultivated varieties
P27
QPHL
Domesticated varieties
P51
NGL
Cultivated varieties
Domesticated varieties
P28
QHL
Domesticated varieties
P52
XXSL
Cultivated varieties
PDXL
Domesticated varieties
P29
YBDJBL
Domesticated varieties
P53
DN5L
Cultivated varieties
P6
PDX-1
Domesticated varieties
P30
HLJHGL
Domesticated varieties
P54
ZXSL
Cultivated varieties
P7
PDX-2
Domesticated varieties
P31
TL
Domesticated varieties
P55
JXSL
Cultivated varieties
P8
HGL
Domesticated varieties
P32
HPXL
Domesticated varieties
P56
DML
Cultivated varieties
P9
HLL
Domesticated varieties
P33
BLXL
Domesticated varieties
P57
TXL
Cultivated varieties
P10
ML
Domesticated varieties
P34
MTHL
Domesticated varieties
P58
JTL
Cultivated varieties
P11
QPCL
Domesticated varieties
P35
AL
Domesticated varieties
P59
SSDL
Wild resources
P12
HXL
Domesticated varieties
P36
HTL
Domesticated varieties
P60
SSHMSL
Wild resources
P13
LHHXSL
Domesticated varieties
P37
TXDYXL
Domesticated varieties
P61
SSTESL
Wild resources
P14
HHGL
Domesticated varieties
P38
SL
Domesticated varieties
P62
SSKWSL
Wild resources
P15
XCZL
Domesticated varieties
P39
DWBKSL
Domesticated varieties
P63
DNSL
Wild resources
P16
CBZL
Domesticated varieties
P40
DNGL
Cultivated varieties
P64
HLSL
Wild resources
P17
BLL
Domesticated varieties
P41
FX
Cultivated varieties
P65
SWSL
Wild resources
P18
LDL
Domesticated varieties
P42
YY351L
Cultivated varieties
P66
ZLTSL-1
Wild resources
P19
HMXSX
Domesticated varieties
P43
QYL
Cultivated varieties
P67
ZLTSL-2
Wild resources
P20
SLGZL
Domesticated varieties
P44
JXL
Cultivated varieties
P68
ZLTSL-3
Wild resources
P21
JBL
Domesticated varieties
P45
HJQL
Cultivated varieties
P69
HEBSL-1
Wild resources
P22
XHTL
Domesticated varieties
P46
LXL
Cultivated varieties
P70
HEBSL-2
Wild resources
P23
DXSL
Domesticated varieties
P47
DXS
Cultivated varieties
P24
YHL
Domesticated varieties
P48
YY1L
Cultivated varieties
2
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Type
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Table 2(on next page)
Content of mineral elements in the pulp of Pyrus ussuriensis (Maxim.)
The vertical axis is the 70 P.ussuriensis varieties, the horizontal axis is the total content of
mineral elements in the pulp of each variety, and the different colors of the columnar
superposition graph are the total content of each mineral element.
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1
Table 2. Reference parameters for microwave-assisted digestion
Step
Power/w
Percentage/%
Heating-up time/min
Controlled temperature/℃
Duration/min
1
600
100
8
100
5
2
600
100
5
150
5
3
600
100
8
190
15
2
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Table 3(on next page)
Linear regression equations and correlation coefficients of the 12 mineral elements
The appropriate concentration range of the standard working curve was selected based on
the characteristic response value of 12 elements and the content conditions of the sample.
Then, the standard curve equation and linear correlation coefficient were obtained.
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Table 3.3 regression ee and correlation coefficients of 12 mineral elements
E
L
Range
Standard Curve E
Correlation Coefficient
Method Detection Limits
Na
0～2000
f(x)=7738.4555*x
R=0.9998
2500
Mg
0～2000
f(x)=3922.2351*x
R=0.9998
2500
Al
0～2000
f(x)=1817.0022*x
R=0.9999
1000
P
0～2000
f(x)=185.2859*x
R=0.9981
1000
K
0～2000
f(x)=8389.8404*x
R=0.9900
2500
Ca
0～2000
f(x)=427.4738*x
R=0.9879
2500
Cr
0～500
f(x)=27376.5899*x
R=0.9998
25
Fe
0～2000
f(x)=354.2781*x
R=0.9981
500
Cu
0～1000
f(x)=4068.2915*x
R=0.9999
100
Zn
0～2000
f(x)=959.5661*x
R=0.9999
500
Cd
0～200
f(x)=1592.8765*x
R=0.9999
2.5
Pb
0～500
f(x)=13304.5313*x
R=0.9999
10
2
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Table 4(on next page)
Difference in mineral element content between the peel and the pulp of 70 Pyrus
ussuriensis (Maxim.) varieties (n=3, mg/kg)
The mean value (MEAN), standard deviation (SD), range of variation (Range) and coefficient
of variation (CV) of the 12 elements were calculated. The average content of each element in
different varieties of P. ussuriensis was analyzed by single factor analysis of variance and
Duncan multiple comparison at 5% and 1% significant levels.
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Table 4 Difference of mineral element content between ss and flesf of 7 PP ussuriensis Maxim.（nn，mg/sm）
Peel
2
>
Pulp
CC（%）
SD
AA !"#
Range
CC（%）
Average
Range
39K
1838.06Aa
885.268&'()*((
547.9
29.81
1615.81Aa
626.008&(6)*((
543.39
33.63
44Ca
226.422+
43.018,&&*-(
132.26
58.41
63.51Cd
23.508&.&*).
42.97
67.66
31P
188.75Cc
73.058&,,*-(
65.61
34.76
141.592+
53.648&5.*)(
57.53
40.63
24Mg
S$
166.90Cc
60.788',/*((
54.14
32.44
78.23Cc
41.4085'.*)/
21.93
28.03
23Na
9.40Dd
4.678/5*-.
7.25
77.13
5.67De
2.4585-*'5
2.28
40.17
27Al
10.18Dd
3.118-6*&/
6.71
65.9
4.22De
0.6285/*5&
2.43
57.5
57Fe
9.23Dd
3.248'6*,&
5.06
54.83
4.27De
1.838,*/)
1.55
36.3
66Zn
1.44Dd
0.678-*&)
0.64
44.71
0.69De
0.2785*-.
0.26
38.13
63Cu
1.27Dd
0.388'*6,
0.55
42.96
0.57De
0.1285*'&
0.23
40.91
52Cr
0.22Dd
0.088(*-6
0.14
65.73
0.11De
0.048(*'(
0.03
28.23
208Pb
0.0435Dd
0.01408(*(,-5
0.0137
31.52
0.0226De
0.01108(*(/5'
0.0091
40.1
111Cd
0.0054Dd
0.00208(*(555
0.0022
41.09
0.0038De
0.00108(*(5.5
0.0023
59.36
Different lol0190 letters in the same column indicate significant differences at the 0.05 le:0;< and different cac=9; letters indicate significant differences
at the 0.01 le:0;?
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Table 5(on next page)
Factor analysis of mineral element content
The factor analysis of 12 mineral elements in the peel and pulp of 70 pear varieties, mainly
extracting common factors through principal component analysis. The results show that the
cumulative variance contribution rate of the first five common factors selected in this study
was greater than 80% of the total detected elements, indicating that these 12 mineral
elements accounted for the majority of the variance between these varieties.
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1
Table 5 Factor analysis of mineral element content
Peel
Pulp
Element
Element
F1
F2
F3
F4
F5
F1
Na
0.843
0.006
0.058
-0.016
-0.081
Cu
0.872
0.227
0.17
-0.148
-0.029
P
0.768
0.39
-0.032
-0.185
0.176
P
0.836
0.113
-0.026
0.194
0.34
Cu
0.744
0.371
0.11
0.056
0.097
K
0.787
0.007
0.031
0.308
0.152
Cr
0.671
0.42
0.35
-0.135
0.007
Zn
0.669
0.291
0.374
0.336
-0.224
K
0.62
0.293
-0.314
-0.375
0.313
Na
0.174
0.836
0.123
0.093
-0.035
Mg
0.565
0.376
0.561
@BDBFG
@BDHBI
Cr
@BDBJF
0.817
@BDHHJ
0.145
0.269
Al
0.114
0.889
0.025
0.197
0.068
Al
0.174
0.794
0.186
@BDHKH
@BDJMI
Fe
0.305
0.796
0.232
0.043
0.036
Ca
@BDBBG
0.174
0.922
0.02
@BDBKF
Zn
0.396
0.773
@BDHMH
@BDBMH
0.187
Mg
0.493
@BDHBH
0.679
0.249
0.119
Ca
0.028
0
0.934
0.043
0.078
Cd
0.197
0.053
0.122
0.92
0.038
Pb
@BDHBF
0.174
0.007
0.953
0.021
Fe
0.342
0.086
0.035
0.091
0.787
Cd
0.046
0.123
0.058
0.013
0.969
Pb
0.246
0.483
0.073
0.454
@BDFIN
VOQROTUW
43.173
13.444
12.006
8.132
5.257
VOQROTUW
36.852
17.236
11.537
8.772
7.135
36.852
54.088
65.624
74.396
81.531
contribution
contribution
(XY
(XY
CumulatiZW
43.173
56.617
68.623
76.755
82.011
CumulatiZW
ZOQROTUW
ZOQROTUW
contribution
contribution
(XY
(XY
2
[
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F2
F3
F4
F5
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Table 6(on next page)
Principal component scores and ranking of 70 Pyrus ussuriensis (Maxim.) varieties
Principal component score, calculated using the ratio of variance contribution rate of each
principal component to cumulative variance contribution rate as the weight. The higher the
comprehensive composition score, the better the comprehensive mineral nutrition of the
selected P. ussuriensis fruit variety.
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Table 6 Principal component scores and ranr\]^ of 70 Pyrus ussuriensis Maxim
Code
Peel
`abdghiji
Pul_
Ranqjbt
`koph
`abdghiji
Ranqjbt
`koph
P1
0.92
15
uvwxx
52
P2
0.02
26
uywx
70
P3
uvwzz
41
uywz{
68
P4
uvwx{
51
uywvz
63
P5
uvwzz
40
uvwyx
43
P6
1.1
13
uvw|x
57
P7
1.32
8
uvw}{
58
P8
0.25
24
uvw}}
60
P9
0.38
21
uvw~{
46
P10
uvwvx
30
uvw
55
P11
uvwy
44
uywv~
62
P12
0.34
22
uywyy
65
P13
0.22
25
uywv}
64
P14
uvwx{
50
0.61
17
P15
uyw~}
68
uvwxx
51
P16
uvw}
48
0.53
20
P17
uywvy
61
0.25
28
P18
uvw}
54
1.24
7
P19
uvwv
29
0.31
27
P20
uyw{{
69
uvw}
59
P21
uvwxy
49
1.92
2
P22
uywy}
66
uvwv
40
P23
uywvz
63
uvwv{
36
P24
uywy{
64
0.13
31
P25
uvwxx
53
uvwv{
35
P26
uvwyx
33
uvwyz
42
P27
uvw~
35
uvw{y
48
P28
uywy|
65
1.5
3
P29
uvwz
43
1.27
6
P30
uvwv}
31
uvwv
39
P31
uyw{
70
0.36
23
P32
uvwy}
34
0.22
29
P33
uvw|x
57
0.09
32
P34
uvw{}
38
uvwyz
41
P35
uvw}
58
uvwx
53
P36
uvwx
47
0.63
15
P37
uvwz
46
0.13
30
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P38
uvwz
45
uvwy|
44
P39
uvw}x
59
uvwzy
49
P40
uvw{
37
uyw{}
67
P41
0.92
14
uywzz
69
P42
0.81
16
uywvy
61
P43
1.41
7
1.93
1
P44
1.74
4
uyw{
66
P45
1.16
11
uvw~z
47
P46
1.19
10
uvwx}
54
P47
uyw~{
67
0.75
13
P48
uvwxz
52
uvwv
38
P49
uvwz
42
1.03
8
P50
uvw~~
36
uvw~
45
P51
uvwv{
28
0.38
22
P52
uvwzz
39
0.33
25
P53
uywv{
62
uvwv
37
P54
uvw|
55
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2
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Enrichment of inflammatory bowel disease and colorectal cancer risk variants in colon expression quantitative trait loci
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RESEARCH ARTICLE Open Access Open Access Hulur et al. BMC Genomics (2015) 16:138
DOI 10.1186/s12864-015-1292-z © 2015 Hulur et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Enrichment of inflammatory bowel disease and
colorectal cancer risk variants in colon expression
quantitative trait loci Imge Hulur1, Eric R Gamazon2,6, Andrew D Skol2, Rosa M Xicola4, Xavier Llor4, Kenan Onel3, Nathan A Ellis5
and Sonia S Kupfer2* Abstract Background: Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs)
associated with diseases of the colon including inflammatory bowel diseases (IBD) and colorectal cancer (CRC). However, the functional role of many of these SNPs is largely unknown and tissue-specific resources are lacking. Expression quantitative trait loci (eQTL) mapping identifies target genes of disease-associated SNPs. This study
provides a comprehensive eQTL map of distal colonic samples obtained from 40 healthy African Americans and
demonstrates their relevance for GWAS of colonic diseases. Results: 8.4 million imputed SNPs were tested for their associations with 16,252 expression probes representing
12,363 unique genes. 1,941 significant cis-eQTL, corresponding to 122 independent signals, were identified at a false
discovery rate (FDR) of 0.01. Overall, among colon cis-eQTL, there was significant enrichment for GWAS variants for
IBD (Crohn’s disease [CD] and ulcerative colitis [UC]) and CRC as well as type 2 diabetes and body mass index. ERAP2, ADCY3, INPP5E, UBA7, SFMBT1, NXPE1 and REXO2 were identified as target genes for IBD-associated variants. The CRC-associated eQTL rs3802842 was associated with the expression of C11orf93 (COLCA2). Enrichment of colon
eQTL near transcription start sites and for active histone marks was demonstrated, and eQTL with high population
differentiation were identified. Conclusions: Through the comprehensive study of eQTL in the human colon, this study identified novel target
genes for IBD- and CRC-associated genetic variants. Moreover, bioinformatic characterization of colon eQTL provides
a tissue-specific tool to improve understanding of biological differences in diseases between different ethnic
groups. Keywords: Expression quantitative trait loci, Colon, Gene expression, African Americans, Regulatory variation,
Transcriptomics, Inflammatory bowel disease, Colorectal cancer, Genomics, Genome-wide association studies * Correspondence: skupfer@medicine.bsd.uchicago.edu
2Department of Medicine, 900 East 57th Street, MB#9, Chicago, IL 60637, USA
Full list of author information is available at the end of the article Background [20-30]. As is the case for GWAS variants in general, a
number of these variants are located in gene deserts and
their functional roles in disease pathogenesis are largely
unknown [31]. Unraveling the functional basis of complex
diseases is a priority as this has implications for under-
standing disease pathogenesis as well as identifying novel
therapeutic targets [32]. Genetic susceptibility is thought to play a role in com-
mon diseases including those affecting the colon such as
inflammatory bowel diseases (IBD) and colorectal cancer
(CRC). Indeed, genome-wide association studies (GWAS),
conducted primarily in populations of European descent,
have identified single nucleotide polymorphisms (SNPs)
associated with IBD, including both ulcerative colitis (UC)
[1-6] and Crohn’s disease (CD) [7-19], as well as CRC Studying the genetics of gene expression is a tool that
can help elucidate the functional consequences of GWAS
variants. Expression quantitative trait loci (eQTL) map-
ping associates genome-wide SNPs with mRNA expres-
sion from the same individuals in a particular tissue to Hulur et al. BMC Genomics (2015) 16:138 Page 2 of 15 significant Hardy-Weinberg equilibrium (HWE) p-values
(n = 3,395, see Methods), non-autosomal SNPs (n = 46,486),
or mapped to the same position (n = 722) as another SNP. Imputation was performed using 1.492 million remaining
SNPs and the 1000 Genomes Project reference panel Phase
I integrated variant set release (v3), which provided a final
dataset of 8.4 million SNPs, after removing imputed SNPs
with MAF < 0.05 or low imputation quality (IMPUTE2-info
score < 0.5) (Figure 1B). Principal components analysis
(PCA) showed that subjects included in this study were
similar to individuals of African ancestry in Southwest USA
(ASW) HapMap population samples in terms of ancestry
(Additional file 1). The proportion of European and African
ancestry among the subjects were represented by the first
principal component (PC); PC1 was thus used as a covari-
ate in the eQTL analyses to control for global ancestry. identify regulatory variation [33]. Previous studies [34-38]
have detected eQTL in modest sample sizes (compared to
typical disease GWAS). These studies have provided im-
portant insights into the architecture of gene regulation in
general [39], as well as across populations [40,41] and tis-
sues [41-43]. Importantly, it has been shown that variants
identified by GWAS as reproducibly associated with com-
plex traits are enriched for eQTL in various cell types
[33,44,45]. Results In order to identify significant colon eQTL, the genotype
and expression data was first subjected to a number of
quality controls. Using the cleaned data, cis- and trans-
eQTL were identified. The overlap between colon eQTL
and disease-associated GWAS SNPs was performed as
well as the overlap with eQTL in other tissue types. As
only AA were used in this study, population differentiated
eQTL were also identified. Finally, using simulations, en-
richment of colon eQTL among IBD- and CRC-associated
variants and for histone marks was performed. Results for
these analyses are presented in this order in the sections
that follow. Identification of colon eQTL eQTL were identified by testing for an association be-
tween each SNP and each gene’s expression probe. The
distribution of eQTL p-values was compared against the
distribution expected by chance separately for cis- and
trans-eQTL. For cis-, but not trans-eQTL, we found a
significant enrichment for small p-values (Additional
file 4). We identified 1,941 cis-eQTL corresponding to
122 genes at a FDR of 0.01, which represent 122 inde-
pendent SNP-gene associations (Table 1). Cis-eQTL
were found to be highly enriched around transcription
start sites (TSS) with no discernible trend toward 3′ or
5′ (Additional file 5). Overlap of cis-eQTL with colonic disease-associated
GWAS variants The National Human Genome Research Institute (NHGRI)
GWAS catalog was searched to identify IBD (i.e. CD and/
or UC)- and CRC-associated GWAS variants that are also
significant cis-eQTL at FDR < 0.10 in the colon samples. Of 8,122 significant cis-eQTL at this threshold, 4 were
previously identified in GWAS of IBD (i.e. both CD and Background While eQTL mapping studies have been performed in
lymphoblastoid cell lines (LCLs) [36,37,40,42,46], liver
[35,47], adipose tissue [42], brain [48], skin [42,46] and
ileum [49], these tissue types may not be relevant for all
disease traits. A recent eQTL mapping study in the hu-
man ileum noted tissue-specific effects as well as enrich-
ment for IBD susceptibility variants [49]. More recently,
Closa et al. conducted an eQTL analysis of CRC loci in
colonic mucosa and found significant cis-eQTL in three
loci [50]. However, comprehensive genome-wide eQTL
mapping has not previously been performed in the
human colon, which is the relevant tissue for colonic
diseases like IBD and CRC. In gene expression analysis, 47,231 probes were pro-
filed. A total of 16,252 probes remained for analysis
after removing probes that mapped to more than one
genomic location (n = 19,278), that contained one or
more SNP (n = 2,343), or that was not expressed in one
or more subjects (n = 9,358). Heat map visualization
and hierarchical clustering of the gene expression data
suggested the removal of eight outliers, leaving 40 indi-
viduals (Additional file 2). Inclusion of the first five
PCs of the gene expression data yielded the maximal
number of significant cis-eQTL probes at various false
discovery rate (FDR) thresholds (Additional file 3). These expression-based PCs were included as covariates
in subsequent eQTL analysis to correct for unmeasured
yet systematic variation in gene expression levels. The goal of the present study was to comprehensively
map
eQTL
in
healthy
human
colon
in
order
to
characterize colon-specific gene regulation and evaluate
its relevance for GWAS of IBD and CRC as well as other
complex phenotypes. Towards this end, genome-wide
genotyping, using a microarray optimized for individuals
of African descent, and gene expression profiling were
performed colonic tissue obtained from 40 African
American (AA) healthy subjects who underwent screening
colonoscopies. These findings and resources will allow for
improved understanding of disease pathogenesis of in-
flammatory and malignant diseases of the colon. Quality control of genotype and expression data UC), CD only, UC only or CRC. When SNPs in high link-
age disequilibrium (LD) (r2 ≥0.8) with the GWAS variants
were included, the overlap between GWAS signals and
significant cis-eQTL increased to 127 variants: 103 com-
mon to CD and UC, 6 for UC-only and 18 for CRC. These significant cis-eQTL corresponded to 8 unique
target genes that co-localize with disease-associated
SNPs (Figure 2; Table 2). target genes in other tissues, including genes in LCLs,
liver, brain, skin, and ileum. As the FDR threshold for de-
fining cis-eQTL in the colon became more stringent, the
percentage overlap of colon cis-eQTL target genes with
cis-eQTL target genes from other tissues increased. Population differentiated colon cis-eQTL Fixation index (FST) values were calculated between
1000 Genomes Project European (EUR) and African
(AFR) populations for all significant colon cis-eQTL (FDR
< 0.20). Out of the 14,177 significant cis-eQTL SNPs, FST
estimates were successfully calculated for 14,135 SNPs. 3,185 cis-eQTL (23% of all cis-eQTL for which FST values
were estimated) had FST > 0.25, indicating high population
differentiation (Figure 3A) which was greater than that ex-
pected under the null at all FDR thresholds tested (0.2,
0.1, 0.05, 0.01; results at FDR < 0.20 in Figure 3B). Popula-
tion differentiated SNPs that are also associated with CD,
UC and CRC were identified among colon cis-eQTL using
the FST statistic. As the mean value of FST between CEU
and YRI has been estimated to be 0.071 across 1000
Genomes Project SNPs [51], FST threshold of 0.10 was
used to define SNPs as population differentiated. For UC,
there was one disease-associated SNP rs9847710 with FST
> 0.10 (Additional file 6). For CD- and CRC-associated
SNPs, there were no GWAS variants with FST > 0.10. Quality control of genotype and expression data (A) Flowchart describing the quality control (QC) process for the SNP genotype and gene
expression data (See Methods for details). The numbers inside the triangles correspond to the numbers of SNPs or probes/genes that are left
after the removal of those (numbers given inside parentheses) that fail to meet the QC criteria outlined in the text next to the arrows. 1,492,955
genotyped SNPs passed the QC filters and were imputed. 16,252 gene expression probes corresponding to 12,363 unique genes passed the QC
filters and were included in the eQTL analysis. (B) Flowchart summarizing the imputation and post-imputation QC steps for the 1,492,955 genotyped
SNPs that passed QC in (A) and were imputed using 1000 Genomes as reference to provide data on 28,156,045 SNPs (see Methods for details). The
description of each step along with the numbers of SNPs that were excluded at each is listed next to the arrows. The final dataset consisted of
8,400,922 imputed SNPs in 48 individuals. Figure 1 Flowchart summarizing the study design. (A) Flowchart describing the quality control (QC) process for the SNP genotype and gene
expression data (See Methods for details). The numbers inside the triangles correspond to the numbers of SNPs or probes/genes that are left
after the removal of those (numbers given inside parentheses) that fail to meet the QC criteria outlined in the text next to the arrows. 1,492,955
genotyped SNPs passed the QC filters and were imputed. 16,252 gene expression probes corresponding to 12,363 unique genes passed the QC
filters and were included in the eQTL analysis. (B) Flowchart summarizing the imputation and post-imputation QC steps for the 1,492,955 genotyped
SNPs that passed QC in (A) and were imputed using 1000 Genomes as reference to provide data on 28,156,045 SNPs (see Methods for details). The
description of each step along with the numbers of SNPs that were excluded at each is listed next to the arrows. The final dataset consisted of
8,400,922 imputed SNPs in 48 individuals. UC), CD only, UC only or CRC. When SNPs in high link-
age disequilibrium (LD) (r2 ≥0.8) with the GWAS variants
were included, the overlap between GWAS signals and
significant cis-eQTL increased to 127 variants: 103 com-
mon to CD and UC, 6 for UC-only and 18 for CRC. These significant cis-eQTL corresponded to 8 unique
target genes that co-localize with disease-associated
SNPs (Figure 2; Table 2). Quality control of genotype and expression data Quality control of genotype and expression data
2.2 million SNPs were genotyped in 48 AA subjects using
the Affymetrix Axiom Pan-African array (Figure 1A). SNPs
were removed if they had a genotyping rate <95% (n =
62,060), minor allele frequency (MAF) < 0.05 (n = 611,784), Hulur et al. BMC Genomics (2015) 16:138 Page 3 of 15 Figure 1 Flowchart summarizing the study design. (A) Flowchart describing the quality control (QC) process for the SNP genotype and gene
expression data (See Methods for details). The numbers inside the triangles correspond to the numbers of SNPs or probes/genes that are left
after the removal of those (numbers given inside parentheses) that fail to meet the QC criteria outlined in the text next to the arrows. 1,492,955
genotyped SNPs passed the QC filters and were imputed. 16,252 gene expression probes corresponding to 12,363 unique genes passed the QC
filters and were included in the eQTL analysis. (B) Flowchart summarizing the imputation and post-imputation QC steps for the 1,492,955 genotyped
SNPs that passed QC in (A) and were imputed using 1000 Genomes as reference to provide data on 28,156,045 SNPs (see Methods for details). The
description of each step along with the numbers of SNPs that were excluded at each is listed next to the arrows. The final dataset consisted of
8,400,922 imputed SNPs in 48 individuals. Figure 1 Flowchart summarizing the study design. (A) Flowchart describing the quality control (QC) process for the SNP genotype and gene
expression data (See Methods for details). The numbers inside the triangles correspond to the numbers of SNPs or probes/genes that are left
after the removal of those (numbers given inside parentheses) that fail to meet the QC criteria outlined in the text next to the arrows. 1,492,955
genotyped SNPs passed the QC filters and were imputed. 16,252 gene expression probes corresponding to 12,363 unique genes passed the QC
filters and were included in the eQTL analysis. (B) Flowchart summarizing the imputation and post-imputation QC steps for the 1,492,955 genotyped
SNPs that passed QC in (A) and were imputed using 1000 Genomes as reference to provide data on 28,156,045 SNPs (see Methods for details). The
description of each step along with the numbers of SNPs that were excluded at each is listed next to the arrows. The final dataset consisted of
8,400,922 imputed SNPs in 48 individuals. Figure 1 Flowchart summarizing the study design. g
p
outlined in Supplementary Methods (Additional file 8). Comparison of colon cis-eQTL with other tissues CD and/or UC) or (B) CRC from the NHGRI GWAS catalog and their target gene’s expression. The x-axes correspond to the SNP genotypes, and the
y-axes represent the log2-normalized gene expression values. The median gene expression level for each genotype is indicated by a horizontal line with the
boxes covering 25th and 75th percentiles and the whiskers extending to 1.5 times the interquartile range. Points outside the whiskers are plotted as outliers. For each target gene, the disease-associated SNP was selected for the box plot even if it is not the most significant cis-eQTL (but must be in r2 ≥0.8 with it). Comparison of colon cis-eQTL with other tissues A comparative analysis was performed to assess the ex-
tent of overlap between colon cis-eQTL and cis-eQTL
for other tissue types (Table 3). Substantial percentages of
colon cis-eQTL target genes were found to be cis-eQTL Table 1 Numbers of significant colon cis-eQTL identified
at four FDR thresholds
Number of cis-eQTL
FDR
SNP-gene pairs
SNPs
Independent*
Genes
0.01
1,941
1,941
122
122
0.05
5,183
5,181
353
352
0.10
8,252
8,122
693
684
0.20
14,255
14,177
1,677
1,614
*These numbers correspond to the numbers of independent SNP-gene
associations that were determined using the stepwise association method
outlined in Supplementary Methods (Additional file 8). Table 1 Numbers of significant colon cis-eQTL identified
at four FDR thresholds Page 4 of 15 Hulur et al. BMC Genomics (2015) 16:138 Figure 2 Colon cis-eQTL that are also associated with colonic diseases. The box plots depict the relationship between SNPs associated with (A) IBD
(i.e. CD and/or UC) or (B) CRC from the NHGRI GWAS catalog and their target gene’s expression. The x-axes correspond to the SNP genotypes, and the
y-axes represent the log2-normalized gene expression values. The median gene expression level for each genotype is indicated by a horizontal line with the
boxes covering 25th and 75th percentiles and the whiskers extending to 1.5 times the interquartile range. Points outside the whiskers are plotted as outliers. For each target gene, the disease-associated SNP was selected for the box plot even if it is not the most significant cis-eQTL (but must be in r2 ≥0.8 with it). Figure 2 Colon cis-eQTL that are also associated with colonic diseases. The box plots depict the relationship between SNPs associated with (A) IBD
(i.e. CD and/or UC) or (B) CRC from the NHGRI GWAS catalog and their target gene’s expression. The x-axes correspond to the SNP genotypes, and the
y-axes represent the log2-normalized gene expression values. The median gene expression level for each genotype is indicated by a horizontal line with the
boxes covering 25th and 75th percentiles and the whiskers extending to 1.5 times the interquartile range. Points outside the whiskers are plotted as outliers. For each target gene, the disease-associated SNP was selected for the box plot even if it is not the most significant cis-eQTL (but must be in r2 ≥0.8 with it). Figure 2 Colon cis-eQTL that are also associated with colonic diseases. The box plots depict the relationship between SNPs associated with (A) IBD
(i.e. Enrichment of colon cis-eQTL among IBD- and
CRC-associated variants for simulation details). Significant enrichment of cis-eQTL
was observed among SNPs associated with CD (n = 251)
and UC (n = 185) from the GWAS catalog, with n corre-
sponding to the number of disease-associated SNPs in-
cluded in the enrichment analysis, with empirical p-values
for enrichment of < 0.001 (Figure 4A; Table 4). Cis-eQTL Simulation-based analysis was performed to investigate
whether IBD- and CRC- associated variants are enriched
for the 150,213 unique cis-eQTL SNPs with p < 0.001
compared with SNPs matched on MAF (see Methods Table 2 Colon cis-eQTL that are associated with Crohn’s disease (CD) and/or ulcerative colitis (UC) as well as colorectal
cancer (CRC)
SNP/gene
Proxy SNP* (r2)
eQTL p-value
Both CD and UC
rs1363907/ERAP2
1.4 × 10−8
rs6545800/ADCY3
rs2384061 (r2 = 0.82)
1.3 × 10−7
rs4077515/INPP5E
rs4266763 (r2 = 0.95)
4.7 × 10−6
rs9858542/UBA7
rs10640 (r2 = 0.92)
8.7 × 10−6
CD only
rs2549794/ERAP2
1.5 × 10−8
UC only
rs9847710/SFMBT1
8.3 × 10−6
rs6781710/NXPE1 & REX02
rs661946 (r2 = 0.94)
8.7 × 10−7
CRC
rs3802842/COLCA2
1.1 × 10−6
*SNPs in LD (r2 ≥0.8) with disease-associated GWAS SNPs from the NHGRI catalog. r2 corresponds to the correlation between the proxy SNP and the GWAS SNP,
calculated based on the 48 African American samples in the colon dataset. L that are associated with Crohn’s disease (CD) and/or ulcerative colitis (UC) as well as colorectal 2 Colon cis-eQTL that are associated with Crohn’s disease (CD) and/or ulcerative colitis (UC) as we CRC *SNPs in LD (r2 ≥0.8) with disease-associated GWAS SNPs from the NHGRI catalog. r2 corresponds to the correlation between the proxy SNP and the GWAS SNP,
calculated based on the 48 African American samples in the colon dataset. Page 5 of 15 Page 5 of 15 Hulur et al. Enrichment of colon cis-eQTL among IBD- and
CRC-associated variants BMC Genomics (2015) 16:138 Table 3 Comparison of colon cis-eQTL results with published studies in different tissues Table 3 Comparison of colon cis-eQTL results with published studies in different tissues
FDR
Samples
0.01
0.05
0.10
0.20
LCL [41]
13,643 genes and ~2.2 million SNPs in 270 HapMap
subjects (CEU, CHB, JPT and YRI populations)
Genes*
25 (20%)
49 (14%)
72 (11%)
119 (7.4%)
SNP-gene pairs**
130
182
298
361
Liver [35]
34,266 genes and 782,476 SNPs in liver samples
acquired from 427 Caucasian subjects
Genes*
41 (34%)
95 (27%)
169 (25%)
346 (21%)
SNP-gene pairs**
8
15
20
33
Brain [48]
22,184 genes and 1,629,853 SNPs in brain tissue
samples from 150 Caucasian subjects
Genes*
17 (14%)
35 (9.9%)
63 (9.2%)
137 (8.5%)
SNP-gene pairs**
139
191
277
327
Skin [46]
14,500 genes and 438,670 SNPs in skin samples
from 57 Caucasian subjects
Genes*
24 (20%)
39 (11%)
59 (8.6%)
92 (5.7%)
SNP-gene pairs**
131
92
71
50
Ileum [49]
19,047 genes and 581,633 SNPs in ileum samples
from 173 mostly Caucasian subjects
Genes*
37 (30%)
83 (24%)
118 (17%)
197 (12%)
SNP-gene pairs**
176
130
94
58
*The number of target genes from other studies overlapping colon cis-eQTL target genes are listed along with the percentages of overlap (in parentheses). To
calculate the percentage of overlap, the number of overlapping target genes was divided by the total number of unique significant colon cis-eQTL target genes at
the corresponding FDR threshold (refer to Table 1 for the actual numbers). **The number of SNP-gene pairs that overlap between the colon and other tissues. *The number of target genes from other studies overlapping colon cis-eQTL target genes are listed along with the percentages of overlap (in parentheses). To
calculate the percentage of overlap, the number of overlapping target genes was divided by the total number of unique significant colon cis-eQTL target genes at
the corresponding FDR threshold (refer to Table 1 for the actual numbers). **The number of SNP-gene pairs that overlap between the colon and other tissues. counts among the CRC-associated SNPs (n = 40) were
also significantly higher than expected based on MAF dis-
tribution (p = 0.031). cancer and melanoma), lipid traits (total cholesterol and tri-
glyceride levels), body mass index (BMI), type 2 diabetes
(T2D), and psychiatric disorders [attention deficit hyper-
activity disorder (ADHD), bipolar disorder (BD) and schizo-
phrenia]. Enrichment of colon cis-eQTL among IBD- and
CRC-associated variants There was statistically significant colon cis-eQTL
enrichment among SNPs associated with T2D (p = 0.034)
and suggestive enrichment among SNPs associated with
BMI (p = 0.055) (Figure 4B; Table 4). SNPs associated with In order to determine whether enrichment of colon
eQTL is specific to particular complex traits or diseases,
enrichment analysis was performed for SNPs associated
with autoimmune disorders [celiac disease, psoriasis,
rheumatoid arthritis (RA)], cancers (breast cancer, prostate Figure 3 Cis-eQTL are enriched for SNPs that are highly differentiated between European and African populations. FST values for the
study SNPs were calculated between 1000 Genomes Project European (EUR) and African (AFR) populations using Weir and Cockerham’s unbiased
estimator. SNPs with FST > 0.25 were defined as population differentiated SNPs. (A) The histogram shows the distribution of FST values for the
significant colon cis-eQTL (FDR < 0.20). Among the 14,135 cis-eQTL for which FST estimates were obtained, 3,185 (23%) were population
differentiated. (B) Enrichment of population differentiated SNPs among significant colon cis-eQTL was evaluated using a simulation-based
method. The box plot depicts the distributions of the number of population differentiated SNPs among 1,000 randomly selected cis-eQTL SNP
sets (left)―generated by randomly selecting a single SNP for each unique cis-eQTL target gene (n = 684) among all cis-eQTL (FDR < 0.20) that are
significantly associated with the expression of that gene―and among 1000 random sets of SNPs (right), each matching the set of 684 significant
cis-eQTL SNPs, based on the distributions of MAF and distance from the nearest TSS. The numbers of population differentiated SNPs among the
eQTL and random SNP sets are indicated by horizontal lines with the boxes covering 25th and 75th percentiles and the whiskers extending to
1.5 times the interquartile range. The numbers of population differentiated SNPs in the eQTL sets were significantly higher than in the random
sets of SNPs (p < 0.001 by Mann–Whitney test). Figure 3 Cis-eQTL are enriched for SNPs that are highly differentiated between European and African populations. FST values for the
study SNPs were calculated between 1000 Genomes Project European (EUR) and African (AFR) populations using Weir and Cockerham’s unbiased
estimator. SNPs with FST > 0.25 were defined as population differentiated SNPs. (A) The histogram shows the distribution of FST values for the
significant colon cis-eQTL (FDR < 0.20). Among the 14,135 cis-eQTL for which FST estimates were obtained, 3,185 (23%) were population
differentiated. Enrichment of colon cis-eQTL among IBD- and
CRC-associated variants (B) Enrichment of population differentiated SNPs among significant colon cis-eQTL was evaluated using a simulation-based
method. The box plot depicts the distributions of the number of population differentiated SNPs among 1,000 randomly selected cis-eQTL SNP
sets (left)―generated by randomly selecting a single SNP for each unique cis-eQTL target gene (n = 684) among all cis-eQTL (FDR < 0.20) that are
significantly associated with the expression of that gene―and among 1000 random sets of SNPs (right), each matching the set of 684 significant
cis-eQTL SNPs, based on the distributions of MAF and distance from the nearest TSS. The numbers of population differentiated SNPs among the
eQTL and random SNP sets are indicated by horizontal lines with the boxes covering 25th and 75th percentiles and the whiskers extending to
1.5 times the interquartile range. The numbers of population differentiated SNPs in the eQTL sets were significantly higher than in the random
sets of SNPs (p < 0.001 by Mann–Whitney test). Figure 3 Cis-eQTL are enriched for SNPs that are highly differentiated between European and African populatio Page 6 of 15 Hulur et al. BMC Genomics (2015) 16:138 Page 6 of 15 the other traits did not show enrichment for colon eQTL
(Addi i
l fil 7 Addi i
l fil 8 T bl S1)
Methods). Significant enrichment of active histone
k
d
i
ifi
i
QTL
i h h
Figure 4 SNPs associated with colonic diseases and type 2 diabetes are enriched for colon cis-eQTL. A simulation-based analysis was
performed to test for the enrichment of colon cis-eQTL among SNPs associated with colonic diseases (A) body mass index (BMI), lipid traits and
type 2 diabetes (T2D) (B) downloaded from the NHGRI GWAS catalog. The distribution of the number of cis-eQTL in 1,000 simulated SNP sets,
each of the same size (n) as the list of trait-associated SNPs and containing SNPs matched on MAF distribution is shown in the histograms. Solid black
circles represent the actual cis-eQTL count (cis-eQTL p-value threshold of 0.001) observed in the trait-associated SNPs. The p-values shown are empirical,
and are calculated as the proportion of sampled SNP sets in which the cis-eQTL count exceeds the actual count observed in the trait-associated SNPs. Enrichment of cis-eQTL among disease-associated SNPs is statistically significant for all colonic diseases. Enrichment of cis-eQTL is statistically significant
for T2D (p = 0.034) and suggestively significant for BMI (p = 0.055). Enrichment of colon cis-eQTL among IBD- and
CRC-associated variants There is no enrichment of cis-eQTL among SNPs associated with lipid traits. Figure 4 SNPs associated with colonic diseases and type 2 diabetes are enriched for colon cis-eQTL. A simulation-based analysis was
performed to test for the enrichment of colon cis-eQTL among SNPs associated with colonic diseases (A) body mass index (BMI), lipid traits and
type 2 diabetes (T2D) (B) downloaded from the NHGRI GWAS catalog. The distribution of the number of cis-eQTL in 1,000 simulated SNP sets,
each of the same size (n) as the list of trait-associated SNPs and containing SNPs matched on MAF distribution is shown in the histograms. Solid black
circles represent the actual cis-eQTL count (cis-eQTL p-value threshold of 0.001) observed in the trait-associated SNPs. The p-values shown are empirical,
and are calculated as the proportion of sampled SNP sets in which the cis-eQTL count exceeds the actual count observed in the trait-associated SNPs. Enrichment of cis-eQTL among disease-associated SNPs is statistically significant for all colonic diseases. Enrichment of cis-eQTL is statistically significant
for T2D (p = 0.034) and suggestively significant for BMI (p = 0.055). There is no enrichment of cis-eQTL among SNPs associated with lipid traits. the other traits did not show enrichment for colon eQTL
(Additional file 7; Additional file 8: Table S1). Methods). Significant enrichment of active histone
marks was noted among significant cis-eQTL with the
following empirical p-values: < 1.0 × 10−6 (H3K4me1,
H3K4me3 and H3K9ac) and 2.0 × 10−5 (H3K36me3)
(Figure 5A). There was no significant enrichment for
the repressive histone mark H3K9me3 among cis-eQTL
(p = 0.46) (Figure 5B). A similar pattern of significant en-
richment of active histone marks but not repressive his-
tone marks among colon cis-eQTL was detected in Enrichment of histone marks in colon cis-eQTL Simulations were conducted to test whether SNPs over-
lapping with activating (H3K4me1, H3K4me3, H3K9ac
and
H3K36me3)
and
repressing
(H3K9me3
and
H3K27me3)
histone
mark
peaks
were
statistically
enriched among cis-eQTL at an FDR of 0.10 (see Page 7 of 15 Page 7 of 15 Hulur et al. BMC Genomics (2015) 16:138 Table 4 Enrichment of colon cis-eQTL among NHGRI
GWAS SNPs
Disease or trait
Number of
GWAS SNPs
Number of GWAS SNPs
that are colon cis-eQTL
p-value*
CD
251
12
<0.001
UC
185
12
<0.001
CRC
40
2
0.031
T2D
65
3
0.034
BMI
100
4
0.055
*P-values were empirically determined using a simulation-based method in
which 1,000 randomized SNP sets, matched in size and MAF distribution to
the NHGRI GWAS SNPs, were generated. These simulations yielded a p-value,
calculated as the proportion of sampled SNP sets in which the cis-eQTL count
exceeds the actual count observed in the GWAS SNPs. For details see
Supplementary Methods (Additional file 8). Table 4 Enrichment of colon cis-eQTL among NHGRI
GWAS SNPs Table 4 Enrichment of colon cis-eQTL among NHGRI colonic smooth muscle, adipose nuclei, adult liver, and
breast myoepithelial cells (data not shown). In human
CRC adenocarcinoma cell line (Caco-2), there was enrich-
ment for H3K4me3 (p < 1.0 x 10−6) and a suggestive en-
richment for H3K36me3 (p = 0.060), but no significant
enrichment for the repressing mark H3K27me3 (p = 0.86)
(Additional file 9). Taken together, these analyses show
that colon cis-eQTL are enriched for active histone marks
in several tissue types including malignant CRC cell lines. *P-values were empirically determined using a simulation-based method in
which 1,000 randomized SNP sets, matched in size and MAF distribution to
the NHGRI GWAS SNPs, were generated. These simulations yielded a p-value,
calculated as the proportion of sampled SNP sets in which the cis-eQTL count
exceeds the actual count observed in the GWAS SNPs. For details see
Supplementary Methods (Additional file 8). Discussion Gene expression in a disease-relevant tissue is a power-
ful intermediate phenotype that could help elucidate the
functional basis of some risk-associated variants identi-
fied in GWAS. Gene expression analysis is especially im-
portant as disease-associated variants are enriched in Figure 5 Cis-eQTL are enriched for active but not repressive histone marks in colonic mucosa. The red histogram in each plot depicts the
distribution of the number of SNPs in histone mark peaks in 1,000 randomly selected cis-eQTL SNP sets, which are generated by randomly
selecting a single SNP for each unique cis-eQTL target gene (n = 684) among all cis-eQTL (FDR < 0.10) that are significantly associated with the
expression of that gene. The blue histograms represent the distributions of the number of SNPs in histone mark peaks in 1,000 randomly
sampled SNP sets, each matching the set of 684 significant cis-eQTL SNPs (chosen at random from the set of 1,000 cis-eQTL SNPs depicted in
red) with respect to MAF and distance from the nearest TSS. Four markers of active chromatin (H3K4me1, H3K4me3, H3K9ac and H3K36me3) are
depicted in (A), while a single marker of inactive chromatin (H3K9me3) is depicted in (B). The p-value in the top right corner of each histogram is
the empirical p-value obtained by comparing the number of SNPs in histone mark peaks in the 1,000 sets of cis-eQTL SNPs (red) to the null
distribution given by the 1,000 sets of matched SNPs (blue). Figure 5 Cis-eQTL are enriched for active but not repressive histone marks in colonic mucosa. The red histogram in each plot depicts the
distribution of the number of SNPs in histone mark peaks in 1,000 randomly selected cis-eQTL SNP sets, which are generated by randomly
selecting a single SNP for each unique cis-eQTL target gene (n = 684) among all cis-eQTL (FDR < 0.10) that are significantly associated with the
expression of that gene. The blue histograms represent the distributions of the number of SNPs in histone mark peaks in 1,000 randomly
sampled SNP sets, each matching the set of 684 significant cis-eQTL SNPs (chosen at random from the set of 1,000 cis-eQTL SNPs depicted in
red) with respect to MAF and distance from the nearest TSS. Four markers of active chromatin (H3K4me1, H3K4me3, H3K9ac and H3K36me3) are
depicted in (A), while a single marker of inactive chromatin (H3K9me3) is depicted in (B). Discussion SFMBT1 is a polycomb protein with transcrip-
tional repressor activity [54,55] that may regulate a num-
ber of genes through epigenetic mechanisms. Additional
SNPs for IBD-associated variants identified in this study
and their gene targets—rs6545800 with adenylate cyclase
(ADCY3), rs4077515 with inositol polyphosphate 5-
phosphatase (INPP5E), rs9858542 with ubiquitin-like
modifier activating enzyme 7 (UBA7), and rs678170 with
neuroexophilin and PC-esterase domain family member 1
(NXPE1) and with RNA exonuclease 2 (REXO2) —were
novel targets and should be validated in a larger study. g
y
A substantial proportion of the gene targets of the
significant colon cis-eQTL were also found to be cis-
regulated target genes in other tissue types notably
liver and ileum – two tissues closely related to the
colon (Table 3). The percentage overlap of cis-eQTL
target genes between colon and other tissue types was
higher at more stringent FDR thresholds for cis-eQTL
associations, which was expected because increased
stringency should filter out false positive results. The
number of significant target genes shared between
studies will also be affected by sample size, significance
thresholds and genotyping platforms. Therefore, the
actual percentage values of overlap should be regarded
as rough estimates of the actual extent of overlap. The
high cross-tissue replicability of the colon cis-eQTL is
consistent with the observation here of active histone
mark enrichment in non-colon tissue types as well as
findings from other studies that showed that a large num-
ber of cis-eQTL are shared across tissues [42,46,65]. The
extent to which eQTL have cell-type or context-specificity
in the human colon remains to be investigated but
could yield important information for further func-
tional characterization. g
g
y
Among CRC-associated GWAS SNPs, rs3802842, was
found to be a cis-eQTL in the colon [26,56], a finding
that was also recently reported by another study [50]. The
target transcript C11orf93 corresponds to an uncharacter-
ized gene known as colorectal cancer associated 2
(COLCA2). A study found that two functional risk vari-
ants (rs7130173 and rs10891246), which are in perfect LD
with rs3802842, lead to decreased expression of C11orf93
[57]. These results were similar to our finding that the C
allele of rs3802842 also results in decreased expression. Discussion The p-value in the top right corner of each histogram is
the empirical p-value obtained by comparing the number of SNPs in histone mark peaks in the 1,000 sets of cis-eQTL SNPs (red) to the null
distribution given by the 1,000 sets of matched SNPs (blue). Hulur et al. BMC Genomics (2015) 16:138 Page 8 of 15 non-coding regions [33,44,52]. The analysis of published
GWAS variants associated with colonic diseases noted
significant enrichment for cis-eQTL in IBD (CD and
UC) as well as CRC. Novel target genes for IBD-
associated variants were identified and several previously
reported eQTL identified in other tissues were validated. Moreover, overlap of colon eQTL in other tissue types,
most notably ileum and liver was noted. Colon cis-eQTL
were located near TSS as well as enriched for active his-
tone marks and variants with high population differenti-
ation, results that underscore the functional role of
eQTL in gene regulation. This analysis could provide in-
sights into the functional consequences of disease-
associated genetic variants. Enrichment of colon cis-eQTL among trait-associated
variants as shown in Table 4 is consistent with other
studies [44,61-63] and adds further evidence for the use-
fulness of eQTL in improving power to detect significant
associations from GWAS. Enrichment of colon cis-eQTL
was observed in diseases with colonic involvement (IBD
and CRC), as well as in T2D and BMI. These results
raise the intriguing hypothesis that gene expression in
the colon could be functionally linked to diabetes and
BMI, though further work is needed to identify specific
mechanisms. No significant enrichment was found for
colon cis-eQTL among variants associated with auto-
immune disorders, cancer or psychiatric disorders. Taken
together, these results support the conclusion that eQTL
most relevant to disease show clear tissue specificity,
highlighting the importance of creating comprehensive
eQTL catalogs in diverse tissue types [64]. Among IBD-associated GWAS SNPs, variants were
associated with differences in the expression of endo-
plasmic reticulum aminopeptidase 2 (ERAP2) and scm-
like with four MBT domains 1 (SFMBT1) (Figure 2A;
Table 2), similar to results in an eQTL mapping study of
the human ileum [49]. ERAP2 encodes an endoplasmic
reticulum aminopeptidase responsible for major histo-
compatibility complex class I (MHC1) ligand trimming
[53] that has in silico support as a functional variant in
CD [10]. Discussion While this variant was first identified in individuals of
European descent, a previous study by our group validated
this SNP as associated with rectal cancer in AA [58],
though other groups have not found evidence for an asso-
ciation of rs3802842 with CRC in AA [59]. A recent trans-
ethnic GWAS identified an additional SNP (rs79453636)
as associated with CRC in AA, independent of rs3802842,
that has not been replicated in other populations [60]. Neither rs79453636, nor any SNPs in LD with it, was
identified as a colon cis-eQTL in the present study. Consistent with the hypothesis that eQTL have regula-
tory functions, enrichment was noted for colon cis-eQTL
near TSS similar to findings in other tissues [34,39,46-48]. Moreover, cis-eQTL were significantly enriched for his-
tone marks associated with active chromatin (H3K4me1,
H3K4me3, H3K9ac and H3K36me3) but not with
transcriptionally inactive chromatin (H3K9me3 and
H3K27me3) in various tissues and in a CRC cell line. These findings are consistent with the widely accepted
theory that cis-eQTL act by affecting transcript stabil-
ity or the rate of transcript degradation [39,66-68],
and other studies that have also noted enrichment of
eQTL among activating cis-elements but not in regions of
repressed chromatin [69-71]. Hulur et al. BMC Genomics (2015) 16:138 Page 9 of 15 resource for future studies. Characterization of the gen-
etic architecture of gene regulation in the human colon
informs the functional impact of GWAS variants and
could benefit understanding of the biological differences
in colonic disease between different ethnic groups. The
findings in this study demonstrate that eQTL are im-
portant in the susceptibility to inflammatory and malig-
nant diseases of the colon, underscoring the utility of
eQTL mapping for elucidating the genetics and biology
underlying colonic diseases. Furthermore the eQTL map
presented here could benefit understanding of the bio-
logical differences in colonic disease between different
ethnic groups. This is especially important given that
there is a paucity of genetic studies of colonic diseases in
AA populations and our understanding of the genetics
and etiology of these diseases is based on Europeans,
which may not be applicable to other populations. Further
efforts should be made to intersect our eQTL data with
IBD and CRC genetic studies, for a better understanding
of the genetic mechanisms and inter-ethnic differences in
these diseases. Discussion A substantial proportion of colon cis-eQTL in this
study were highly differentiated between European and
African populations (FST > 0.25) as shown in Figure 3. The degree of population differentiation among these
eQTL suggests that they may well reflect local adapta-
tion to environment. A similar finding was previously
described for LCL eQTL [72]. It remains to be investi-
gated whether such colon eQTL, which exhibit large
allele frequency differences between European and African
populations, contribute to inter-ethnic gene expression
differences in the colon that translate into differences in
disease risk. Integration of GWAS results with population
genetic approaches may prove instrumental in under-
standing the genetic basis of differences in IBD and CRC
risk between Europeans and AA [73-76]. A limitation of this study is the moderate sample size,
which reduces power to detect significant eQTL associa-
tions (especially those acting in trans), thereby resulting
in a high false negative rate. Further investigation in a
larger cohort is likely to result in more significant eQTL
associations, and is warranted to confirm findings from
this study. Another caveat of this study is that the GWAS
SNPs used in the analyses were identified in Europeans,
whereas the eQTL were identified in AA. Although many
of the variants identified through GWAS in European
populations generalize to other populations, there are
differences in genetic susceptibility between populations,
due to several factors including epistasis, gene-environment
interactions, population-specific polymorphisms and differ-
ential LD patterns [77]. Therefore, it is possible that there
are differences in genetic associations between European
and AA populations. As IBD and CRC GWAS in popula-
tions of African descent become available, the overlap be-
tween eQTL identified in this study and GWAS SNPs in
these populations should be investigated. Ethics statement
h
h This research was approved by the Institutional Review
Boards at the University of Illinois at Chicago and the
University of Chicago. Methods
Subjects A total of 48 AA subjects were included. All subjects
underwent colonoscopy at the University of Illinois
Chicago for screening purposes. Blood samples were
obtained at the time of colonoscopy. Colonic biopsies
were obtained using standard forceps (Boston Scientific,
Natick, MA) in all subjects at 20 cm from the anal verge
at the recto-sigmoid junction. Biopsies were placed im-
mediately into RNAlater (Life Technologies Corporation,
Grand Island, NY), and cryopreserved according to the
manufacturer’s instructions. Subjects’ mean age (stand-
ard deviation) was 59.0 years (8.5). While the intention
was to include only males in order to reduce heterogen-
eity, two female subjects were included due to sample
mislabeling. Written informed consent was obtained
from participants. Conclusions In the present study, a comprehensive map of eQTL in
the healthy colon of AA was generated as a resource for
genetic studies of colonic diseases. The analysis showing
the enrichment of colon eQTL among SNPs associated
with colonic diseases supported the usefulness of colon
eQTL as a tissue-specific tool to improve understanding
of colonic disease susceptibility. The utility of colon
eQTL for studying the genetic basis of inter-ethnic dif-
ferences in colonic disease risk was demonstrated by
showing their enrichment for SNPs that exhibit high al-
lele frequency differences between European and African
populations. These SNPs could mediate population-
specific gene expression responses, which could translate
into differences in disease risk. Genotyping and imputation Colon samples from study subjects were genotyped on
the Affymetrix Axiom Genome-Wide Pan-African array
that includes 2,217,402 probes (Affymetrix, Santa Clara,
CA). Genotypes were called using the Illumina Genome
Studio software package (Illumina, San Diego, CA). QC
filters applied to the data are outlined in Figure 1. The
analysis was limited to SNPs that map to unique
chromosomal locations based on the latest annotation This study offers novel insights into the functional
basis of genetic susceptibility for colonic inflammatory
and malignant diseases and provides a tissue-specific Page 10 of 15 Hulur et al. BMC Genomics (2015) 16:138 file for the genotyping array (Release 33). Markers
with <95% genotype call rates, MAF < 0.05 or HWE
p < 10−6 were excluded from analysis. Markers with
HWE p < 10−4 for which there were no heterozygotes
or homozygotes for the minor allele, as well as
markers with HWE p < 10−3 for which there were <6
heterozygotes or homozygotes for the minor allele
were also excluded. Finally, all non-autosomal SNPs
were omitted. The final genotyped dataset contained
1,492,955 autosomal SNPs in 48 subjects with an
average call rate of 99.69%. common SNP (MAF ≥0.05) in either 1000 Genomes
AFR or EUR populations were further excluded, leaving
25,610 probes with unique perfect matches to autosomal
genes that do not contain common SNPs. Furthermore
only probes with an Illumina detection p < 0.01 in at
least one individual were included in further analysis. Eight individuals were identified as outliers based their
poor clustering with other subjects on gene expression
heat map (Additional file 2). There was no correlation
between any of the demographic and clinical characteris-
tics and outlier status. The separation of the outliers
from the rest of the samples seemed to be driven by sys-
tematic differences in gene expression rather than by
expression differences of only a few genes. Four of the
outlier samples had low RNA integrity numbers (RIN)
(<8). As the outliers with RIN > 8 seemed to cluster with
the four outliers with the low RIN scores, the outliers
were likely driven by poor RNA quality. Upon the exclu-
sion of the eight outlier samples, there were no further
outliers on the gene expression heat map. The outliers
were excluded from the gene expression analysis prior
to the microarray pre-processing steps in Bioconductor. Genotyping and imputation After the removing probes that were not significantly
expressed, the final expression dataset included 16,252
probes interrogating 12,363 unique genes in 40 subjects. The gene expression data is available at Gene Expression
Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo) with
the accession number GSE 56789. Imputation was performed with IMPUTE2 version
2.2.2 [78,79] using the 1000 Genomes Phase I integrated
variant set release (v3) (https://mathgen.stats.ox.ac.uk/
impute/impute_v2.html) [51] as reference panel. IM-
PUTE2 uses a reference panel of known haplotypes and
a fine-scale recombination map to infer missing geno-
types in a study dataset that has been typed at a relatively
sparse set of markers. IMPUTE2 assigns each imputed
SNP an INFO score on the basis of imputation quality. INFO scores can take values from 0 to 1, where values
closer to 1 indicate that there is little uncertainty in the
imputed genotype. Imputed SNPs with MAF < 0.05 and
IMPUTE2-info
score < 0.5
were
excluded
from
the
analysis, leaving 8,400,922 imputed SNPs in 48 subjects. Imputed
genotypes
were
coded
as
allelic
dosages
(i.e. estimated fractional counts of the non-reference
allele ranging continuously from 0 to 2). The linear model that is used in the eQTL analysis as-
sumes that the expression values for each probe follow a
normal distribution, and it may be sensitive to the pres-
ence of outliers and non-normality. Thus the log2-
transformed and quantile-normalized expression values
for each probe were transformed into a standard normal
distribution using the rank-based inverse normal trans-
formation implemented in the qqnorm function in R
prior to the eQTL analysis. Transcriptional profiling RNA was extracted from manually ground tissue using
the Maxwell 16 Tissue LEV Total RNA Purification Kit
(Promega, Fitchburg, WI) for automated purification on
the Maxwell 16 Instrument (Promega, Fitchburg, WI). Gene expression levels were measured using the Illu-
mina HT-12v4 Expression BeadChip whole-genome ex-
pression array containing 47,231 gene probes (Illumina,
San Diego, CA), according to manufacturer’s instruc-
tions. The Illumina GenomeStudio software was used to
extract the signal intensity for each probe, which were
then further preprocessed using the lumi package of Bio-
conductor (http://www.bioconductor.com) [80,81] in R
version 3.0.2. The preprocessing included background
correction of the expression data, followed by variance
stabilization transformation (VST), log2-transformation
and quantile-normalization. Colon biopsies were composed of primarily epithelial
cells with a small amount of stromal cells. At least 2 biop-
sies from the colon were used per individual to minimize
tissue heterogeneity. Overall, expression of an epithelial
marker E-cadherin (CDH1) did not show significant inter-
individual variation when normalized to a housekeeping
gene GAPDH across the 40 individuals (data not shown). Colon cis-eQTL associated with colonic diseases Colon cis-eQTL associated with colonic diseases
All NHGRI GWAS SNPs associated with colonic dis-
eases (CD, UC and CRC) that were either significant
cis-eQTL (FDR < 0.10) or in high LD with a cis-eQTL
were identified. All SNPs in high LD (r2 ≥0.8) with the
cis-eQTL were obtained from SNAP database (http://
www.broadinstitute.org/mpg/snap) [86] using CEU as
the reference. Associations were visualized using box
plots that show expression as a function of eQTL
genotype. For details, refer to Supplementary Methods
(Additional file 8). Ancestry estimates BLAST-like alignment tool (BLAT) pairwise sequence
alignment algorithm was used to map the probe se-
quences onto the RefSeq transcriptome (downloaded
from the UCSC Genome Browser: http://hgdownload. cse.ucsc.edu/goldenPath/hg19/bigZips). All probes map-
ping to multiple Entrez Gene IDs were omitted and only
probes that map uniquely to autosomal genes with no
mismatches were included. Probes that encompass a PCA of the genotype data was used to quantify the pro-
portions of European and African ancestry in the AA
subjects. Samples from HapMap ASW, CEU, and YRI
populations were used as reference populations. PCA
analysis was performed in EIGENSTRAT [82] using an
LD-pruned dataset of 48,553 SNPs. The first PC from
the analysis was included as a covariate to account for
ancestry in subsequent analyses. Hulur et al. BMC Genomics (2015) 16:138 Page 11 of 15 (4) BMI (n = 100), lipid traits (n = 210) and T2D (n =
65) and (5) psychiatric disorders (ADHD [n = 179], BD
[n = 257] and schizophrenia [n = 247]). The number of
GWAS SNPs for each trait that fit the aforementioned
criteria and were thus included in the enrichment ana-
lyses are indicated by n and listed inside the brackets. Bioinformatic analysis of cis-eQTL The genomic locations of cis-eQTL were characterized
by plotting their position relative to the TSS of genes. The enrichment of histone marks among significant cis-
eQTL at an FDR threshold of 0.10 was evaluated using a
modified version of the simulation-based strategy that
was used to test for eQTL enrichment among trait-
associated SNPs. Briefly, the number of SNPs in histone
marks peaks among 1,000 sets of independent cis-eQTL
were compared to that among 1,000 randomized SNP
sets, matched based on the distributions of MAF and
distance from the nearest TSS to the independent cis-
eQTL. For details and adaptations, see Supplementary
Methods (Additional file 8). Evaluation of cis-eQTL enrichment among trait-associated
SNPs eQTL mapping
QTL
i pp
g
eQTL mapping was performed using the Matrix eQTL R
package [83]. Associations between SNP genotype and
probe expression level were analyzed using a linear re-
gression model with additive genotype effects. Cis-eQTL
were defined as associations between SNPs within 1
mega base pair (Mb) of the TSS or transcription end site
(TES) of a gene. Trans-eQTL were defined as associ-
ation signals from SNPs located greater than 1 Mb
from the TSS and TES of a gene or on a different
chromosome. FDR calculations were performed separ-
ately for cis- and trans-eQTL in Matrix eQTL accord-
ing to the Benjamini and Hochberg method [84]. A
PCA-based approach was used to correct for con-
founding variability due to hidden factors in the gene
expression data [85] as outlined in the Supplementary
Methods (Additional file 8). Simulation-based enrichment analyses were conducted
as previously described [44], to evaluate if the trait-
associated SNPs were enriched for colon cis-eQTL. Briefly, 1,000 randomized SNP sets were generated sam-
pled without replacement from the 8,400,922 study
SNPs. Since there was an overrepresentation of higher-
frequency variants among the significant cis-eQTL com-
pared to all study SNPs (Additional file 10), SNPs were
matched on MAF distribution. SNPs were also matched
on distance to the nearest TSS since cis-eQTL were
more enriched around TSS compared to other SNPs
(Additional file 11). For each sampled SNP set and the
set of NHGRI SNPs, the number of SNPs that were
also cis-eQTL was determined. The relatively relaxed
significance threshold of p < 0.001 was used for eQTL
p-values since the numbers of trait-associated cis-eQTL
were too low to quantify enrichment at higher thresh-
olds of significance. An empirical p-value for cis-eQTL
enrichment was obtained based on the simulations, cal-
culated as the proportion of sampled SNP sets in which
the cis-eQTL count exceeds the actual cis-eQTL count
observed in the NHGRI SNPs. To determine the number of independent cis-eQTL
associations at the different FDR thresholds, a stepwise
association model was applied that involves repeating
the eQTL association analysis conditioned on the most
significant cis-eQTL for each gene expression probe,
until there were no more significant cis-associations left
as described in the Supplementary Methods (Additional
file 8). Evaluation of cis-eQTL enrichment among trait-associated
SNPs The enrichment of significant cis-eQTL among SNPs
associated with various complex traits and diseases were
investigated. For the enrichment analyses, only trait-
associated SNPs that were among the 8,400,922 SNPs
analyzed were considered. Trait-associated GWAS SNPs
were downloaded from the NHGRI Catalog of Published
GWAS using the default p-value threshold of 10−5 [87]. SNPs that were not identified in populations of Euro-
pean descent were excluded. The complex traits/diseases
were classified into 5 broad categories: (1) autoimmune
disorders (celiac disease [n = 57], psoriasis [n = 35] and
RA [n =54]) (2) cancers (breast cancer [n = 100], pros-
tate cancer [n = 121] and melanoma [n = 16]), (3) colonic
diseases (CD [n = 251], UC [n = 185] and CRC [n = 40]), Fixation index analysis of cis-eQTL To estimate the level of genetic differentiation between
European and African populations at the colon cis-eQTL,
FST values for all colon cis-eQTL (n = 14,177; FDR < 0.20)
were calculated by using Weir and Cockerham’s unbiased
estimator [88]. For the FST calculations, genotype data for
EUR and AFR populations from the 1000 Genomes Pro-
ject was used [54]. To test whether the number of inde-
pendent colon cis-eQTL that show high population
differentiation between European and African populations
is greater than that expected by chance, we performed a
simulation-based analysis which we describe in detail in
the Supplementary Methods (Additional file 8). SNPs
that show high population differentiation between EUR
and AFR were defined as those with FST > 0.25. The
number of highly population differentiated SNPs among
all 1,000 randomized SNP sets was compared to the
number of highly population differentiated SNPs among
the sets of independent cis-eQTL using the Mann–
Whitney test. High FST SNPs (FST > 0.10) were identified
among cis-eQTL that were associated with CD, UC or
CRC and allele frequency plots showing the worldwide
distributions of ancestral and derived alleles in human Additional file 4: Figure S4. The colon eQTL dataset is enriched for
cis- but not trans-eQTL. (A) The Q-Q plot shows the eQTL association
p-values for all SNP-probe pairs (8,400,922 SNPs x 16,252 probes)
evaluated in the study. Red dots represent cis- and blue dots represent
trans-association p-values. The gray line is the identity line (y = x)
representing the null distribution under which there is no association
between SNP genotype and probe expression level. Cis-association
p-values deviate strongly from the identity line, demonstrating an
enrichment of significant associations. There is no such enrichment for
trans-associations. (B) The histogram shows the eQTL association
p-values for all cis SNP-probe pairs. There is a clear enrichment of
cis-associations with small p-values. Additional file 5: Figure S5. Cis-eQTL cluster roughly symmetrically
around TSS. The scatter plot depicts the distribution of cis-eQTL relative
to TSS. Each dot represents the most significant cis-associated SNP for
each gene expression probe. –log10(p-value) for the SNP-probe association
(y-axis) is plotted against the base pair (bp) distance of the associated SNP
from the TSS of the transcript that the probe is interrogating (x-axis). Negative and positive values of the distance denote SNPs 5′ and 3′ of TSS
(set at 0), respectively. Comparative analysis with cis-eQTL from other tissues
h
l
b
h
l
d Comparative analysis with cis-eQTL from other tissues
The overlap between cis-eQTL in the colon and cis-eQTL
in other tissue was determined using the Genotype-Tissue
Expression (GTEx) eQTL browser (available at http://www. ncbi.nlm.nih.gov/gtex/GTEX2/gtex.cgi), which allows the
query of eQTL results from multiple previous studies by
SNP or gene name. To evaluate the overlap of the colon
cis-eQTL with cis-eQTL from another tissue, the GTEx
eQTL browser was queried to obtain cis-eQTL p-values for Hulur et al. BMC Genomics (2015) 16:138 Page 12 of 15 genes regulated by a significant colon cis-eQTL identified
in this study (at four different FDR thresholds: 0.01, 0.05,
0.10 and 0.20). The number of genes that were also
cis-regulated, defined as within 1 Mb of a gene, in
other studies was determined at a p-value cut-off of 10−5. These studies included the following tissue types: LCLs
[37], liver [35] and brain [48]. Cis-eQTL target genes were
considered to be overlapping between the colon and an-
other tissue if both studies had at least one cis-eQTL for
that target gene. Overlap of colon with skin cis-eQTL
(defined as within 1 Mb of a gene and with p < 10−5)
[46] and colon with ileum cis-eQTL (defined as within
50 kilo base pairs (kb) of a gene and with p < 10−5) [49]
was also investigated using the same approach. Skin cis-
eQTL data was obtained from the University of Michigan,
Center for Statistical Genetics website (http://www.sph. umich.edu/csg/junding/eQTL/). Ileum cis-eQTL data was
downloaded from the Additional file of the article [49]. For each tissue type, the total number of target genes
that overlap between colon and other tissues was re-
ported along with the number of overlapping target
genes that are associated with the same cis-eQTL SNP
(or a proxy with r2 ≥0.8) in both tissues. The number of
SNP-gene pairs that overlap between the present study
and other eQTL studies was reported as well. Our ra-
tionale for focusing on the overlap of eQTL target genes
rather than the overlap of eQTL SNPs, is that genes are
more comparable and less biased across different stud-
ies that use different genotyping platforms with different
marker sets. populations were constructed. The data used in the gen-
eration of these plots are described in detail in a previ-
ous publication [89]. Additional file 2: Figure S2. Additional file 2: Figure S2. Heat map visualization and hierarchical
clustering of the gene expression data. Study samples are organized based on
the similarity of log2-transformed and quantile-normalized gene expression
levels across all probes evaluated in the study. The heat maps represent the
pairwise correlation matrix of similarity among the study subjects across all
probes. Each row and column represents one of the subjects. The color scale
indicates the degree of correlation from lowest (white) to highest (red). (A)
Heat map corresponding to the gene expression data for the 48 subjects prior
to the removal of outliers. The first eight subjects from the left side of the plot
were deemed as outliers and excluded from the eQTL analysis. (B) Heat map
corresponding to the gene expression data for the 40 remaining subjects after
the removal of outliers. Additional file 2: Figure S2. Heat map visualization and hierarchical
clustering of the gene expression data. Study samples are organized based on
the similarity of log2-transformed and quantile-normalized gene expression
levels across all probes evaluated in the study. The heat maps represent the
pairwise correlation matrix of similarity among the study subjects across all
probes. Each row and column represents one of the subjects. The color scale
indicates the degree of correlation from lowest (white) to highest (red). (A)
Heat map corresponding to the gene expression data for the 48 subjects prior
to the removal of outliers. The first eight subjects from the left side of the plot
were deemed as outliers and excluded from the eQTL analysis. (B) Heat map
corresponding to the gene expression data for the 40 remaining subjects after
the removal of outliers. Additional file 3: Figure S3. The numbers of significant cis-eQTL probes
were maximized with the inclusion of the first five PCs of the gene
expression data as covariates. Each of the first 20 PCs from the gene
expression data were sequentially included as covariates in the analysis and
the numbers of gene expression probes that were associated with a cis-eQTL
were determined for each at two different FDR thresholds for SNP-gene
associations. The maximum numbers of gene expression probes associated
with a cis-eQTL were obtained when the first five PCs from the gene expression
data were used as covariates in the eQTL analysis. Thus the first five PCs
were included as covariates in subsequent analyses, to correct for
unmeasured variation in the gene expression data. Additional files Additional file 1: Figure S1. PCA plot of the genotype data from the
study samples along with HapMap reference populations. The 48 African
American subjects cluster with HapMap African American samples on the
principal components analysis (PCA) plot, indicating mixed African and
European ancestry. A PCA-based analysis was performed in EIGENSTRAT
to estimate the proportions of European and African ancestry for each
subject (represented by PC1). HapMap populations of African ancestry in
Southwest USA (ASW), Utah residents of Northern and Western European
ancestry (CEU) and Yoruba in Ibadan, Nigeria (YRI) were used as references
in the PCA plot and are color-coded as indicated in the legend. PC1 was
included as a covariate in the eQTL analysis to account for ancestry. References Additional file 10: Figure S9. MAF distribution of colon cis-eQTL is
skewed towards higher frequencies compared with the complete set of
study SNPs. MAF distribution of the complete set of study SNPs (top
graph) is presented, along with the MAF distributions of cis-eQTL at four
different FDR thresholds. The skew towards higher frequencies becomes
more pronounced as the FDR threshold becomes more stringent. Additional file 10: Figure S9. MAF distribution of colon cis-eQTL is
skewed towards higher frequencies compared with the complete set of
study SNPs. MAF distribution of the complete set of study SNPs (top
graph) is presented, along with the MAF distributions of cis-eQTL at four
different FDR thresholds. The skew towards higher frequencies becomes
more pronounced as the FDR threshold becomes more stringent. 1. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, Taylor KD, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing
the number of confirmed associations to 47. Nat Genet. 2011;43(3):246–52. 1. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, Taylor KD, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing
the number of confirmed associations to 47. Nat Genet. 2011;43(3):246–52. 2. Franke A, Balschun T, Sina C, Ellinghaus D, Hasler R, Mayr G, et al. Genome-wide
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(IL17REL). Nat Genet. 2010;42(4):292–4. 2. Franke A, Balschun T, Sina C, Ellinghaus D, Hasler R, Mayr G, et al. Genome-wide
association study for ulcerative colitis identifies risk loci at 7q22 and 22q13
(IL17REL). Nat Genet. 2010;42(4):292–4. Additional file 11: Figure S10. Colon cis-eQTL are closer to TSS than
other SNPs. The histograms show the distributions of the base pair (bp)
distances to the nearest TSS for all study SNPs that are within 1 mega
base pair (Mb) of a TSS and significant cis-eQTL at various FDR thresholds. Negative distances refer to SNPs upstream (5′) of the TSS (set at 0) while
positive distances refer to those downstream (3′). 3. Franke A, Balschun T, Karlsen TH, Hedderich J, May S, Lu T, et al. Replication
of signals from recent studies of Crohn’s disease identifies previously
unknown disease loci for ulcerative colitis. Nat Genet. 2008;40(6):713–5. 4. McGovern DP, Gardet A, Torkvist L, Goyette P, Essers J, Taylor KD, et al. Genome-wide association identifies multiple ulcerative colitis susceptibility
loci. Nat Genet. 2010;42(4):332–7. 5. Abbreviations AA: African American; ADCY3: Adenylate cyclase 3; ADHD: Attention deficit
hyperactivity disorder; AFR: 1000 Genomes Project African population;
ASW: African ancestry in Southwest USA; BD: Bipolar disorder; BLAT: BLAST-like
alignment tool; BMI: Body mass index; CD: Crohn’s disease; CEU: Utah residents
with Northern and Western European ancestry; ChIP: Chromatin
immunoprecipitation; COLCA2: Colorectal cancer associated 2; CRC: Colorectal
cancer; eQTL: Expression quantitative trait loci; ERAP2: Endoplasmic reticulum
aminopeptidase 2; EUR: 1000 Genomes Project European population; FDR: False
discovery rate; FST: Fixation index; GEO: Gene Expression Omnibus;
GTEx: Genotype-Tissue Expression; GWAS: Genome-wide association study;
HWE: Hardy-Weinberg equilibrium; IBD: Inflammatory bowel disease;
INPP5E: Inositol polyphosphate-5-phospatase; Kb: Kilo base pair;
LCL: Lymphoblastoid cell line; LD: Linkage disequilibrium; MAF: Minor allele
frequency; Mb: Mega base pair; MHC1: Major histocompatibility complex class I;
mRNA: Messenger ribonucleic acid; NHGRI: National Human Genome Research
Institute; NXPE1: Neuroexophilin and PC-esterase domain family member 1;
PC: Principal component; PCA: Principal components analysis; QC: Quality
control; Q-Q: Quantile-quantile; RA: Rheumatoid arthritis; RIN: RNA integrity
number; REXO2: RNA exonuclease 2; SFMBT1: Scm-like with four MBT domains 1;
SNP: Single nucleotide polymorphism; T2D: Type 2 diabetes; TES: Transcription
end site; TSS: Transcription start site; UBA7: Ubiquitin-like modifier activating
enzyme 7; UC: Ulcerative colitis; VST: Variance stabilizing transformation;
YRI: Yoruba in Ibadan, Nigeria. 6. Silverberg MS, Cho JH, Rioux JD, McGovern DP, Wu J, Annese V, et al. Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by
genome-wide association study. Nat Genet. 2009;41(2):216–20. 6. Silverberg MS, Cho JH, Rioux JD, McGovern DP, Wu J, Annese V, et al. Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by
genome-wide association study. Nat Genet. 2009;41(2):216–20. 7. Julia A, Domenech E, Ricart E, Tortosa R, Garcia-Sanchez V, Gisbert JP, et al. A genome-wide association study on a southern European population
identifies a new Crohn’s disease susceptibility locus at RBX1-EP300. Gut. 2013;62(10):1440–5. 7. Julia A, Domenech E, Ricart E, Tortosa R, Garcia-Sanchez V, Gisbert JP, et al. A genome-wide association study on a southern European population
identifies a new Crohn’s disease susceptibility locus at RBX1-EP300. Gut. 2013;62(10):1440–5. 8. Kenny EE, Pe’er I, Karban A, Ozelius L, Mitchell AA, Ng SM, et al. A genome-wide
scan of Ashkenazi Jewish Crohn’s disease suggests novel susceptibility loci. PLoS
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imputation identifies novel and refined associations for the Wellcome Trust
Case Control Consortium phase 1 Data. Acknowledgments
h
k
h different human populations are depicted in pie charts where the ancestral
allele (C) is shown in blue and the derived allele (T) is shown in orange. different human populations are depicted in pie charts where the ancestral
allele (C) is shown in blue and the derived allele (T) is shown in orange. different human populations are depicted in pie charts where the ancestral
allele (C) is shown in blue and the derived allele (T) is shown in orange. different human populations are depicted in pie charts where the ancestral
allele (C) is shown in blue and the derived allele (T) is shown in orange. Additional file 7: Figure S7. SNPs associated with diseases that do not
involve the colon are not enriched for colon cis-eQTL. This is similar to
Figure 4, except the plots depict the enrichment of colon cis-eQTL
among SNPs associated with (A) autoimmune disorders, (B) cancers and
(C) psychiatric disorders. There is no significant enrichment for colon
cis-eQTL among SNPs associated with these disorders. different human populations are depicted in pie charts where the ancestral
allele (C) is shown in blue and the derived allele (T) is shown in orange. Additional file 7: Figure S7. SNPs associated with diseases that do not
involve the colon are not enriched for colon cis-eQTL. This is similar to
Figure 4, except the plots depict the enrichment of colon cis-eQTL
among SNPs associated with (A) autoimmune disorders, (B) cancers and
(C) psychiatric disorders. There is no significant enrichment for colon
cis-eQTL among SNPs associated with these disorders. g
We thank Joseph Maranville for critical review of the manuscript and Barbara
Stranger for helpful discussions. This work was supported by the National
Institutes of Health (R01CA 140804 and U01CA 153060 to NAE, K08CA 142892
to SSK) and the American Cancer Society Illinois Division (223187) to XL. Additional file 7: Figure S7. SNPs associated with diseases that do not
involve the colon are not enriched for colon cis-eQTL. This is similar to
Figure 4, except the plots depict the enrichment of colon cis-eQTL among SNPs associated with (A) autoimmune disorders, (B) cancers and
(C) psychiatric disorders. There is no significant enrichment for colon
cis-eQTL among SNPs associated with these disorders. Abbreviations Eur J Hum Genet. 2012;20(7):801–5. 10. Franke A, McGovern DP, Barrett JC, Wang K, Radford-Smith GL, Ahmad T,
et al. Genome-wide meta-analysis increases to 71 the number of confirmed
Crohn’s disease susceptibility loci. Nat Genet. 2010;42(12):1118–25. 11. McGovern DP, Jones MR, Taylor KD, Marciante K, Yan X, Dubinsky M, et al. Fucosyltransferase 2 (FUT2) non-secretor status is associated with Crohn’s
disease. Hum Mol Genet. 2010;19(17):3468–76. 12. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, et al. Genome-wide association defines more than 30 distinct susceptibility loci
for Crohn’s disease. Nat Genet. 2008;40(8):955–62. 13. Raelson JV, Little RD, Ruether A, Fournier H, Paquin B, Van Eerdewegh P,
et al. Genome-wide association study for Crohn’s disease in the Quebec
Founder Population identifies multiple validated disease loci. Proc Natl Acad
Sci U S A. 2007;104(37):14747–52. Received: 3 September 2014 Accepted: 29 January 2015 Received: 3 September 2014 Accepted: 29 January 2015 Received: 3 September 2014 Accepted: 29 January 2015 Fixation index analysis of cis-eQTL The majority of significant cis-eQTL are found within
100 kb of TSS. Additional file 6: Figure S6. Human populations across the world
differ in the designations of ancestral and derived alleles as major alleles
for UC-associated colon cis-eQTL rs9847710. rs9847710 exhibits a relatively
high level of population differentiation between 1000 Genomes EUR and
AFR populations (FST > 0.10). The plot shows the distribution of rs9847710
alleles in multiple populations worldwide. The haplotype frequencies for Page 13 of 15 Page 13 of 15 Hulur et al. BMC Genomics (2015) 16:138 References Barrett JC, Lee JC, Lees CW, Prescott NJ, Anderson CA, Phillips A, et al. Genome-wide association study of ulcerative colitis identifies three new sus-
ceptibility loci, including the HNF4A region. Nat Genet. 2009;41(12):1330–4. Competing interests The authors declare that they have no competing interests. 14. Franke A, Hampe J, Rosenstiel P, Becker C, Wagner F, Hasler R, et al. Systematic association mapping identifies NELL1 as a novel IBD disease
gene. PLoS One. 2007;2(8):e691. Additional file 9: Figure S8. Additional file 9: Figure S8. Cis-eQTL are enriched for active but not
repressive histone marks in a colorectal adenocarcinoma cell line. This is
similar to Figure 5, except the histone mark data is from a colorectal
adenocarcinoma cell line (Caco-2) and only two active histone marks
(H3K4me3 and H3K36me3) and one repressive histone mark (H3K27me3)
are depicted. Colon cis-eQTL are enriched for active histone marks with the
enrichment reaching statistical significance for H3K4me3 (p < 1.0 × 10-6)
and being highly suggestive for H3K36me3 (p = 0.060). There is no
statistically significant enrichment of the repressive histone mark H3K27me3. Author details
1 1Committee on Genetics, Genomics and Systems Biology, Chicago, IL 60637,
USA. 2Department of Medicine, 900 East 57th Street, MB#9, Chicago, IL
60637, USA. 3Department of Pediatrics, University of Chicago, Chicago, IL
60637, USA. 4Department of Medicine, Yale University, New Haven, CT 06510,
USA. 5University of Arizona Cancer Center, Tucson, AZ 85724, USA. 6Division
of Genetic Medicine, Department of Medicine, Vanderbilt University,
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Submit your manuscript at
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
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Urban Traffic Travel Time Short-Term Prediction Model Based on Spatio-Temporal Feature Extraction
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Hindawi
Journal of Advanced Transportation
Volume 2020, Article ID 3247847, 16 pages
https://doi.org/10.1155/2020/3247847
Research Article
Urban Traffic Travel Time Short-Term Prediction Model Based on
Spatio-Temporal Feature Extraction
Leilei Kang,1 Guojing Hu,2 Hao Huang,1 Weike Lu,3 and Lan Liu
1,4
1
School of Transportation and Logistics, Southwest Jiaotong University, Chengdu, Sichuan 610031, China
Department of Mathematics and Statistical Sciences, Jackson State University, Jackson 39217, MS, USA
3
Alabama Transportation Institute, The University of Alabama, Tuscaloosa 35487, AL, USA
4
National United Engineering Laboratory of Integrated and Intelligent Transportation, Chengdu, Sichuan 610031, China
2
Correspondence should be addressed to Lan Liu; jianan_l@swjtu.edu.cn
Received 1 February 2020; Revised 4 July 2020; Accepted 30 July 2020; Published 14 August 2020
Academic Editor: Ludovic Leclercq
Copyright © 2020 Leilei Kang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In order to improve the accuracy of short-term travel time prediction in an urban road network, a hybrid model for spatiotemporal feature extraction and prediction of urban road network travel time is proposed in this research, which combines
empirical dynamic modeling (EDM) and complex networks (CN) with an XGBoost prediction model. Due to the highly nonlinear
and dynamic nature of travel time series, it is necessary to consider time dependence and the spatial reliance of travel time series
for predicting the travel time of road networks. The dynamic feature of the travel time series can be revealed by the EDM method, a
nonlinear approach based on Chaos theory. Further, the spatial characteristic of urban traffic topology can be reflected from the
perspective of complex networks. To fully guarantee the reasonability and validity of spatio-temporal features, which are dug by
empirical dynamic modeling and complex networks (EDMCN), for urban traffic travel time prediction, an XGBoost prediction
model is established for those characteristics. Through the in-depth exploration of the travel time and topology of a particular road
network in Guiyang, the EDMCN-XGBoost prediction model’s performance is verified. The results show that, compared with the
single XGBoost, autoregressive moving average, artificial neural network, support vector machine, and other models, the
proposed EDMCN-XGBoost prediction model presents a better performance in forecasting.
1. Introduction
Intelligent transportation system (ITS) is currently the most
effective technical solution to improve public transportation
service and management [1, 2]. The successful application of
ITS is inseparable from the accurate identification and
prediction of urban traffic status, usually measured by travel
time. The advantage of travel time lies in that it can be easily
understood by road administrators and users [3]. Also,
managers can take precautions and actively intervene in the
state of urban traffic from the overall situation, and users can
choose their driving routes according to the released information. Previous studies have proved that using travel
time as an indicator can better guide users and make the
traffic flow in the peak period reach a state of balanced
distribution in the road network [4, 5]. However, the prediction of urban road network travel time is a complicated
and challenging work, mainly reflected in the acquisition
and analysis of the spatio-temporal characteristics of the
road network. Therefore, the prediction of urban traffic
conditions has attracted much attention from scholars
committed to fully exploring public transportation information, grasping the law of urban traffic situation and
development and providing real-time and accurate traffic
information for decision-makers [6]. Initially, traditional
statistical models, including autoregressive integrated
moving average (ARIMA) [7] and Kalman filter [8], were
leveraged to forecast the traffic state. However, the statistical
methods are based on linear assumptions about data, while
the actual traffic data are usually nonlinear. Thus, state
prediction has gradually shifted from the previous statistical
models to the current data-driven models [9], which mainly
includes machine learning and deep learning methods. Both
machine learning algorithms such as artificial neural
2
network [10], Bayesian network [11], the k-nearest neighbor
[12], and support vector machine [13], as well as deep
learning models such as deep confidence network [14], and
long short-term memory models [15], have achieved excellent results in the field of traffic prediction. Furthermore,
some prediction approaches have evolved in recent years
based on multiinput multioutput supervised regression (MSVR and M-KNN, for instance), and these approaches strive
to capture the spatio-temporal relationship within the regression process [16, 17]. Some studies also intend to capture
traffic states’ spatial dependencies through the graph convolutional network and learn the dynamic changes of traffic
data by the recursive neural network to capture the time
dependence of traffic states [18]. Although these models
perform well in describing the influence of spatio-temporal
correlation on traffic state evolution, detailed explanations of
temporal and spatial feature information are still lacking
[19–21].
Because various factors affect the travel time of the road
network, only by thoroughly understanding these factors
can we better plan and manage urban traffic. To better
complement the point, this study conducts an exploratory
analysis of traffic factors to understand the physical
mechanisms of the system by introducing a spatio-temporal
approach that only uses the nonlinear method, which includes empirical dynamic modeling (EDM) [22, 23] and
complex networks (CN) [24]. The method provides a perspective of highlighting in an understanding way the spatiotemporal features without binding assumptions. EDM has
been verified promising in nonlinear testing of time series,
external driving factors for the evolution of mining systems
[25]. Therefore, EDM can be adopted to analyze travel time’s
influencing factors and quantify the causal relationship
between these factors. Simultaneously, the urban road
network’s topological characteristics and spatial structure
play an irreplaceable role in the analysis of urban traffic
characteristics. To reveal it better, the study uses the relevant
knowledge of complex networks to deeply explore the spatial
structure characteristics of urban road networks and their
impact on urban traffic conditions.
Recently, extreme gradient boosting (XGBoost)
[26, 27], as an efficient implementation of gradient enhancement tree algorithm, has become promising candidates for short-term forecasting not only because it can
flexibly represent complex nonlinear traffic systems but
also because of its ability to map from input variables to
output variables directly. Simultaneously, it also has significant advantages to explain the features imported into a
predictive model. For example, Zhang and Haghani [28]
obtained quite good experimental results when applying
the gradient enhancement tree algorithm to predict the
travel time of road sections, and the model was well
interpreted. Nevertheless, the shortcomings in his paper are
only a few road sections which were concerned, the prediction of road network level was not considered, and the
selection of features was not explained in detail. To overcome these shortages and maximize the XGBoost forecast
method’s advantages, a hybrid prediction model named
EDMCN-XGBoost for spatio-temporal feature extraction
Journal of Advanced Transportation
and prediction of urban road network travel time is proposed in this research.
2. Methodology
2.1. Research Thinking and Architecture. Figure 1 shows the
complete pipeline of our proposed approach, consisting of
four interacting steps. First, data preprocessing is conducted
to transform raw data into understandable format. Second,
the complete mining process of EDM on road segment data
is carried out to explore the temporal dependence and
influencing factors of travel time. Third, the complex network theory is used to extract the spatial statistical features
of traffic networks. Finally, the linkages between spatialtemporal features and corresponding travel time of the road
network are built using XGBoost, and key features are
identified according to importance ranking and recursive
elimination. Afterwards, we establish the XGBoost predictive model.
2.2. EDMCN
2.2.1. Empirical Dynamic Modeling. Due to the stochastic
and nonlinear nature of actual traffic conditions, linear-based
methods are no longer suitable for the study of urban traffic
data, while the application of nonparametric methods in
traffic data mining has gained more attention. Empirical
dynamic modeling (EDM) is a data-driven nonparametric
modeling framework for nonlinear dynamic systems, including simplex projection, S-map, multivariate embedding,
convergent cross-mapping (CCM), and multiview embedding
[22, 29–33]. In contrast to fitting parametric equations used
by many pieces of research, EDM instead relies on data to
infer the operating mechanism of the dynamic system and
reveal the relationship among variables, because of the unknown mechanism equation. The main idea of EDM is to
regard time series as projections of complex dynamic system
behaviors. The state of the system is described as a point in the
high-dimensional space whose axis can be regarded as a
fundamental state variable, also known as an attractor. An
analogy would be with the traffic system. The travel time
variable is the projection point of the high-dimensional space
of the complex traffic system. The coordinate axis is the
primary state variable such as the lagging variable, the speed
variable, and the indicator variable. In general, many state
variables in complex systems are unobservable, but some
systems can recover their evolution using only time series. As
Takens’ theorem [33] tells us, although the behavior of a
system is nominally determined by the high-dimensional state
space, it can replace some unknown variables with its lag
variable, that is, construct the attractor using the lag variable.
Therefore, this paper studies the dimension and nonlinearity of the time series using EDM quantitative analysis.
Based on the nonlinear system’s dimension, the causal relationship between other external primary state variables
and the target variable is determined by the optimal lag
variable of the time series and the EDM convergent crossmapping test.
Journal of Advanced Transportation
3
Raw data
Input
Data preprocessing
Step 1: data preprocessing
EDM analysis for road segments
Step 2: temporal dependence and
quantitative analysis of influencing factors
Simplex projection
S-map test
Whether the self-lag effect can
reveal its own dynamic evolution
Step 3: spatial dependence and
quantitative analysis using complex network
Extract features
Road network topology
The number of lag
CCM test
Complex traffic network feature
Temporal features
Spatio-temporal features
Link space attribute
characteristics
The key spatio-temporal features
Step 4: train model
and forecast
Train XGBoost model
Output
Forecast result
Figure 1: Framework of the proposed method.
(1) System Dimension and Nonlinearity Testing. The univariate time series of the target variable (travel-time) is
analyzed and tested to find its optimal dimension and the
nonlinear degree. The optimal dimension (E) of the target
variable, determined by simple projection, is defined as the
lag number that can maximize the prediction ability. When
using the simplex projection method, the time series is
divided into two parts, one of which is used as a sample to
predict the remaining part. It is noteworthy that the simplex
projection method does not divide the training set and the
prediction set, and the prediction is performed outside the
sample. Simplex projection is a nonparametric analysis
method in the state space of dynamic systems that obtains
the prediction set by mapping the neighbors of the predicted
points. The nonlinearity of the dynamic system can be
quantified by the S-map analysis method (S-map represents
sequence-local weighted global linear mapping) to obtain
the nonlinear parameter (θ), which is the state dependence
of nonlinear systems.
(2) Causality Analysis. EDM can be used to reveal the relationship between different variables. According to the
Takens’ theorem, the univariate reconstructed attractor XZ is
the shadow version of the original multivariate attractor X,
i.e., topological invariance. Therefore, X s reconstructed
′
shadow version gives a one-to-one mapping with X. Furthermore, when there is a causal relationship between two
variables, it will lead to a one-to-one mapping of their
corresponding reconstructed attractors (e.g., XZ′, Xp′) [23].
Based on the principle and the simplex projection method,
Sugihara et al. [30] proposed a convergent cross-mapping
(CCM) method for testing the causal relationship between a
pair of variables in a dynamic system. If two variables belong
to the same variable system with causal links, it must be
convergent. The convergence means that the cross-mapping
technique (p) increases as the size of the library increases,
because the more data in the library, the greater the density
of reconstructed polylines, and then the higher resolution
attractors can improve the prediction accuracy based on
neighbors (i.e., simplex projection).
2.2.2. Complex Networks. Originated from graph theory,
complex network is a large-scale complex system theory tool
applied to the real world. In fact, the urban traffic state has
spatial autocorrelation characteristics, which are affected by
4
Journal of Advanced Transportation
spatial structure and topology features of road networks.
Previous studies have identified the prominent impact of
network spatial features and hierarchical features [34, 35] on
the network traffic flow. Therefore, this paper mainly applies
the theory of complex networks to mine the topological
structure characteristics and network structure characteristics of urban traffic networks. For details, please refer to
Section 4.3.
2.3. Extreme Gradient Boosting Prediction Model. XGBoost
[27], an integrated learning parallel processing algorithm
based on tree structure, is nonparameterized and can deal
with the complex nonlinear relationships between features.
Compared to other algorithms, XGBoost has higher interpretability, predictive accuracy, and computational speed.
The idea of this algorithm is to continuously add trees,
continuously perform feature splitting to grow a tree and
each time to add a tree, needing to learn a new function to fit
the last predicted residual. When we intend to forecast a
sample score, we should get K trees through a training
sample dataset. This process corresponds to the characteristics of the sample, each characteristic will fall into a corresponding leaf node, and each leaf node will correspond to a
score. Finally, we need to add each tree’s corresponding
score to obtain the predicted value of the sample. XGBoost’s
tree integration model is essentially a set of classification
regression trees (CARTs). Each tree (CART) means a decision model f(·). The XGBoost algorithm uses model integration; to be precise, it uses k decision tree models to
output results based on input xi and sums the K output
. The set of decision tree models is called F.
results to obtain y
K
1
Ω(f) � cT + λ‖w‖2 .
2
(4)
Equation (4) represents the regularization term for the
decision tree function f, where c, λ are regularization coefficients, corresponding to gamma and lambda in XGBoost’s
parameters; T represents the number of leaf nodes of the
decision tree function f; and ‖w‖2 represents the sum of the
output squares of all leaf nodes of the decision tree function f.
Equation (4) is used to control the variance of the fitting
in order to enhance the flexibility of learning task and to
obtain a better prediction model for unknown data.
Therefore, the over-fitting of training data is avoided and the
complexity of prediction model is penalized.
As can be seen from the above, the newly generated tree
is the new mapping relationship to fit the residual of the last
prediction, so when after generating t trees, the predicted
(t)
(t−1)
value can be written as y
+ ft (xi ), so the target
i � y
i
function can be rewritten as equation (3).
In order to get the minimum value of the objective
function, XGBoost approximates it with Taylor’s secondorder expansion, so that the objective function can be approximated as
n
1
(t−1)
obj(t) ≈ lyi , y
+ gi ft xi + hi f2t xi + Ω ft ,
i
2
i�1
(5)
(t−1)
where gi � zy(t−1) l(yi , y
) is
i
i
(t−1)
z2 (t−1) l(yi , y
) is the second
i
y
the first derivative and hi �
derivative.
Since the prediction score of the former t − 1 tree does
not affect the optimization of the objective function, it can be
neglected, so that the objective function can be simplified as
i
� ∅ xi � fk xi ,
y
(1)
k�1
F � f(x) � wq(x) ,
function; and Ω is the regularization term. The regularization term is shown as
m
T
q: R ⟶ T, w ∈ R ,
n
where F is a set that incorporates k decision tree models,
among them F � f1 (·), f2 (·), . . . , fk (·); fk (·) represents
the input/output function relationship of the kth regression
tree; andfk (·) corresponds to the structure q and leaf node
weight w of the kth regression tree.wi represents the score of
the ith leaf node. The first few steps of actually fitting the
(0) � 0; (2) y
(1) � f1 (xi ); (3)
predicted value include (1) y
(2) � f1 (xi ) + f2 (xi ) � y
(1) + f2 (xi ). Therefore, the tth
y
iteration can be displayed as
t
(t− 1) + ft xi .
(t) � fk xi � y
y
(2)
k�1
Further optimization of the objective function of the
prediction model can be obtained by fd3
n
(t−1)
obj(t) � lyi , y
+ ft xi + Ω ft ,
i
(3)
i
(t−1)
where y
represents the prediction of the actual value i at
i
(t−1)
iteration t − 1; l(yi , y
) corresponds to the training loss
i
1
(t−1)
(t) ≈ ly , y
obj
+ gi ft xi + hi f2t xi + Ω ft .
i i
2
i�1
(6)
Equation (6) sums the loss function values of each
sample. According to the abovementioned analysis, each
sample will eventually fall to a leaf node, so all the samples of
the same leaf node can be reorganized as follows:
n
1
obj(t) ≈ gi ft xi + hi f2t xi + Ω ft
2
i�1
n
1
1 T
� gi wq(xi ) + hi w2q(xi ) + cT + λ w2j
2
2 j�1
i�1
(7)
T
⎟
⎜
⎜
⎠w2j ] + cT.
⎝ gi )wj + 1 ⎛
⎝ hi + λ⎞
� ⎡⎢⎢⎣⎛
2
j�1
i∈I
i∈I
j
j
It can be seen from the abovementioned analysis that the
objective function can be transformed into a unary quadratic
function about the leaf node score w, and the optimal w and
Journal of Advanced Transportation
5
the objective function obj can be directly solved by using the
vertex formula of the unary quadratic function.
wj∗ � −
Gj
,
Hj + λ
1 T Gj
obj � −
+ cT,
2 j�1 Hj + λ
(8)
∗
where Gj � i∈Ij gi ; Hj � i∈Ij hi .
In the previous lifting tree model, the classification regression trees are generally arranged in a sequence, but the
trees generated by the XGBoost training are arranged in a
parallel manner. When the XGBoost training data fits a
model, all the cores of the computer CPU are called to
construct the tree itself in a parallel way, which in turn
increases the speed of calculation. For more details on the
XGBoost model, see literature [27].
3. Data Description
The dataset used in this study comes from the Alibaba Cloud
Tianchi dataset platform, the actual desensitization data of a
specific location in Guiyang, providing the attributes of
urban road segments and travel time of each segment during
the historical period (April 2017). The research dataset is
from 132 roads of a particular place in Guiyang, including
the average travel time of motor vehicles passing the road
sections. The period is 6:00–9:00, 14:00–16:00, and 17:00–19:
00 in April 2017, and the time interval is 2 minutes. It should
be noted that the original data is not aggregated. Some
attributes of the road network are shown in Figure 2.
3.1. Data Preprocessing. The target variable shows a long tail
distribution through statistical analysis of the dataset, indicating that the overall quality of the dataset is good, but
there is still a small number of unreasonable extreme values.
To eliminate the influence of abnormal data, the paper first
carried out noise reduction processing on data. Next, the
target variable is transformed by log; after the log transformation, the target variable presents a normal distribution,
which is more suitable for input into the prediction model to
fit.
Another problem with the dataset is the existence of
missing values. In April 2017, there were 1069200 sample
data, but the actual data were only 973978, so there were
95222 missing data. Although XGBoost has a good handle
on missing values, due to the nature of time-series data,
the values at first few moments have a significant impact on
the values at the next moments. Therefore, to minimize the
impact of missing values, this study uses the trend of the data
to complete the missing data.
3.2. Road Network Topology. The topological characteristics
and spatial structure of the urban road network play an
irreplaceable role in the analysis of urban traffic characteristics. In this study, the relevant knowledge of complex
network is employed to deeply explore the spatial structure
characteristics of urban road networks and their impact on
urban traffic conditions. The road network studied in this
paper includes 132 road sections and their upstream and
downstream relationships. The actual dataset does not
contain the geographic coordinates of the real road sections
and only provides the adjacency relations of 132 road sections. The topology structure of the road network is
expressed according to the adjacency relation of road sections [36, 37], as shown in Figure 3.
4. Spatio-Temporal Features Extraction
and Analysis
4.1. Basic Spatio-Temporal Characteristics. The attributes of
the road network dataset include the link-ID of each road
section, travel time of the two-minute slice, and the relationship between the upstream and downstream sections. A
large number of basic spatio-temporal features can be
extracted from the raw data, as shown in Table 1. Nos. 1–3
are the lagging values of the target variable; Nos. 4–6 represent the timing variables; Nos. 7–10 are the time and space
shift variable of the target variable; Nos. 11–16 are the indicator variable; Nos. 17–19 are some basic spatial properties
of the road segment.
Features are derived values from raw data and used as
input to a machine learning algorithm. High-quality features
(e.g., being informative, relevant, interpretable, and nonredundant) are the basis for modeling and problem-solving
and generating reliable and convincing results. Due to the
spatio-temporal characteristics of urban road network travel
time, this study focuses on two kinds of features: time-related and spatial correlation features. First, the EDM method
is used to analyze the basic time-series features quantitatively. Then, the theory of complex network is leveraged to
deeply dig the urban road network to obtain its spatial
characteristics.
4.2. Quantitative Analysis by Empirical Dynamic Modeling.
The results of the simplex projection of the dataset show that
the optimal embedding dimension (E) of the univariate
analysis of the travel time sequence of the road segment is six
(see Figure 4(a)). The nonlinear test of the travel time series
by S-map shows that the dataset exhibits a relatively high
nonlinearity in θ � 2 (see Figure 4(b)). Forecast skill (ρ)
represents the Pearson correlation coefficient (ρ) between
the observed value and the predicted value by the EDM
method, which is used to maximize the prediction performance. The above indicates that it is reasonable to use the
EDM method to process and analyze the data. It should be
reminded that before using the empirical dynamic modeling,
the dataset needs to be normalized to eliminate the influence
of different data units.
4.2.1. EDM Multivariate Analysis. Firstly, a prediction model
is established by using EDM. The prediction of travel time is to
use the maximum available time lag determined by the simplex
projection. The target variable (travel time at time t) is fixed
time coordinates, and new coordinates (1, 2, . . ., or (E−1)) are
6
Journal of Advanced Transportation
Traffic direction
Link 1
Width
Link 2
Length
(a)
Link 3
Link 1
Link 4
Link 2
Link 2
Link 5
(b)
Figure 2: Main spatial information of roads. (a) The attribute information of links. (b) The upstream and downstream relationship of road
segments.
Figure 3: Road network topology map (desensitized data).
considered as the construction model predictor coordinates.
Correctly, the delay effect of the target variable itself is used as a
predictor, and the “block-lnlp” function in EDM can be used
to accurately show whether the lagging effect of the target
variable itself can fully explain the complex system dynamics
of the target variable. The result in Figure 5 shows that the lag
coordinates of travel time itself cannot reveal its complex
dynamic evolution. After adding the external driving factor of
the indication, the model prediction skill is not significantly
improved, which indicates that the evolution of travel time
series is a highly nonlinear complex phenomenon, and the lag
variable of the target variable is a very crucial driving factor
that is of greater importance than other external driving
factors such as a vacation.
The driving factors affecting travel time evolution have
been proved to be diverse and complex, so if we want to
restore the complex dynamic behavior of travel time series as
much as possible, it is necessary to find more external
driving factors.
4.2.2. Nonlinear Causality Test. It can be seen from the
abovementioned analysis that the evolution of urban road
network travel time is a highly nonlinear complex
phenomenon. According to the results of EDM multivariate analysis, the five important driving factors of travel
time evolution can be preliminarily determined as TTt−1,
TTt−2, TTt−3, TTt−4, and TTt−5. On this basis, the nonlinear
causality relationship (CCM) test is applied to identify the
travel time-driven time series variables, time-space timeshifting features, time-indicating features, and state-indicating features of the target road segments. Sugihara
et al. [29, 30] developed a cross-mapping algorithm to test
the causation between a pair of variables in dynamic
systems. If there is a causal relationship between two
variables, the cross-mapping between them shall be
“convergent.” Convergence means that the cross-mapping
skill (ρ) improves with increasing library size [23]. A
typical road segment determines the causal association
between the following characteristics and the target
variable, specifically Minute, Hour, Dayofweek, Dayofyear, Vacation, Average_speed, out_link_lagging, T1, etc.
It should be noted that the black-marked numbers in
Table 2 are identified as causal variables. The detailed
quantitative analysis results are shown in Table 2 and
Figure 6.
Note that, the direction of cross-mapping is opposite to
the direction of cause-effect in Figure 6. According to the
cross-convergence test of some external driving factors, the
magnitude of the cross-mapping rise of external factors is
not high. Therefore, based on the quantitative analysis results, it can be determined that there is no strong causal
relationship between these features and target variables, only
a medium causal or weak relationship.
The interesting point is the first picture and the last
picture of Figure 6. The CCM test does not treat time-space
time-shifting features (in_link_lagging) in some road
segments as a powerful causal, even weak causal association features. By contrast, another time-space timeshifting feature (out_link_lagging) is regarded as a causal
connection feature. Maybe the time step is quite long
regarding the urban traffic dynamic. It can explain that no
relationship between the upstream and downstream sections is statistically highlighted. From the perspective of
urban traffic congestion, the downstream section’s causal
Journal of Advanced Transportation
7
Table 1: The basic spatio-temporal features.
Symbol
TTt−1
TTt−2
TTt−3
T1
H1
Average_speed
in_link1_lagging1
in_link2_lagging1
out_link1_lagging1
out_link2_lagging1
Minute
Hour
Dayofweek
Dayofyear
Vacation
Link-ID
Length
Width
Area
Description
The first lagging values of target variable
The second lagging values of target variable
The third lagging values of target variable
The first values before the start of the forecast period
The true values of the hour before the forecast period
The first speed before the prediction point
The true values of the ith upstream road of the target
Road before the predicted point
The true values of the ith downstream road of the target
Road before the predicted point
The minute of the forecast point
The hour of the forecast point
The forecast point represents the day of the week
The date of the forecast point
Holiday or not
The ID of each road segment
The length of each road segment
The width of each road segment
The area of each road segment
0.62
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
0.70
0.60
Forecast skill (rho)
Forecast skill (rho)
0.68
0.58
0.66
0.64
0.62
0.56
5
Embedding dimension (E)
10
4
Nonlinearity (theta)
(a)
8
(b)
Figure 4: The embedding dimension and nonlinear analysis of travel time series. (a) Simplex projection. (b) S-map nonlinear analysis.
3
Predicted
2
1
0
–1
–2
–2
–1
0
1
2
Observed
(Lagging alone) rho = 0.61
(Lagging and vacation) rho = 0.61
Figure 5: The influence of lagging variables on the evolution of target variable sequences.
3
8
Journal of Advanced Transportation
Table 2: CCM test of external driving factor and its correlation with target variables.
Candidate variable (xi)
Minute
Hour
Dayofweek
Dayofyear
Vacation
T1
H1
Average_speed
in_link_lagging
out_link_lagging
Cross-map skill (travel_time∼xi)
0.1030
0.0824
0.0718
−0.0364
0.0303
0.0681
0.0082
0.0540
−0.0240
0.1171
0.225
0.12
0.12
0.200
0.08
0.06
0.04
0.02
0.175
Predictive skill (p)
Predictive skill (p)
0.10
Predictive skill (p)
Linear correlation (travel_time∼xi)
−0.0143
0.0072
−0.1250
0.0125
−0.0484
0.6939
0.6701
−0.2800
0.0140
0.1644
0.150
0.125
0.100
0.075
0.00
0.050
–0.02
0.025
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap in_link_lagging
In_link_lagging xmap travel_time
0.10
0.08
0.06
0.04
0.02
0.00
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap t1
T1 xmap travel_time
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap minute
Minute xmap travel_time
0.08
0.10
0.06
0.04
0.02
0.00
0.10
Predictive skill (p)
0.12
Predictive skill (p)
Predictive skill (p)
0.14
0.10
0.08
0.06
0.04
0.02
0.08
0.06
0.04
0.02
0.00
0.00
–0.02
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap dayofweek
Dayofweek xmap travel_time
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap hour
Hour xmap travel_time
1.0
0.06
0.04
0.150
0.00
–0.02
–0.04
–0.06
Predictive skill (p)
0.9
0.02
Predictive skill (p)
Predictive skill (p)
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap dayofyear
Dayofyear xmap travel_time
0.8
0.7
0.6
–0.08
0.5
–0.10
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap vacation
Vacation xmap travel_time
0.125
0.100
0.075
0.050
0.025
0.000
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap average_speed
Average_speed xmap travel_time
200 400 600 800 1000 1200 1400
LibSize
Travel_time xmap out_link_lagging
Out_link_lagging xmap travel_time
Figure 6: External driver factor CCM test for the travel time series.
impact on the target section is following the traffic jam
propagation law. It can be seen from Table 2 that the linear
relationship cannot be related as the basis for judging the
causal connection.
4.3. Complex Traffic Network Spatial Characteristics. To
further reveal the spatial dependence of the road network, it
is necessary to dig out more valuable information from the
perspective of spatial characteristics. Based on the raw
Journal of Advanced Transportation
9
dataset, we can extract some essential spatial characteristics,
that is, length, width, area, and link-ID of different road
segments, but the above four features are only the primary
attributes of a road segment and do not involve the spatial
correlation characteristics between road segments. Therefore, this study tries to understand the traffic network from
the perspective of the complex network to mine useful
features and obtain the quantitative characteristics of
mathematical statistics of a large number of complex traffic
networks such as degree, in-degree, out-degree, closeness
centrality, the importance of nodes, betweenness-centrality,
degree-centrality, and PageRank. To explore the spatial
aggregation behavior of different road segments, the community structure is introduced into the complex traffic
network. The following is a detailed explanation of some
spatial features.
4.3.1. Complex Traffic Network Characteristic Parameters.
Complex networks have been widely concerned by traffic
scholars. A large number of studies have found that
transportation networks have the same complex network
structural characteristics as social networks and computer
networks. Therefore, a road network of a certain place in
Guiyang city modeled by the dual-method is taken as the
research object [37], and the complex network is adopted to
study the interaction between the structural characteristics
and the topology of the traffic network. The research is
shown below.
(1) Degree of node. The degree of a node represents the
number of nodes directly connected to the node. In a
directed graph, the degree of a node is divided into
the degree of out (out_degree) and the progress
(in_degree), indicating the intensity of the road
segment in the road network to some extent. Outdegree is expressed as the number of road segments
connected downstream of the road segment, and indegree means the number of road segments connected upstream. Thus, the higher the degree (in or
out), the node has a more prominent connection
effect in the road network.
(2) Closeness centrality. The closeness centrality of a
node in the network measures the proximity of the
node to all other nodes. The larger the value of
closeness centrality, the more central the node is in
the road network, and the faster it can reach other
nodes. Therefore, it is used to emphasize the values of
the different nodes in a traffic network; the specific
calculation method is as follows:
Cc (i) �
N−1
,
N
j�1 dij
(9)
where dij means the shortest distance from node i to
node j and N represents the number of road
segments.
(3) Node importance ordering. The typical node importance ranking methods are PageRank, LeaderRank,
Figure 7: Community structure of the traffic network.
and HITS algorithms, among which PageRank [38] is
the core sorting algorithm of Google search. The
main idea of the PageRank algorithm is that if the
quality of webpage A is high, and page A points to
page B, then the quality of page B is also high. Because the actual page links are much more complex,
iterations are required to get the final result of the
page sort. It is mapped to a complex traffic network
with specific quantized PageRank values (referred to
as PR) after repeated iterations. The specific calculation steps are mainly referenced in [34].
4.3.2. Complex Networks Community Detection.
Community detection, also known as community discovery,
is a way to reveal network aggregation behavior. Commonly
used community discovery algorithms include Louvain,
label propagation, and infomap. After comprehensive
comparisons, this study adopts the infomap algorithm to
identify community clusters of road network topology, and
the specific calculation steps have mainly referenced the
literature [31].
Taking the complex traffic network topology (dual-map
structure) of a certain place in Guiyang city as input, the
community division result of the traffic network is obtained
in Figure 7, where nodes of the same color represent the
same community. Each node represents a specific road
segment, and each arrow indicates the upstream and
downstream relationship of adjacent nodes.
4.4. Feature Extraction Results. Quantitative analysis characteristics of each road segment are obtained by the EDM
method; it has obtained a large number of spatial statistical
features of urban traffic networks through complex network
theory. The abovementioned steps make deep excavation
and quantitative description of the spatio-temporal features,
which greatly enhance the interpretability and richness of
the features. However, through the EDM method, the
10
Journal of Advanced Transportation
Table 3: The key spatio-temporal features of the traffic network.
TTt−1
T1
Vacation
in_degree
out_link1_lag1
TTt−2
Average_speed
Length
out_degree
out_link1_lag2
Predictors (symbol)
TTt−3
Minute
Width
closeness_centrality
quantitative analysis results of each road segment are not
precisely the same. If the analysis result of each road segment
is taken as the feature input of the road network, it will cause
a lot of information redundancy and lead to conflicting
results. Therefore, it is necessary to extract the road network
characteristics that conform to most road segments. The
characteristics of the road network extracted through the
complex network are plentiful and specific, but at the same
time, there is also redundancy. It is necessary to streamline
the complex network special features to seek the causality
between those features with the target variable.
The relationship between spatio-temporal features and
travel time of road segments is established by XGBoost, and
redundant variables are removed by its feature importance
ordering and recursive elimination methods. Table 3 shows
the road network spatio-temporal features finally determined for this study.
5. Analysis of Experimental Results
The travel time prediction of the traffic network is a critical
step in constructing an urban intelligent transportation
system. According to the above empirical dynamic modeling
and quantification results, the evolution of travel time in an
urban road network is proved to be highly nonlinear, which
is difficult to predict by purely mechanical equations. Thus,
to accurately forecast the evolution of complex time series, it
is necessary to extract the key driving factors of system
evolution from the data itself. Combining the broad prospects of data-driven methods in traffic prediction applications in recent years and the considerable advantage of tree
models based on parallel integration processing in dealing
with the relationship between a large number of feature data
and processing features, this study decides to adopt XGBoost
as the predictive model.
5.1. Dataset Description and Division. In the experiment,
80% of the data is used as the training set and 20% as the test
set. In other words, the data from April 1 to April 24, 2017, is
taken as the training set to predict the travel time in the
traffic network from April 25 to 30, 2017. The main target
period in this study is the morning peak (8:00–9:00), the
noon stable period (15:00–16:00), and the evening peak (18:
00–19:00). The topology of the road network is detailed in
Section 3.2.
Table 4 shows the model input and output, the first 21
lines (f0∼f21) in the table are the eigenvalue data as input, and
the last line is the output value of the XGBoost model. It
should be pointed out that some of the data in the model are
TTt−4
Hour
Area
PageRank
TTt−5
Dayofweek
Link_ID
infomap
No.
1∼5
6∼10
11∼15
16∼20
21–22
log-transformed, and the output data also experience the log
conversion. So, when we verify the prediction result, the
transformed data should be restored to the original scale.
5.2. Model Optimization. In the machine learning model,
data quality is the root cause of the prediction accuracy, but
different parameter combinations will also have a particular
impact on the prediction model. Therefore, the model is
compared several times through the experiment, and the
evaluation index used in the comparison process is the root
mean square error (RMSE). The derived optimal parameter
combination of the research dataset is determined, as shown
in Table 5.
5.3. Model Interpretation. It is well known that the prediction accuracy of the XGBoost model is very demanding
on the predictor variables (i.e., feature vectors). In order to
further analyze the influence of the characteristic variables of
EDMCN on the model output (response variables), the
feature importance is understood by the trained XGBoost
model (see Figure 8).
It can be seen from Figure 8 that TTt−1 is the most critical
characteristic variable, indicating that the value at the
previous moment of the prediction point has the most
considerable influence on the next point. It is consistent with
our common sense, and the performance of other lagging
variables demonstrates that the lagging variables selected
before the model input are also reasonable. It is proved that
the evolution of travel time series is highly time-dependent,
and the time dependence is highly valued in urban traffic
management. Average_speed is also a vital feature. In urban
traffic management, attention should be paid to the driving
speed of road sections. Surprisingly, the T1 feature performed well. The inspiration to us in this article is to pay
attention to before making predictions the first values before
the start of the forecast period, which may be significant.
Simultaneously, the space-time time-shifted variables,
out_link1_lag1 and out_link1_lag2, we need to the critical
focus on spatio-temporal variable, are outstanding, which is
also in line with the law of congestion propagation of roads.
Among the time indication features, Minute performs the
best, which indicates that in the management and control of
urban traffic, attention should be paid to the excellent time
granularity. Among the spatial indication features, link_ID
and length are the most prominent, which means that each
road needs careful management and detailed analysis of its
characteristics in urban traffic management. Among the
sophisticated traffic network features, closeness_centrality,
PageRank, and infomap perform better. It means that the
Journal of Advanced Transportation
11
Table 4: XGBoost model input and output.
Symbol
Minute
Hour
Dayofweek
Vacation
in_degree
out_degree
Length
Width
Area
Link_ID
T1
closeness_centrality
PageRank
infomap
TTt−1
TTt−2
TTt−3
TTt−4
TTt−5
Average_speed
out_link1_lag1
out_link2_lag1
TTt
0
8
2
0
1
1
48
3
144
43
1.931
0.053
0.004
5
2.197
1.995
2.197
2.197
2.140
6.000
2.360
−999
2.042
2
8
2
0
1
1
48
3
144
43
1.931
0.053
0.004
5
1.903
2.197
1.995
2.197
2.197
8.403
2.379
−999
1.932
The values of features
6
8
10
8
8
8
2
2
2
0
0
0
1
1
1
1
1
1
48
48
48
3
3
3
144
144
144
43
43
43
1.931
1.931
1.931
0.053
0.053
0.053
0.004
0.004
0.004
5
5
5
1.918
1.921
1.918
1.911
1.918
1.921
1.903
1.911
1.918
2.197
1.903
1.911
1.995
2.197
1.903
8.262
8.231
8.264
2.370
2.476
2.595
−999
−999
−999
1.948
1.948
1.950
4
8
2
0
1
1
48
3
144
43
1.931
0.053
0.004
5
1.911
1.903
2.197
1.995
2.197
8.329
2.360
−999
1.949
12
8
2
0
1
1
48
3
144
43
1.931
0.053
0.004
5
1.906
1.918
1.921
1.918
1.911
8.378
2.803
−999
1.945
14
8
2
0
1
1
48
3
144
43
1.931
0.053
0.004
5
1.899
1.906
1.918
1.921
1.918
8.447
2.687
−999
1.937
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
...
No.
f0
f1
f2
f3
f4
f5
f6
f7
f8
f9
f10
f11
f12
f13
f14
f15
f16
f17
f18
f19
f20
f21
...
Table 5: The optimal parameter combination of the XGBoost prediction model.
n_estimators
200
eta
0.26
min_child_weight
66
max_depth
7
colsample_bytree
0.7
Lambda
1
Features
Feature importance
f14
f19
f10
f15
f16
f0
f18
f20
f17
f9
f21
f6
f13
f12
f2
f1
f11
f8
f3
f5
f7
f4
1989
1915
1747
1745
1474
1304
1303
1275
1157
1102
1023
662
471
430
418
348
324
304
186
135
130
44
0
250
500
750
1000
1250
1500
1750
F score
Figure 8: F score of feature vector in the EDMCN-XGBoost model.
2000
Gamma
0.2
Journal of Advanced Transportation
Travel_time (s)
12
80
The morning peak
8:00-9:00
The noon stable period
15:00-16:00
60
The evening peak
18:00-19:00
40
20
0
20
40
60
80
Time (4/25/2017, one unit = 2 min)
Original value
Predicted value
Travel_time (s)
(a)
60
The morning peak
8:00-9:00
40
The evening peak
18:00-19:00
The noon stable period
15:00-16:00
20
0
20
40
60
80
Time (4/26/2017, one unit = 2 min)
Original value
Predicted value
(b)
Travel_time (s)
100
75
The morning peak
8:00-9:00
50
The noon stable period
15:00-16:00
The evening peak 18:00-19:00
25
0
20
40
60
80
Time (4/27/2017, one unit = 2 min)
Original value
Predicted value
(c)
Travel_time (s)
60
The morning peak
8:00-9:00
40
The noon stable period
15:00-16:00
The evening peak
18:00-19:00
20
0
20
40
Time (4/28/2017, one unit = 2 min)
Original value
Predicted value
(d)
Figure 9: Continued.
60
80
Journal of Advanced Transportation
13
Travel_time (s)
40
The noon stable period
15:00-16:00
30
The evening peak
18:00-19:00
The morning peak
8:00-9:00
20
10
0
20
40
60
80
Time (4/29/2017, one unit = 2 min)
Original value
Predicted value
(e)
Travel_time (s)
Vacation
100
The morning peak
8:00-9:00
50
The evening peak
18:00-19:00
The noon stable period
15:00-16:00
0
20
40
60
80
Time (4/25/2017, one unit = 2 min)
Original value
Predicted value
(f )
Figure 9: The prediction effect of the EDMCN-XGBoost model.
evolution of travel time in the urban road network is spatially dependent. Therefore, urban traffic management and
control need to consider the specific characteristics of each
road and the spatial connection between roads. The unexpected point is that in_degree has the worst performance in
this study.
Figure 9 shows the comparison between the travel times
of the 132 roads in Guiyang and the travel times predicted by
the EDMCN-XGBoost model from April 25, 2017, to April
30, 2017. The blue line represents the actual travel time, and
the cross represents the travel time predicted by the forecast
model. The marked morning peak, noon stable period, and
evening peak in the figure represent the time concept of
urban traffic commuting and does not mean the road’s
actual travel status. The horizontal axis 0–29 represents the
morning peak 8:00–9:00, 30–59 represents the noon stable
period 15:00–16:00, and 60–89 stands for the evening peak
18:00–19:00. Each unit in the horizontal axis denotes two
minutes.
The time scale of the vertical axis in Figure 9 is measured
in seconds to reflect the real-time variation law of the traffic
state. It can be seen that the overall performance of XGBoost
is quite well, and the prediction effect of different roads in
different periods is useful. It is worth noting that some roads
of the road network are not strictly following the morning
Table 6: Comparison between the forecasting model and baseline
model evaluation index.
Forecast model
ARIMA
ANN
SVR
XGBoost
CN-XGBoost
EDM-XGBoost
EDMCN-XGBoost
MAPE (%)
20.627
20.653
20.265
19.262
19.111
18.744
18.695
MAE
3.576
3.740
3.381
3.319
3.305
3.256
3.230
RMSE
7.688
8.080
7.477
6.858
6.840
6.728
6.710
R2
0.879
0.866
0.885
0.903
0.904
0.908
0.909
and evening peak travel rules, and the holiday travel rules are
also different from the usual. Although the travel time rules
of different road segments at different periods are different,
the spatio-temporal dynamics of this travel time can still be
captured by the model and accurately predicted.
5.4. Model Comparison. In order to test the validity of the
EDMCN-XGBoost model, the currently accepted traffic
prediction baseline models ARIMA [8], ANN [10], and SVR
[13] and some hybrid models are comprehensively evaluated, and four classical evaluation index functions MAPE,
MAE, RMSE, and R2 are used to evaluate the prediction
models.
14
Journal of Advanced Transportation
140
120
120
Travel_time (s)
Travel_time (s)
100
80
60
40
20
100
80
60
40
20
0
0
0
10 20 30 40 50 60 70 80 90 100 110 120 130
Link
Original
ARIMA
ANN
SVM
EDMCNXGB
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Link
Original
XGB
CNXGB
(a)
EDMXGB
EDMCNXGB
(b)
Figure 10: The actual operation status of the road network at a specific moment and the predicted values of different models.
(a) Longitudinal comparison of predictive models. (b) Horizontal comparison of predictive models.
1 Q ti − ti
The mean absolute percentage error : MAPE �
,
Q i�1 ti
The mean absolute error : MAE �
1 Q
t − t ,
Q i�1 i i
�����������
2
1 Q
The root mean square error : RMSE �
t − t ,
Q i�1 i i
The coefficient of determination : R2 � 1 −
2
i ti − ti
2 ,
i ti − ti
(10)
where ti represents the actual value; ti is the predicted value;
ti represents the mean of the test set; and Q represents the
number of test sets.
In this study, data from the first 24 days of April (see
Section 5.1) were selected as the training set, and data from
the last six days of April were used as the verification set. To
thoroughly verify the validity of the EDMCN-XGBoost
model, it was compared with other prediction models from
vertical and horizontal directions. For ensuring the fairness
of the input of different predicted models. In the longitudinal comparison, the (p, d, q) parameters obtained by the
ARIMA model based on the dataset optimization in the
longitudinal comparison are (4, 0, 2); the inputs to the ANN
and SVR models are based on the features of the EDMCN
screening. In the horizontal comparison, when the study
relies solely on XGBoost, the input of the lagging variables is
determined by ARIMA’s 4; the spatial features of road
segments are length, width, area, and link-ID; XGBoost’s
feature importance ranking selects features variables. The
input of CN-XGBoost adds complex network features to the
XGBoost input, increasing the spatial connectivity between
roads. The input of EDM-XGBoost aims to select and filter
the feature vector of the roads by EDM quantitative analysis
before the input. It is to notice that the dataset is a bit short of
producing an efficient neural network, but it remains useful
for comparison [39].
It can be seen from the experimental results that the
effect of EDMCN-XGBoost is superior to other models from
a vertical and horizontal direction. From comparison, result
further demonstrates the scientific validity of spatio-temporal features from EDMCN and more explanatory. One
thing to add is that it is useful to compare the prediction
performance according to the considered peak period instead of the full day-period, especially when aggregated
indicators are considered. The off-peak period is not of
paramount importance for the prediction as it is a stable
state. The specific experimental results are shown in Table 6.
Figure 10 compares the actual operating conditions of
the road network at a specific time interval (2 min) and the
forecast performance of different models. The x-axis indicates the link_ID of each road segment of the road network.
It can be seen from the comparison that the EDMCNXGBoost model performs best in prediction accuracy.
From the experimental results in Figures 8–10, and Table 6, the following conclusions can be drawn: (a) the
EDMCN-XGBoost forecast model is useful for forecasting the
travel time of the urban road network and superior to the
baseline models. (b) The evolution of travel time series is
dynamic and highly nonlinear, and the influencing factors are
relatively diverse. (c) Travel time prediction at the network
level must consider both spatial and temporal dependencies.
(d) During the peak period, the change of travel time in the
road network is more apparent, and the travel rules of the
traffic network on working days, weekends, and holidays are
not exactly the same. (e) It is necessary to construct highquality feature engineering as much as possible, making it
rich, interpretable, and nonredundant. Only in this way can
the evolution of time series be restored to the greatest extent.
Journal of Advanced Transportation
6. Conclusion
This study designed a framework for analyzing, mining,
quantifying, and predicting spatio-temporal travel time of
the urban road network. The framework is mainly divided
into the following steps: (a) firstly, a large amount of primary
feature data is obtained from the original data. (b) The EDM
is used to quantify the dynamic and highly nonlinear evolution of the travel time series. Meanwhile, the time series
lagging variable (TTt−i) is determined as the crucial variable
of travel time evolution for the strong causal relationship
between TTt−i and TTt. (c) The complex network theory is
adopted to explore the topological structure of urban traffic
networks deeply, and a large number of spatial statistical
features of the road network is obtained. (d) The feature
importance ordering and recursive elimination principle of
XGBoost are used to remove the redundancy of acquired
spatio-temporal features and finally establish the prediction
model. The example shows that the framework proposed in
this study performs well in obtaining high-quality features
that are rich, interpretable, and nonredundant to establish a
predictive model. The established XGBoost prediction
model is superior to other comparable models in both interpretability and prediction accuracy.
Overall, the road network travel rules are complex and
changeable, challenging to capture. By digging out the information behind raw data as much as possible, we can
better seize the evolution rules of urban traffic travel time. To
further improve the prediction accuracy of road network
travel time, based on the framework of this paper, we will
continue to refine the feature engineering of road network
travel time and try to combine more forecasting methods.
Data Availability
The terms of use of the data used in this study do not allow
the authors to distribute or publish the data directly.
However, these data can be obtained directly from Tianchi
dataset platform via the following webpage: https://tianchi.
aliyun.com/dataset/dataDetail?dataId�1079.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
The authors would like to thank the Alibaba Cloud Tianchi
platform which provided the traffic dataset for them and the
fund from the National Natural Science Foundation of
China (61873216).
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W2018186360.txt
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https://zenodo.org/records/2491637/files/article.pdf
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de
|
Über den Untersehied yon tierisehem und pflanzliehem Zwittertum
|
Molecular genetics and genomics
| 1,917
|
public-domain
| 537
|
266
Referate.
Correns~ C.~ 1916. Uber den Untersehied yon tierisehem und pflanzliehem
Zwittertum. (Biol. Ctbl. 36, S. 12--24,)
He r t w i g und D e m o 11 haben versucht, die Erklarung fiir Geschlechtstrennung und Vererbung, welchc die cytologischen Untersuchungen bei Anglostoma ergaben, auf dim Pflanzen zu tibertragen. Der Veff. bringt einen Beweis daftir, daft bei den Pflanzen eine Geschlechtsbestimmung nach dem
Angiostoma-Schema nicht erfolgt.
Die Verhaltnisse bei Angiostoma sind kurz die folgenden: Die 9
der getrenntgeschlechtlichen Generation haben 12, die c~c~ 11 Chromosomen;
alle Eier erhalten 6, yon den Spermatozoiden die Halfte 6, die Halfte 5 Chromosomen. Die letzte Gruppe geht zugrunde, es kommen nur die Spermatozoiden mit 6 Chromosomen zur Befruchtung und geben mit den Eiern zu
6 Chromosomen weibchentihnliche Zwitter. Bei den Reifeteilungen dieser
Zwitter erhalten wieder alle Eier sowie die Halfte der Spermatozoiden
6 Chromosomen; bei der bleibenden HMfte wird 1 Chromosom ausgeschaltet;
beide Arten yon 8permatozoiden sind befruchtungsfiihig und geben 9 9 mit
12 und c~c~ mit 11 Chromosomen.
In gleieher Weise nehmen die oben gen. Autoren an, daft bei
den Pflanzen die Zwitterbliite und die 9 Bltite eines MonSeisten in
ihrem Fruchtknoten nur einerlei Samenanlagen mit weiblichem Chromosomenbestand haben, dagegen die Staubbeutel zweierlei PollenkSrner, zu
gleichen Teilen mit mannlichem und weiblichem Chromosomenbestand enthalten, yon denen die letzteren allein funktionsfahig sind - - d.h. n u r die
H a l f t e d e r P o l l e n k S r n e r f i i h r t e i n e B e f r u c h t u n g aus.
Um diese Theorie zu priifen, hat tier Veff. mit Pflanzen gearbeitet,
deren Pollentetraden nicht in ihre einzelnen KSrner zerfallen; so zunachst
mit Epilobium hirsutis, das indessen tiberhaupt nicht ansetzte, hierauf effolgreich mit der Solanacee 8alpiglossis variabilis.
Der Fruchtknoten enthiilt etwa 300--400 Samenanlagen; die hTarbe der
kastrierten Bliiten wurde mit 1--10 Tetraden belegt. So behandelt entwickelten sich, wenn man nur die gelungenen Versuche beriicksichtigt,
einmal bei 1 Tetrade 4 Samen, bei 3 Tetraden 9, bei 5 Tetraden 13, 13 und
16, bei 10 Tetraden 22, 24, 24, 25, 25 und 26 Samen. D . h . bei einer ganzen
Reihe yon Bltiten hatte m e h r als die doppelte Zahl der verwendeten Tetraden,
also mehr als die Halfte der verwendeten PollenkCirner befruchtend gewirkt;
ja in 2 Fallen (4 bei 1 Tetrade, 16 bei 5 Tetraden) miissen sogar alle 4 K(irner
einer Tetrade funktionstiichtig gewesen sein.
Damit ist der obigen Annahme der Boden entzogen. Andererseits
erhalt die schon friiher 1) ausgesprochene theoretische Erwagung des Verf.
dadurch erneut Gewicht, derzufolge vom philogenetischen Standpunkte aus
das Zwittertum bei Tieren und Pflanzen nicht in Parallele zu setzen ist.
Bei den l~lanzen geht die Entwicklung vom zwittrigen (Moos)zum getrenntgeschlechtlichen Organismus (Populus); bei den Tieren ist die Getrenntgeschlechtlichkeit philogenetisch alter, das Zwittertum erst ein abgeleiteter
Modus. Nimmt man mit S c h l e i p die MSglichkeit einer voraufgegangenen
primaren Zwittrigkeit bei den Tieren an, so ware die normale Zwittrigkeit
der Tiere philogenetiseh der sekundaren Zwittrigkeit bei einer infizierten
Melandryumpflanze zu vergleichen.
E. S c h i e m a n n .
1) Correns u. Goldschmidt, Die Vererbung und Bestimmung des Geschlechts.
1913.
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https://openalex.org/W4324129908
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https://healtheconomicsreview.biomedcentral.com/counter/pdf/10.1186/s13561-023-00428-9
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English
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The level of countries’ preparedness to health risks during Covid-19 and pre-pandemic: the differential response to health systems building blocks and socioeconomic indicators
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Health economics review
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cc-by
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Abstract The global health security (GHS) Index assesses countries’ level of preparedness to health risks. However, there is no
evidence on how and whether the effects of health systems building blocks and socioeconomic indicators on the
level of preparedness differ for low and high prepared countries. The aim of this study was to examine the contribu-
tions of health systems building blocks and socioeconomic indicators to show differences in the level of preparedness
to health risks. The study also aimed to examine trends in the level of preparedness and the World Health Organiza-
tion (WHO) regional differences before and during the Covid-19 pandemic. We used the 2021 GHS index report
data and employed quantile regression, log-linear, double-logarithmic, and time-fixed effects models. As robustness
checks, these functional form specifications corroborated with one another, and interval validity tests confirmed. The
results show that increases in effective governance, supply chain capacity in terms of medicines and technologies,
and health financing had positive effects on countries’ level of preparedness to health risks. These effects were con-
siderably larger for countries with higher levels of preparedness to health risks. The positive gradient trends signaled a
sense of capacity on the part of countries with higher global health security. However, the health workforce including
doctors, and health services including hospital beds, were not statistically significant in explaining variations in coun-
tries’ level of preparedness. While economic factors had positive effects on the level of preparedness to health risks,
their impacts across the distribution of countries’ level of preparedness to health risks were mixed. The effects of Social
Development Goals (SDGs) were greater for countries with higher levels of preparedness to health risks. The effect of
the Human Development Index (HDI) was greatest for countries whose overall GHS index lies at the midpoint of the
distribution of countries’ level of preparedness. High-income levels were associated with a negative effect on the level
of preparedness, especially if countries were in the lower quantiles across the distributions of preparedness. Relative
to poor countries, middle- and high-income groups had lower levels of preparedness to health risks, an indication
of a sense of complacency. We find the pandemic period (year 2021) was associated with a decrease in the level of
preparedness to health risks in comparison to the pre-pandemic period. There were significant WHO regional differ-
ences. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Da’ar and Kalmey Health Economics Review (2023) 13:16
https://doi.org/10.1186/s13561-023-00428-9 Da’ar and Kalmey Health Economics Review (2023) 13:16
https://doi.org/10.1186/s13561-023-00428-9 (2023) 13:16 Health Economics Review Open Access The level of countries’ preparedness
to health risks during Covid‑19
and pre‑pandemic: the differential
response to health systems building blocks
and socioeconomic indicators Omar B. Da’ar1,2,3* and Farah Kalmey3,4,5 Abstract Apart from the Eastern Mediterranean, the rest of the regions were more prepared to health risks compared *Correspondence:
Omar B. Da’ar
obdaar@gmail.com
Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 2 of 14 to Africa. There was a negative trend in the level of preparedness to health risks from 2019 to 2021 although regional
differences in changes over time were not statistically significant. In conclusion, attempts to strengthen countries’
level of preparedness to health shocks should be more focused on enhancing essentials such as supply chain capacity
in terms of medicines and technologies; health financing, and communication infrastructure. Countries should also
strengthen their already existing health workforce and health services. Together, strengthening these health systems
essentials will be beneficial to less prepared countries where their impact we find to be weaker. Similarly, boosting
SDGs, particularly health-related sub-scales, will be helpful to less prepared countries. Moreover, there is a need to
curb complacency in preparedness to health risks during pandemics by high-income countries. The negative trend in
the level of preparedness to health risks would suggest that there is a need for better preparedness during pandemics
by conflating national health with global health risks. This will ensure the imperative of having a synergistic response
to global health risks, which is understood by and communicated to all countries and regions. Backgroundh This paper examines the relationship between global
health security (GHS) versus health system building
blocks, socioeconomic indicators, and the World Health
Organization (WHO) regional differentials. The overall
global health risk preparedness index is constructed as
a composite measure, which is a linear combination of
the pillars of GHS, including prevention, detection, rapid
response, robust health system, compliance with inter-
national norms, and overall risk environment and vul-
nerability [1]. Specifically, we consider the questions: (a)
What are the levels of contributions of a country’s health
system building blocks and socioeconomic indicators to
the level of preparedness to epidemics and pandemics? (b) Do the effects of the health systems building blocks
and socioeconomic indicators differ for countries with
weak and strong levels of preparedness to epidemics and
pandemics? (c) What is WHO regional differentials in
GHS during Covid-19 and pre-pandemic periods?i The emergence and spread of infectious diseases with
pandemic potential occurred regularly throughout his-
tory. Although public health control efforts have been in
place for more than a century [7], protecting the health
and safety of people remains an imperative for govern-
ments [8]. Despite improvements in technologies, com-
munication, and health systems, countries around the
world still face a perfect storm of converging threats that
might substantially increase the risk of infectious disease
epidemics [8]. In particular, the last two decades saw the
emergence and reemergence of more deadly outbreaks,
epidemics, and pandemics of infectious diseases causing
widespread disruptions to all aspects of global health sys-
tems. Some of the diseases the world witnessed in recent
decades include severe acute respiratory syndrome
(SARS) coronavirus outbreak [9], H1N1 influenza [10],
cholera [11], Middle East respiratory syndrome coronavi-
rus (MERSCoV) [12], Ebola [13], Chikungunya and Zika
[14], and yellow fever [15]. Consequently, health security
has become increasingly important within the broader
context of health systems-strengthening, enhancing
responses to public health emergencies, and global cata-
strophic biological risks [16, 17]. WHO defines global health security as the prevention,
detection, and response to naturally emerging, acciden-
tal, and deliberate biological threats [2]. Keywords Global health security Health systems building blocks Pandemic preparedness And quantile regression Keywords Global health security, Health systems building blocks, Pandemic preparedness, And quantile regression
JEL classifications I100, I140, I150, I180 of the overall score of a country’s level of preparedness
against socioeconomic factors. These factors include
income level, the Human Development Index (HDI), and
Sustainable Development Goals (SDGs) [1]. The value
preposition of improvements in socioeconomic indica-
tors in protecting population health cannot be denied. The SDGs, for instance, emphasize strengthening of early
warning, risk reduction, and management of health risks
by the year 2030 [5]. HDI remains a valuable tool for
guiding decision making and monitoring policymaking at
both national and subnational levels, especially on health
security capacities and capabilities in many countries [6].h Conceptual frameworkh The framework for analyzing health systems building
blocks, socioeconomics, geography, and preparedness to
health risks comes from the following generic aggregate
function: (1)
G = f (P, D, R) G = f (P, D, R) (1) While the GHS index provides for a framework to
assess the capacity of countries to prevent and mitigate
emerging health risk, the extent of the variation and
the levels of contributions of a country’s health system
building blocks and socioeconomic indicators to level
of preparedness to epidemics and pandemics is not well
known. Additionally, there is absence of evidence on
whether the effects of the health system essentials differ
for countries with weak and strong levels of prepared-
ness. Studies underscore the importance of these health
systems building blocks, especially in acting as a first line
of defense and signaling resilience during outbreaks of
health risks [27–30]. On the other hand, evidence shows
how a poor health system that lacks basic inputs or build-
ing blocks cannot prepare for or respond to crises such as
pandemics, posing a threat to health security [27, 31–33]. The absence of a robust health system has been shown
to impede effective response during health crises across
high and low income countries [27]. The main goals of
this paper were to provide empirical evidence in filling
this research gap on whether the effects of health sys-
tems building blocks and socioeconomic indicators differ
across the distribution of countries’ levels of prepared-
ness to health risks and examine WHO regional differen-
tials during Covid-19 and pre-pandemic period. where G is a measure of GHS index as a function of a vec-
tor of health system building blocks, including improved
health service delivery e.g., beds per 1000 population, the
health workforce development e.g. doctors and nurses
per 1000 population, information systems e.g., commu-
nications infrastructure, access to essential medicines
for supply chain in medicine distribution, health system
financing and leadership and governance effectiveness:
D is socioeconomic and development measures includ-
ing SDGs, HDI, and level of income categories per World
Bank classification; R represents the WHO regional
offices. Backgroundh The Center for
Disease Control (CDC) considers GHS as the existence
of a strong and resilient public health system that can
prevent, detect, and respond to infectious disease threats
wherever they occur in the world [3].h The motivation for this research is that with increas-
ing epidemics and pandemics globally, the level of coun-
tries’ health risk preparedness is now being measured to
provide a framework to assess the capacity of countries
to prevent and mitigate emerging health risks [1, 4]. The
GHS Index was released prior to the Covid-19 pandemic
in 2019. Recent work by the John Hopkins Center for
Health Security 2021 Global Health Security Report pro-
vides a new composite measure of the level of prepared-
ness to epidemics and pandemic threats for 195 countries
[1]. The GHS Index also allows for the benchmarking The prioritization of GHS interventions is done both at
national public health systems and in coordination with Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 3 of 14 using a regression model with time-fixed effects account-
ing for unobserved heterogeneity.h multilateral institutions including WHO, Global Health
Institute, and the World Bank Group, among other institu-
tions [2, 18–22]. The global disruption of the COVID-19
pandemic has once again reminded the world of the need
to conflate national health with global health risks. WHO
requires member countries to improve capacity in emerging
epidemic threats as part of their commitment to safeguard-
ing health [23]. However, the COVID-19 pandemic exposed
capacity gaps which indicate that many countries were not
sufficiently ready for major health risks [24, 25]. The com-
mitment to safeguarding health often needs the measure-
ment of GHS to evaluate countries’ capacities [17]. Despite
the serious threats posed by pandemics globally, compliance
to commitments to health protection remains low [26]. The rest of the study is organized as follows: Section 2
presents the methods including conceptual framework,
specifications, and data. Section 3 reports results. Sec-
tion 4 discusses results. Section 5 concludes. Datah The data used were publicly available [1]. The main out-
come variable we analyzed is the overall 2021 GHS Index,
which measures the capacities of 195 countries to prepare
for epidemics and pandemics, including threats potentially
more devastating than COVID-19. However, we report
and analyze for both 2019 and 2021. We recognize that
the GHS index for each country is assumed to be corre- (3) We specify and estimate the following explicit baseline
linear statistical equations. We ran both ordinary least
square (OLS) and quantile regression for this specifica-
tion as a comparison to illustrate how conclusions can
differ when understanding effects across the entire distri-
bution of the GHS index. (4) j+β3Bj+β4Sj+β5Gj+β6Fj+β7Ci+β8SDGj+β9HDIj+β10Yj+φyear2021+εj GHSj = β0+β1Dj+β2Nj+β3Bj+β4Sj+β5Gj+β6Fj+β7Ci+β8SDGj+β9HDIj+β10Yj+φyear2021+εj lated over time such that the two periods can control for
unobserved characteristics that do not change or change
slowly over time. Of interest were also several explanatory
variables, including health systems building blocks such as
human and capital resources, supply chain, public health
spending, effective governance, and communication infra-
structure. We draw these data from the GHS index report
and the global health observatory of the WHO [39]. Other
independent variables considered were socioeconomic and
development measures including SDGs, HDI, and level of
income category per World Bank classification as well as
WHO region classification based on geography. We filtered
countries by region and income level. where GHS is the overall global health security of coun-
try j; D is doctors per 1000 persons, N is nurses per 1000
persons, B is beds per 1000 persons, S is supply chain
capacity, G is governance effectiveness, F is public health
financing, C is communications infrastructure, SDG is
social development goals, HDI is human development
index, Y is World Bank’s development income level,
implying Y = 1 for high income and Y = 0 for low income;
Year is a dummy taking one for the Covid-19 pandemic
year 2021 and zero for 2019. Next, to estimate the share of contributions of pillars of
health security to the overall GHS score, we implemented
a double logarithmic regression model. Additionally, we
used a log-linear model as a robustness check and to nor-
malize the skewed distribution of the global health secu-
rity index. The fitted estimable double logarithmic model
is as follows: All variables were normalized to a scale of 0 to 100. Estimable models To examine the differential effects of global health sys-
tems building blocks and socioeconomic factors along
the distribution of levels of preparedness to health risks,
we specified a quantile regression model equation. We
also specified a log-linear and double logarithmic models. Quantile regression differentially weights the distances
between the values predicted by the regression line and the
observed values, then tries to minimize the weighted dis-
tances [34]. The method has the advantage in that it allows
for understanding relationships between variables outside
of the mean of the data. Quantile regression weights dif-
ferent portions of the sample to generate coefficient esti-
mates, thus increasing the power to detect differences in
the upper and lower tails. This approach has previously
been used in health services and health economics stud-
ies [34–38]. We report median regression given that it is
more robust to outliers than least squares regression. The
quantile regression model equation for the 𝜏th quantile as: First, using quantile regression, we examine the role of
health system building blocks and show their differential
effects along the distribution of the levels of prepared-
ness to health risks. The GHS index was generated from
37 indicators and 96 sub-indicators [1]. Second, using
logarithmic transformed data, we examined the respec-
tive share of contributions of the health system building
blocks and socioeconomic factors to the overall global
health security score. We interpret these share contri-
butions as percentage changes or elasticities, establish-
ing whether protecting GHS is a normal and necessary
endeavor, consistent with public health as a collective
benefit. Third, we assessed the regional differential effects
of GHS during the Covid-19 and pre-pandemic periods (2)
Q
(
gi
)
= 훽0(휏) + 훽1 (휏)xi1 + … … … … ⋯+ 훽1(휏)xip I = 1, … .., n (2) where gi is a measure of GHS index and xi is a vector of
explanatory variables, including pillars of health sys-
tems building blocks, socioeconomic and development Page 4 of 14 Da’ar and Kalmey Health Economics Review (2023) 13:16 where φ is time trend; λ denotes a vector of WHO
regional differences in GHS compared to the reference
region; and π denotes difference in changes over time. measures, and geography. The coefficients, β, are func-
tions of the quantiles, τ and are determined by minimiz-
ing the median absolute deviation. Datah Other
data of interest include time trend from 2019 to 2021 and
WHO regional offices. fi
We analyzed the data using STATA® version 16 (STATA fi
analyzed the data using STATA® version 16 (STA (5)
LnGHSj =훽0 + 훽1 ln (Dj
) + 훽2 ln (Nj
) + 훽3 ln (Bj
) + 훽4 ln (Sj
) + 훽5 ln (Gj
) + 훽6 ln (Fj
)
+ 훽7 ln
(
Cj
)
+ 훽8 ln
(
SDGj
)
+ 훽9 ln
(
HDIj
)
+ 휶
∑4−1
j=1 Yij + 훟year2021 + 흀
∑6−1
j=1 Regionij + 휀j (5) Cooperation, TX). We present descriptive and regression
results. We grouped countries into WHO regions and
the World Bank income categories. where the variables are as defined earlier; Yij are income
categories – low, lower middle, upper middle, and high;
and regions are the WHO region groups. Next, we implemented a regression model with time-
fixed effects. The effects of the health systems building
blocks and socioeconomic indicators on GHS are medi-
ated by the differences across WHO regions and the
shock of the Covid-19 pandemic as proxied by year 2021,
accounting for unobserved heterogeneity. Estimable models (3)
MAD = 1
p
∑p
n=1p휏
(
gi −
(
훽0(휏) + 훽1xi1(휏) + ⋯+ 훽pxip(휏)
) Resultsh This section presents the results of the descriptive analy-
sis and various regression models for 195 countries for the
data of the years 2019 and 2021 data (N = 390). lnGHSj =훽0 + 훽1ln(Dj
) + 훽2ln(Nj
) + 훽3ln(Bj
) + 훽4ln(Sj
) + 훽5ln(Gj
) + 훽6ln(Fj
)) + 훽7ln(Cj
) + 훽8ln(SDGj
) + 훽9ln(HDIj
)
+ 훼
∑4−1
i=1 Yij + ϕyear2021 + 휆r
∑6−1
r=1Regionjr + 휋Year2021 ∗
∑6−1
r=1Regionjr + 휀j lnGHSj =훽0 + 훽1ln(Dj
) + 훽2ln(Nj
) + 훽3ln(Bj
) + 훽4ln(Sj
) + 훽5ln(Gj
) + 훽6ln(Fj
)) + 훽7ln(Cj
) + 훽8ln(SDGj
) + 훽9ln(HDIj
)
+ 훼
∑4−1
i=1 Yij + ϕyear2021 + 휆r
∑6−1
r=1Regionjr + 휋Year2021 ∗
∑6−1
r=1Regionjr + 휀j (6) Page 5 of 14 Da’ar and Kalmey Health Economics Review (2023) 13:16 Log‑linear and double‑logarithmic models results g
g
Table 2 shows the results of log-linear and double-loga-
rithmic regressions. According to the log-linear regres-
sion, a unit increase in each of the health system building
blocks such as supply chain, public health spending,
effective governance, and communication infrastructure
were associated with a 0.59, 0.18, 0.41, and 0.41% increase
in the geometric mean of the GHS index. A unit increase
in the SDG score was associated with a 0.37% increase in
the geometric mean of the GHS index. A unit increase
in the HDI score was associated with a 24% increase in
the GHS index. However, the pandemic year (2021) was
associated with a 7.4% decrease in the geometric mean
of the GHS index (p < 0.001). There is a negative gradi-
ent between level of income and the level of preparedness
to health risks. Relative to poor countries, middle- and
high-income countries had lower levels of preparedness
to health risks, an indication of a sense of complacency. Descriptive analysis significant at higher quantiles. While positive effects of
SDGs are greater for countries with higher levels of pre-
paredness to health risks, the effect of HDI on the level of
preparedness is greatest for countries whose overall GHS
index lies at midpoint of the frequency distribution of
observed values Fig. 2. Compared to pre-pandemic period, countries with low
SDGs, HDI, and income were less prepared for health
risks during Covid-19 pandemic (Fig. 1). In the next sec-
tion, we assess whether these effects varied with quan-
tiles of health security. Quantile regression model results
bl
d
h
l
f g
Table 1 depicts the results of a level-level OLS and
quantile regression analyses. The results suggest that
health system building blocks such as supply chains,
public health spending, and effective governance were
associated with levels of preparedness to health risks. A unit increase in the score of each of these health sys-
tems building blocks was associated with an increase
of between 0.08 and 0.27 in the GHS index. Increases
in effective governance, supply chain capacity in terms
of medicines and technologies, and financing had posi-
tive effects on the level of preparedness to health risks. These effects differ considerably, having a strong impact
at higher quantiles. However, while an increase in com-
munications infrastructure had positive effects on the
level of preparedness to health risks at the 25th and 50th
quantiles, a negative effect was associated at higher quan-
tiles of preparedness to health risks. In the log-log model, the marginal contributions to the
level of preparedness to health risks of human resources,
supply chains, public health financing and govern-
ance of the healthcare systems were significant. A 10%
increase in each of the health system building blocks
was associated with an increase of between 0.11 to 1.4%
in the GHS index (all p < 0.001). Thus, as expected, the
stronger the health system building blocks, the stronger A unit score increases in the SDG score had a posi-
tive effect on the level of preparedness to health risks. This effect differs considerably, having a strong impact
on the GHS index at higher quantiles (p < 0.001). Com-
pared with countries with lower income, countries with
higher income had 2.2 lower GHS scores at the 25th and
50th quantile (p < 0.001). This differential effect was not Fig. 1 Level of preparedness to health risk by socioeconomic indicators (Mean, 0 to 100), N=390: Compared to pre-pandemic period, countries
with low SDGs, HDI, and income were less prepared for health risks during Covid-19 pandemic Fig. Quantile regression model results
bl
d
h
l
f 1 Level of preparedness to health risk by socioeconomic indicators (Mean, 0 to 100), N=390: Compared to pre-pandemic period, countries
with low SDGs, HDI, and income were less prepared for health risks during Covid-19 pandemic Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 6 of 14 Table 1 Level OLS and quantile regressions on global health security index, N = 390 Table 1 Level OLS and quantile regressions on global health security index, N = 390
Standard errors in parentheses; *** p < 0.01, ** p < 0.05, * p < 0.1
Quantile regression (GHS index)
VARIABLES
OLS
10th Percentile
25th Percentile
50th Percentile
75th Percentile
90th Percentile
Year (Pre-pandemic, 2019 = reference)
Pandemic (2021)
−2.910***
−3.633***
− 3.557***
− 2.596***
−1.953**
− 1.715*
(0.734)
(1.383)
(0.945)
(0.801)
(0.884)
(0.995)
Doctors per 1000 persons
0.00562
−0.0189
0.0213
0.0527
0.0401
− 0.0244
(0.0325)
(0.0825)
(0.0404)
(0.0655)
(0.0424)
(0.0427)
Nurses per 1000 persons
0.0248
0.0102
0.00879
0.0439
0.0514
−0.0109
(0.0357)
(0.113)
(0.0398)
(0.0522)
(0.0583)
(0.0393)
Beds per 1000 persons
−0.0342
−0.0119
− 0.000324
−0.0734*
− 0.0774
0.0114
(0.0327)
(0.121)
(0.0396)
(0.0435)
(0.0530)
(0.0409)
Supply chain capacity
0.231***
0.170***
0.230***
0.193***
0.268***
0.275***
(0.0203)
(0.0332)
(0.0265)
(0.0262)
(0.0360)
(0.0279)
Govt health spending % public spending
0.0900***
0.0932
0.105***
0.109**
0.109***
0.0848***
(0.0294)
(0.0829)
(0.0353)
(0.0425)
(0.0348)
(0.0251)
Governance effectiveness
0.138***
0.142***
0.1000***
0.116***
0.133***
0.214***
(0.0281)
(0.0452)
(0.0345)
(0.0335)
(0.0416)
(0.0377)
Communication infrastructure capacity
0.0549*
0.0576
0.0942**
0.0719**
−0.0135
−0.000723
(0.3090)
(0.0649)
(0.0419)
(0.0351)
(0.0550)
(0.0355)
Human development index (HDI)
4.821**
2.740
3.137
6.715**
5.849
3.783
(2.068)
(4.462)
(2.454)
(2.709)
(4.355)
(4.261)
Social development goals (SDGs)
0.147***
0.119***
0.111***
0.127***
0.143***
0.166***
(0.0190)
(0.0415)
(0.0224)
(0.0209)
(0.0229)
(0.0229)
World Bank Development level (Low-income = reference)
High-income
−0.512
−1.808
−2.286*
−2.273*
0.759
1.868
(1.020)
(1.490)
(1.224)
(1.259)
(1.395)
(1.940)
Constant
11.52***
9.306***
10.14***
11.92***
17.18***
17.41***
(1.600)
(3.109)
(1.997)
(2.168)
(2.924)
(1.620)
Observations
390
390
390
390
390
390
R-squared
0.779
0.768
0.771
0.772
0.772
0.769 Standard errors in parentheses; *** p < 0.01, ** p < 0.05, * p < 0.1 Americas, and West Pacific regions than in Africa. Although not statistically significant, the parameter esti-
mate of the Eastern Mediterranean region implies that it
also had higher GHS. This would suggest that the African
region was the least prepared to health risks across all the
regions. These results corroborate with the depiction of
Fig. 3. Discussionh This study aimed to examine the differential effects of
health systems building blocks and socioeconomic fac-
tors along the distribution of countries’ level of prepar-
edness to health risks. The study also examined WHO
regional offices’ preparedness level differentials during
Covid-19 and pre-pandemic period.hf Quantile regression model results
bl
d
h
l
f the preparedness of countries to threats of epidemics and
pandemics. A 10% increase in the SDG index was asso-
ciated with a 0.15% increase in the GHS index (< 0.001). The year 2021 (during the pandemic) was associated
with a lower GHS index compared to the pre-pandemic
period. Compared to African region, the Americas, Euro-
pean, and Southeast Asia regions were associated with
higher GHS. There was statistically significant difference
between African region and the rest of the WHO regions. A regression model with time fixed effectshf The results of the effects of health systems building
blocks and socioeconomic indicators in the time fixed-
effects model corroborate with the results of the previous
specifications. The results in Table 3 show that there were
significant regional differences in GHS. Specifically, GHS
was higher on average in the Southeast Asia, Europe, The quantile regression results suggest that the effect
of effective governance, supply chain capacity in terms of
medicines and technologies, and financing had positive Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 7 of 14 Fig. 2 indicates comparison of the different effects of the factors controlled for in the OLS and quantile regressions Fig. 2 indicates comparison of the different effects of the factors controlled for in the OLS and quantile regressions inefficient inter-sectoral relationships, parallel deci-
sions, inefficient distribution of the human resources,
lack of applied education, lack of integrated health
protocols, and lack of appropriate evaluation of perfor-
mance [28]. effects on the level of preparedness to health risks, with
impact being considerably larger for countries with
higher levels of preparedness to health risks. These posi-
tive gradient trends signal a sense of capacity on the part
of countries with higher global health security. More generally, evidence shows that a well-function-
ing health system act as a first line of defense during
outbreaks of health risks [27]. With increasingly global
health risks, there is a need to have well-integrated and
locally grounded health systems that are more resil-
ient to shocks. Such a need should include designing
efficient health information systems, financing mecha-
nisms, and health workforces. These building blocks
imply having the information and knowledge to make
a decision on what needs to be done, and investing or
mobilizing resources to fund a response [28]. To ensure
interventions in all health system building blocks are
successful, such designs should also recognize and
include promoting effective governance and wider
systems values [28]. Governance challenges in health
systems were remarkably noticeable during Covid-19
pandemic, including weak organizational coordination, Our results suggest that health-related workforces
as building blocks of a well-functioning health system
were not associated with commensurate levels of health
risk preparedness. While an increase in nurses per 1000
population was the only factor in this category to be
associated with increased preparedness to health risk,
even then, we find no statistically significant difference
between low and high prepared countries. Robust standard errors in parentheses; *** p < 0.01, ** p < 0.05, * p < 0.1 a implies variable in natural logarithm A regression model with time fixed effectshf The health
workforce is crucial for a health system because it is
the component that determines how plans for response
to shocks are implemented [28]. However, our results
indicate that although an increase in doctors per 1000
population enhanced level of preparedness to health
risks, that impact was not statistically significant. Frontline workers in communities were found to be
important assets in the capacity building and prepared-
ness strategies during Covid-19 pandemic [29]. Health Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 8 of 14 Table 2 Log-linear and Log-log regression on global health security index, N = 390
Robust standard errors in parentheses; *** p < 0.01, ** p < 0.05, * p < 0.1
a implies variable in natural logarithm
Log-linear
Log-log
Year (reference = Pre-pandemic, 2019)
Year (reference = Pre-pandemic, 2019)
Pandemic (2021)
− 0.0767***
Pandemic (2021)
− 0.0696***
(0.018)
(0.019)
Doctors per 1000 persons
0.001
Doctors per 1000 persons a
−0.005
(0.001)
(0.005)
Nurses per 1000 persons
0.000
Nurses per 1000 persons a
0.0161***
(0.001)
(0.004)
Beds per 1000 persons
−0.001
Beds per 1000 persons a
0.002
(0.001)
(0.005)
Supply chain capacity
0.00586***
Supply chain capacity a
0.0125***
(0.001)
(0.001)
Govt health exp. % public exp. 0.00182**
Govt health exp. % public exp. A regression model with time fixed effectshf It has been
noted that the ability for rapid development of medi-
cal products and being able to take procurement and
manufacture of new products to scale in a very short
time period was a key resilience and health systems
preparedness lesson during Covid-19 pandemic [40]. However, our results suggest that while an increase in
communications infrastructure had positive effects on
the level of preparedness to health risks at lower quan-
tiles, a negative effect was associated at higher quantiles
of preparedness to health risks. The impact was consid-
erably greater for countries with lower levels of prepar-
edness to health risks. Generally, while the emphasis of
a robust health system is on good disease surveillance
systems and their integration with health management
information systems [40], it appears this was impactful
in less prepared countries than in more prepared econ-
omies. Communications infrastructures were much
needed in weak health systems, given that the dearth
of well-coordinated communication channels can bode
ill for the successful fight against pandemics [41]. Evi-
dence shows a lack of communication could jeopardize
effective interventions to mitigate exposure and man-
agement of health risks, especially in weak health sys-
tems [42]. Further, our analysis suggests positive gradient effects
of SDGs on GHS that are smaller when countries have
lower global health security, but much larger when global
health security is higher. Compared to pre-pandemic
period, countries with low SDG index were underpre-
pared for health risks during Covid-19 pandemic. These
results affirm the interconnectedness of protection of
population health and SDGs. In the world’s agenda for
SDGs by 2030, ensuring good health and wellbeing
remains a central goal [5, 43], and that improving public
health is a central pillar for the SDGs agenda [44]. A sub-
point of the SDG health goal is the strengthening of early
warning, risk reduction, and management of health risks
[5]. Almost 16 of the SDGs goals are related to health
or their achievement will contribute to health indirectly
[43] and should be a priority in global health policy
dialog [45–47]. The contribution of HDI to GHS index. HDI has the greatest positive effect on countries whose
overall GHS index lies at the midpoint of a frequency
distribution of observed values. Compared to pre-pan-
demic period, countries with low HDI were underpre-
pared for health risks during Covid-19 pandemic. A regression model with time fixed effectshf a
0.0111***
(0.001)
(0.004)
Governance effectiveness
0.00414***
Governance effectiveness a
0.144***
(0.001)
(0.021)
Communication infrastructure
0.00415***
Communication infrastructure a
0.011
(0.001)
(0.011)
HDI
0.215***
HDI a
0.008
(0.060)
(0.005)
SDGs
0.00372***
SDGs a
0.0155***
(0.001)
(0.002)
World Bank income category (Low income = reference)
Low-middle income
−0.0647*
−0.0768**
(0.033)
(0.036)
High-middle income
−0.108**
−0.038
(0.042)
(0.047)
High-income
−0.204***
0.022
(0.051)
(0.053)
WHO regions (Africa = reference)
Americas
0.017
0.135***
(0.036)
(0.041)
Eastern Mediterranean
−0.029
0.053
(0.036)
(0.037)
European
−0.012
0.225***
(0.042)
(0.039)
Southeast Asia
0.126***
0.205***
(0.044)
(0.055)
West Pacific
−0.030
0.061
(0.036)
(0.045)
Constant
2.743***
2.936***
(0.045)
(0.082)
Observations (n)
390
390
R-squared
0.798
0.731 Table 2 Log-linear and Log-log regression on global health security index, N = 390 Robust standard errors in parentheses; *** p < 0.01, ** p < 0.05, * p < 0.1 Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 9 of 14 Page 9 of 14 Page 9 of 14 Table 3 A regression model with time fixed effects, N = 390
Robust standard errors in parentheses
*** p < 0.01, ** p < 0.05, * p < 0.1
VARIABLES
Dependent
variable = Natural
logarithm
of GHS index
Robust S.E
Doctors per 1000 persons
−0.00540
(0.00522)
Nurses per 1000 persons
0.0163***
(0.00400)
Beds per 1000 persons
0.00190
(0.00533)
Supply chain capacity
0.0124***
(0.00142)
Govt health spending % public
spending
0.0109**
(0.00442)
Governance effectiveness
0.142***
(0.0206)
Communication infrastructure
capacity
0.0101
(0.0106)
Human development index (HDI)
0.00871
(0.00548)
Social development goals (SDGs)
0.0168***
(0.00241
World Bank income category (low-income = reference)
Low-middle income
−0.0763**
(0.0367)
High-middle income
−0.0357
(0.0469)
High-income
0.0264
(0.0528)
Year (2021)
−0.0571*
(0.0330)
WHO regions (Africa = reference)
Americas
0.142***
(0.0500)
Eastern Mediterranean
0.0613
(0.0519)
European
0.211***
(0.0452)
Southeast Asia
0.246***
(0.0694)
West Pacific
0.122**
(0.0498)
Year * Americas
−0.0126
(0.0613)
Year * Eastern Mediterranean
−0.0208
(0.0612)
Year * European
0.0255
(0.0447)
Year * Southeast Asia
− 0.0776
(0.0975)
Year * West Pacific
−0.113
(0.0774)
Constant
2.931***
(0.0840)
Observations
390
R-squared
0.735 larger for countries with higher quantiles of prepared-
ness. Increase in supply chains, notably medicines and
technologies appear to have enhanced the level of pre-
paredness to health risks, with impact being consid-
erably higher in more prepared countries. A regression model with time fixed effectshf If
the health and economic burden of a local issue such as
unsafe food can be avoided through preventive meas-
ures, investments, and behavioral changes adopted
from farm to fork, [52] countries should take a global
matter as important as GHS more seriously than the
current complacency and cavalier attitude. The 2021
GHS Index report showed that countries are continu-
ing to neglect the preparedness needs of vulnerable
populations, which exacerbates the impact of health
security emergencies [1]. Thus, as a necessary endeavor
and imperative, the promotion of GHS requires local,
national, regional, and global responses to establish
how an outbreak becomes a pandemic and to prepare
for future health threats [53]. Thus, it is imperative to
build accountability for national preparedness and in
coordination with multilateral institutions including
WHO, Global Health Institute, the National Academy
of Medicine, and the World Bank Group [2, 18–22].h provides information on the development of countries,
considering essential issues that influence people’s well-
being [48]. In showing the value proposition of the GHS
Index, HDI has been shown as a valuable tool for guid-
ing decision making and monitoring policymaking at
both national and subnational levels, especially on health
security capacities and capabilities in many countries [6]. Moreover, the results showed a negative income dif-
ferential effect on the GHS index, with high-income
countries being associated with lower levels of prepar-
edness to health risks than low-income countries. How-
ever, countries with less capacity to respond to health
threats generally tend to be low-income. This result is
intuitive given that any increase in income in these coun-
tries is likely to be used to improve food and nutrition. Improvement in food and nutrition can in turn enhance
health. Evidence shows that food and health security are
attainable only when the underlying social inequities are
addressed [49]. Improvements in incomes in developing
countries do not necessarily translate to enhancements in
GHS in the short run because these countries face chal-
lenges of food security, nutrition, and poverty. Interestingly, while quantile regression reveals nega-
tive and positive gradient differential impact of health
systems building blocks and socioeconomic indicators,
these impacts are masked the OLS estimation. The log-
arithmic regression showed the marginal contributions
to the level of preparedness to health risks of nurses per
1000 population, supply chain, public health financ-
ing and governance of the healthcare systems were
positive and significant. A regression model with time fixed effectshf This
result underscores the importance of HDI to countries
with median GHS index capabilities. Improving HDI systems faced several health workforce challenges,
including inefficient distribution, employee turnover,
lack of clear approaches for staffing, and shortage of
specialized manpower [30]. Many personnel-related
challenges were noted, including insufficient knowledge
of the employees, psychological disorders, reduction of
self-confidence, burnout, workload increase, reduced
level of job satisfaction, effects of colleague and patients
bereavement and unsafety sense against the work place
[30]. A notable finding in our study is that compared to
other building blocks, the supply chain was associ-
ated with a higher impact on the level of preparedness
to health risks, with the impact being considerably Page 10 of 14 Da’ar and Kalmey Health Economics Review (2023) 13:16 Fig. 3 Predictive margins of trends in preparedness to health risks and WHO regional differentials: A negative trend in the level of preparedness
to health risks from 2019 to 2021 for all regions is shown, implying all regions were less prepared during Covid-19 pandemic compared to
pre-pandemic period Fig. 3 Predictive margins of trends in preparedness to health risks and WHO regional differentials: A negative trend in the level of preparedness
to health risks from 2019 to 2021 for all regions is shown, implying all regions were less prepared during Covid-19 pandemic compared to
d
i
i d Fig. 3 Predictive margins of trends in preparedness to health risks and WHO regional differentials: A negative trend in the level of preparedness
to health risks from 2019 to 2021 for all regions is shown, implying all regions were less prepared during Covid-19 pandemic compared to
pre-pandemic period to health risks is a normal and necessary endeavor. Given the increasing regularity with which infectious
disease threats happen, the results underscore the fact
that it is imperative upon countries across the globe to
enhance GHS preparedness regardless of the changes
in health systems building blocks. The idea that pro-
motion of GHS is a necessary endeavor is also con-
sistent with healthcare as a necessity, especially when
delivered through the public sector [50, 51]. Enhanc-
ing GHS as a collective benefit is also consistent with
the good health and safe food imperatives argument. A regression model with time fixed effectshf The percentage changes in the
GHS index with respect to percentage changes in the
health systems building blocks imply that preparedness The foregoing results indicate the importance of the
contributions of health systems building blocks, affirm-
ing their value proposition in enhancing health secu-
rity capacity. The results further indicated that the year Da’ar and Kalmey Health Economics Review (2023) 13:16 Page 11 of 14 Page 11 of 14 the study assessed the regional differential effect of GHS
during Covid-19 and pre-pandemic period However, the
study has limitations. It relied on macro data from the
2021 global health security index. The index has been
criticized for showing a discrepancy between the GHS
index rating and the actual performance of countries
during pandemic, overestimating the preparedness of
some and underestimating others [57]. A more micro-
data disaggregating the preventive and responsive meas-
ure of countries as well as robustness of health systems,
commitments and overall risks would have provided a
more accurate behaviors of individuals in communities
in different countries in preparing to and response to
global health risks. Broadly, microdata can be beneficial
in exploring the rich sources of heterogeneity shaping
the behaviors of participants at the micro level of soci-
ety. Microdata also help in netting out a large array of
individual-level factors that may contribute to geographic
variation in health care utilization [58]. The use of more
microdata can improve on aggregate time-series meth-
ods by building models that link economic models for
individuals to data on individual behavior [59]. 2021 (during the pandemic) was associated with a sta-
tistically significant less preparedness to global health
risks compared to the pre-pandemic period. Again, this
result affirms that, as has been shown during Covid-19
outbreak, the health security of countries remains frag-
ile and that no countries were sufficiently ready for a
major biological [24, 25, 54]. The results showed statistically significant regional
differences in the level of preparedness to health risks. Compared to African region, the Americas, European,
and Southeast Asia regions were associated with higher
GHS. There was statistically significant difference
between African region and the rest of the WHO regions. The results of the time-fixed effect comparison of the
changes in GHS from 2019 to 2021 show the positive
effects of health systems building blocks and socioeco-
nomic indicators. Concluding remarks In conclusion, we revisit my original queries. What is the
impact of health systems building blocks and socioeco-
nomic indicators on level of preparedness to epidemics
and pandemics, and whether such effects differ for less
and more prepared countries? Our analysis of the relationship between health sys-
tems building blocks, socioeconomics, and regional dif-
ferences versus preparedness to health risks consistently
confirmed the robustness of the models estimated. The
choice of the specifications also corroborates with model
statistics and internal validity assessments, including mis-
specification test tool (Linktest) available in Stata, the
econometric software used in the research. The direction
and magnitude of the coefficients reveal the contribu-
tion of each of the health systems building blocks, soci-
oeconomic indicators, and regional differentials to the
overall level of preparedness to health risks. The results
show that increases in effective governance, supply chain
capacity in terms of medicines and technologies, and
financing had positive effects on the level of preparedness
to health risks. However, the health workforce including
doctors, and health services including hospital beds were
not statistically significant in explaining the variations in
countries’ level of preparedness.f A regression model with time fixed effectshf Apart from the Eastern Mediterranean
region, the rest of the WHO regions were more prepared
to health risks compared to Africa. There was a negative
time trend in the level of preparedness to health risks,
although regional differences in changes over time were
not statistically significant. The 2021 GHS index report
showed that most countries saw little or no improvement
in maintaining a robust, capable, and accessible health
system for outbreak detection and response [1]. The sug-
gestion that some regions performed better than other
regions is consistent with WHO data during the Covid-
19 pandemic. For instance, the Western Pacific region
recorded the highest total vaccine doses administered
of 222.52 per 100 population, while European region
registered 166.89 per 100 population. Western Pacific
countries also recorded the highest in terms of persons
boosted, registering 46.78 per 100 population. Euro-
pean region was the third after Western Pacific and the
Americas, registering 27.55 per 100 population. The two
regions were way above the global average, both in terms
of total vaccine doses administered and persons boosted
per 100 population [55]. There is evidence that countries
in Asia-Pacific region such as Taiwan and New Zealand
had global successes in strategies to control COVID-19
compared to countries in Western Europe. Countries in
this region took urgent action to eliminate community
transmission through a series of non-pharmaceutical
interventions: a ‘zero-COVID’ strategy. At the same time,
they kept their economies afloat and avoided longer,
harsher lockdown measures [56]. Consent for publication
Not applicable. Consent for publication
Not applicable. Not applicable. Funding No funding received. Availability of data and materialsi Availability of data and materials
Data supporting study findings are available upon request. y
Data supporting study findings are available upon request. Competing interests
h
h
d
l
h The authors declare that they have no competing interests. Author details 1 Department of Health Systems Management, College of Public Health
and Health Informatics, King Saud bin Abdulaziz University for Health Sciences,
Riyadh, Saudi Arabia. 2 King Abdullah International Medical Research Center,
Riyadh, Saudi Arabia. 3 Institute for Cost Analysis and Research Evaluation, Min-
neapolis, MN, USA. 4 College of Science and Health Professions, King Saud bin
Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia. 5 Organizational
Health and Wellbeing at the Division of Health Research, Lancaster University,
Lancaster, UK. Received: 10 August 2022 Accepted: 6 March 2023 Declarations Ethics approval and consent to participate Authors’ contributions OBD: conceptualizing the idea, methodology design, data analysis, writ-
ing and reviewing the manuscript. FK: Data coding and shaping, writing,
and reviewing the manuscript. Both authors have read and approved the
manuscript. All the models reveal that the pandemic period (year
2021) was associated with a decrease in the level of pre-
paredness to health risks compared to the pre-pandemic
period. There were significant regional differences, and
apart from the Eastern Mediterranean region, the rest
of the WHO regions were more prepared to health risks
compared to Africa. There was a negative time trend in
the level of preparedness to health risks from 2019 to
2021. However, regional differences in changes over time
were not statistically significant. Contribution and limitationshi Using a quantile regression, we show that the effect
of effective governance, supply chain capacity in terms
of medicines and technologies, and financing had posi-
tive effects on the level of preparedness to health risks,
with impact being considerably larger for countries with This study is the first to examine whether the effects or
contributions and strength of the health systems build-
ing blocks and socioeconomic indicators on countries’
level of preparedness to health risks differ for countries
with weak and strong health securities. At the same time, Page 12 of 14 Da’ar and Kalmey Health Economics Review (2023) 13:16 Da’ar and Kalmey Health Economics Review (2023) 13:16 higher levels of preparedness to health risks. These posi-
tive gradient trends signal a sense of capacity on the part
of countries with higher global health security. While
socioeconomic factors had positive effects on the level
of preparedness to health risks, their impacts on the dis-
tribution of countries’ level of preparedness to health
risks were mixed. The effects of SDGs were greater for
countries with higher levels of preparedness to health
risks. The effect of HDI on the level of preparedness was
greatest for countries whose overall GHS index lie at the
midpoint of the distribution of the level of prepareness. High-income was associated with a negative effect on
the level of preparedness, especially if countries were in
lower quantiles across the distributions of preparedness. Relative to poor countries, middle- and high-income
countries had lower levels of preparedness to health
risks, an indication of a sense of complacency. 3.
Prevention C for DC and. Global Health–CDC and the Global Health
Security Agenda. 2017. 2.
Organization WH. Report of the Ebola interim assessment panel.
2015; 4.
Khalifa BA, Abbey EJ, Ayeh SK, Yusuf HE, Nudotor RD, Osuji N, et al.
The Global Health security index is not predictive of vaccine rollout
responses among OECD countries. Int J Infect Dis. 2021;113:7–11. References
1.
Center for Health Security. Global health security index. Baltimore:
Bloomberg School of Public Health. Baltimore, Maryland, USA; 2021.
2.
Organization WH. Report of the Ebola interim assessment panel.
2015;
3.
Prevention C for DC and. Global Health–CDC and the Global Health
Security Agenda. 2017.
4.
Khalifa BA, Abbey EJ, Ayeh SK, Yusuf HE, Nudotor RD, Osuji N, et al.
The Global Health security index is not predictive of vaccine rollout
responses among OECD countries. Int J Infect Dis. 2021;113:7–11. Ethics approval and consent to participate This study was approved by the International Review Board in King Abdullah
International Medical Research Center with reference number NRC22/063/01. No consent to participate or publish was needed. y
gi
We conclude with implications and recommenda-
tions for practical actions for addressing health systems
building blocks and socioeconomic indicators impacting
on GHS preparedness. Our results would suggest that
attempts to strengthen countries’ level of preparedness
to health shocks should be focused more on enhanc-
ing essentials such as supply chain capacity in terms of
medicines and technologies; health financing, commu-
nication infrastructure, while maintaining their already
existing health workforce and health services. Strength-
ening health systems building blocks would be beneficial
to less prepared countries where their impacts we find to
be weaker. Similarly, boosting SDG, particularly health-
related sub-scales, would be beneficial to less prepared
countries. There is a need to curb complacency in prepar-
edness to health risks during pandemics by high income
and countries with better capacity for protecting popu-
lation health. The negative trend in the level of prepar-
edness to health risks would suggest that there is a need
for better preparedness during pandemics by conflating
national health with global health risks. This will ensure
the imperative of having synergistic response is apparent
to all countries and regions. Consent for publication
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https://www.researchsquare.com/article/rs-2987954/latest.pdf
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English
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A fast Chebyshev wavelet numerical method for the solution of the Advection partial differential equations
|
Research Square (Research Square)
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Vivek . Institute of Engineering and Technology, Lucknow Abstract In this article, we present a computationally efficient technique based
on the method of Chebyshev wavelets and collocation technique for
the solution of linear and nonlinear the Advection partial differential
equations. We transform these problems into a system of algebraic
equations using truncated Chebyshev wavelet expansions and then sim-
plified using a suitable method. The suggested Chebyshev wavelet
approach is worked out for the convergence analysis it is demon-
strated that the estimation of a function using Chebyshev wavelets
converges uniformly to itself. It is also anticipated that the proposed
approach would be more efficient and suitable for solving a variety
of nonlinear partial differential equations that occur in science and
engineering. Examples are given to show how the suggested wavelet
method provides enhanced accuracy for a wide range of problems. Keywords: Taylor wavelet, Collocation points, Advection equation, Newton
iterative method, MATLAB Keywords: Taylor wavelet, Collocation points, Advection equation, Newton
iterative method, MATLAB Research Article License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Vivek1, Manoj Kumar† and Suyash Narayan Mishra†
Applied Sciences and Humanities Department, Institute of
Engineering and Technology, Sitapur Road, Lucknow, 226021,
Uttar Pradesh, India. Vivek1, Manoj Kumar† and Suyash Narayan Mishra†
Applied Sciences and Humanities Department, Institute of
Engineering and Technology, Sitapur Road, Lucknow, 226021,
Uttar Pradesh, India. 1 Introduction Nonlinear problems are the formulation of a scientific theory for many real-
world and physical problems. In recent decades, nonlinear partial differential
equations have drawn a lot of attention since they are utilized in several 1 2 technical and scientific applications, like gas dynamics, atomic structure,
chemical processes, and nuclear physics. Sometimes it is impossible to obtain
the analytical solutions to given nonlinear problems using analytical methods. We use numerical approaches like the Tau method, wavelet based collocation
method and operational matrix of integration method, etc for such problems. Localized or tiny waves are referred to as wavelets. They tend to zero,
as opposed to fluctuating indefinitely. The research area of wavelet analysis
has been used in various engineering disciplines, including signal analysis,
harmonic analysis, temporal frequency analysis, and others. The multiresolu-
tion analytical feature of wavelets allows for the accurate representation of a
wide number of functions as well as operators. In continuous time, wavelets
are considered to be basis functions of the form ζi,j and every such function
in this basis is built from a single function ζ(ρ), which is known as mother
wavelet. This is a unique property of the wavelets basis. Several experts from the field of numerical analysis have emphasised the
importance of examining nonlinear partial differential equation solutions using
a variety of techniques like Variational iteration method (VIM) [5–7], ado-
mian decomposition method (ADM) [3], Adomian decomposition approach
[15], Laplace decomposition algorithm [16], homotopy perturbation method
(HPM) [1], differential transform method, and so many others. The Laplace
transform is absolutely unable to handle nonlinear equations due to the diffi-
culties created by the nonlinear variables. Recently, a significant number of researchers in several branch of sciences and
engineering , have utilised wavelet analysis to solve various types of Advection
partial differential equations (PDEs). Wavelet analysis has a significant advan-
tage over other existing numerical approaches due to its compactness, locality,
multiresolution analysis, density, compact support, and many other charac-
teristics. The wavelet basis differentiation using the collocation method is one
of the most used numerical techniques for handling both linear and nonlinear
differential equations. This method relies on eliminating the coefficients of the
approximate solution in order to transfer the problem into a set of algebraic
equations. 2 Wavelets Wavelets are generated from a single function known as ”mother wavelet” by
dilating and translating it. When both the translational and dilational vari-
ables are continuous, the family of continuous wavelets are defined as follows
[26]: ζνη(ρ) = |ν|−1
2 ζ
ρ −η
ν
,
ν, η ∈R,
ν ̸= 0. By limiting the variables ν and η to discrete quantities, the discrete wavelets
family is defined as ν = ν−θ
0 , η = ωη0ν−θ
0 , ζ0 > 1, and η0 > 1, where ω and θ
are natural numbers. Therefore, ζθω(ρ) = |ν0|
θ
2 ζ
νθ
0ρ −ωη0
, where ζθω is a basis(wavelet basis) of L2(R). In particular, when ν0 = 2 and
η0 = 1, then ζθω(ρ) forms an orthonormal basis. where ζθω is a basis(wavelet basis) of L2(R). In particular, when ν0 = 2 and
η0 = 1, then ζθω(ρ) forms an orthonormal basis. 1 Introduction The purpose of this research is to apply the chebyshev wavelet collocation
method to obtain an approximate solution to the nonlinear partial differential
equation which is describing the physical phenomenon that is the Advection
problem, having the standard form as, uβ + uuα = R(α, β), u(α, 0) = f(α),
(1) (1) Eq. (1) is said to be homogeneous Advection equation if r(α, β) = 0. Chebyshev wavelet collocation method is straightforward, quick, and ensures
the required accuracy for a relatively small number of grid points. Eq. (1) is said to be homogeneous Advection equation if r(α, β) = 0. Chebyshev wavelet collocation method is straightforward, quick, and ensures
the required accuracy for a relatively small number of grid points. 3 2.1 Chebyshev wavelets Four arguments exist for Chebyshev wavelets ζωr(ρ) = ζ(θ, ˆω, r, ρ): ˆω = 2ω −
1, ω = 1, 2, . . . , 2θ−1, where r is the order of the Chebyshev polynomials.The
Chebyshev wavelets are defined in the range [0, 1] as follows [25]: ζωr(ρ) =
(
2
θ
2 ˜Lr
2θρ −ˆω
,
ˆω
2θ−1 ≤ρ < ˆω+1
2θ−1
0,
otherwise
(2) (2) otherwise where r = 0, 1, 2, . . . , µ −1 and ˜Lr(ρ) is given by 3 Function approximation Any continuous function u(α, β) ∈L2(R × R) on [0, 1) × [0, 1) can be written
as:
∞
∞ as: u(α, β) =
∞
X
ω=1
∞
X
r=0
cωrζωr(α)φω(β)
(3) (3) where φω(β) = βω−1 and cωr = (2ω −1)
R 1
0
R 1
0 u(α, β)ζωr(α)φω(β)dαdβ. If
the infinite series in (3) is truncated then it can be written as 0
0
the infinite series in (3) is truncated then it can be written as u(α, β) ∼=
2θ−1
X
ω=1
µ−1
X
r=0
cωrφωr(α)φω(β) = CT ζ(α)φ(β)
(4) (4) where C and ζ(β) are 2θ−1µ × 1 matrices given by where C and ζ(β) are 2θ−1µ × 1 matrices given by C =
c10, c11, . . . c1µ−1, c20, . . . , c2µ−1, . . . , c2θ−10, . . . , c2θ−1µ−1
T
ζ(β) =[ζ10(β), ζ11(β), . . . , ζ1µ−1(β), ζ20(β), . . . , ζ2µ−1(β) . . . , ζ2µ−10(β), . . . , ζ2θ−1µ−1(β)
T where r = 0, 1, 2, . . . , µ −1 and ˜Lr(ρ) is given by ˜Lr(ρ) =
1
√π,
r = 0,
q
2
πLr(ρ),
elsewhere. while, Lr(ρ) are the Chebyshev polynomials [27] of order r given by:
L0(ρ) = 1
L1(ρ) = ρ
Lr+1(ρ) = 2ρLr(ρ) −Lr−1(ρ),
r = 1, 2, . . . , µ −1.
1(ρ)
ρ
Lr+1(ρ) = 2ρLr(ρ) −Lr−1(ρ),
r = 1, 2, . . . , µ −1. It is noted that the Chebyshev polynomials are orthogonal with respect to
the weight function z(α) =
1
√
1−α2 on the interval [−1, 1]. Also, the Cheby-
shev wavelets are orthogonal with respect to the weight function zω(α) =
z
2θα −2ω + 1
on the interval [0, 1]. 4 4 Methodology Let us consider the Advection equation uβ + uuα = f(α, β) in (1). On integrating this equation with respect to ’ β ’ limit form 0 and β we get, Z β
0
uβdβ +
Z β
0
uuαdβ =
Z β
0
f(α, β)dβ
u(α, β) −u(α, 0) +
Z β
0
uuαdβ =
Z β
0
f(α, β)dβ,
u(α, β) = u(α, 0) −
Z β
0
uuαdβ +
Z β
0
f(α, β)dβ. Z β
0
uβdβ +
Z β
0
uuαdβ =
Z β
0
f(α, β)dβ
u(α, β) −u(α, 0) +
Z β
0
uuαdβ =
Z β
0
f(α, β)dβ, u(α, β) = u(α, 0) −
Z β
0
uuαdβ +
Z β
0
f(α, β)dβ. Let u(α, β) =
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β), 5 Therefore, (8) becomes, Therefore, (8) becomes, 2θ−1
X
ω =1
µ−1
X
r =0
cωrζωr(α)φω(β)
θ
1 X
ω =1
X
r =0
= u(α, 0)
−
Z β
0
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β)
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β)
α
dβ
+
Z β
0
f(α, β)dβ. (6) (6) (7)
2θ−1
X
ω =1
µ−1
X
r =0
cωrζωr(α)φω(β) −u(α, 0) +
Z β
0
2θ−1
X
ω =1
µ−1
X
r =0
cωrζωr(α)φω(β)
×
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β)
α
dβ −
Z β
0
f(α, β)dβ = 0. 2θ−1
X
ω =1
µ−1
X
r =0
c (7) Collocate (7) at 2θ−1µ points αi we get a system of 2θ−1µ equations. Solving
these equations using Newton’s iterative method in MATLAB, we can obtain
the value of the unknown vector C. Finally, substitute these values of ci,j into
(5) we can get the Taylor wavelet approximate solution for the given problem Collocate (7) at 2θ−1µ points αi we get a system of 2θ−1µ equations. Solving
these equations using Newton’s iterative method in MATLAB, we can obtain
the value of the unknown vector C. Finally, substitute these values of ci,j into
(5) we can get the Taylor wavelet approximate solution for the given problem. ,j
(5) we can get the Taylor wavelet approximate solution for the given problem. 5 Convergence analysis In this, section convergence analysis of the method is discussed along with an
error estimation for the given function approximation. (Convergence Theorem). A continuous bounded function u(α, β) with
bounded second derivative say
∂2u
∂α2
⩽M expressed as in Eq. (3) converges
uniformly to the exact solution u(α, β). Proof Let u(α, β) be a continuous function defined on [0, 1) × [0, 1) such that
∂2u
∂α2
⩽M, and u(α, β) =
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β), u(α, β) =
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β),
where cωr = ⟨u(α, β), ζωr(α)φω(β)⟩zω are the Chebyshev wavelet coefficients then,
cωr =
Z 1
0
Z 1
0
u(α, β), ζωr(α)φω(β)zω(α)dαdβ. (8) ω=1 r=0
where cωr = ⟨u(α, β), ζωr(α)φω(β)⟩zω are the Chebyshev wavelet coefficients then,
cωr =
Z 1
0
Z 1
0
u(α, β), ζωr(α)φω(β)zω(α)dαdβ. (8) (8) cωr =
Z
0
Z
0
u(α, β), ζωr(α)φω(β)zω(α)dαdβ. (8) 6 Using the definition of Chebyshev wavelets Eq. (8) reduces as, cωr =
Z 1
0
Z 1
0
u(α, β)2
θ
2 ˜Lr
2θα −2ω + 1
φω(β)dαdβ. Consider Cωr = (2ω −1)
R 1
0 u(α, β)ζωr(α)dα. θ 0
t 2θα −2ω + 1=cos x, we obtain, Put 2θα −2ω + 1=cos x, we obtain, Cωr =
1
√
2θ−1π
Z π
0
u
cos x + 2ω −1
2θ
, β
cos rxdθ. Applying integration by parts, we obtain Applying integration by parts, we obtain Cωr =
1
√
2θ−1π
u
cos x + 2ω −1
2θ
, β
sin rx
r
π
0
,
+
1
r
√
23θ−1π
Z π
0
∂
∂xu
cos x + 2ω −1
2θ
, β
sin rx sin xdx
=
1
r
√
23θ+1π
∂2
∂2xu
cos x + 2ω −1
2θ
, β
εr(x)
π
0
+
1
r
√
25θ+1π
Z π
0
∂2
∂2xu
cos x + 2ω −1
2θ
, β
(sin x)εr(x)dx,
(9) (9) where εr(x) = sin(r −1)x
r −1
−sin(r + 1)x
r + 1
. (10) (10) r
r +
Applying modulus on both sides of Eq. (9), we have Applying modulus on both sides of Eq. (9), we have |Cωr| =
1
r
√
25θ+1π
Z π
0
∂2
∂2xu
cos x + 2ω −1
2θ
, β
(sin x) εr(x)dx
. ≤
2rπM
r (r2 −1)
√
25θ+1π
=
√
2πM
2
5θ
2 (r2 −1)
. 5 Convergence analysis A continuous function u(α, β) defined on [0, 1) such
that | ∂2u
∂2x|⩽M then the accuracy estimate is as follows: En ⩽
∞
X
ω=2θ
∞
X
r=µ
√
2πM
(2ω)
5
2 (r2 −1)
2ω −1
ω
,
(12) En ⩽
∞
X
ω=2θ
∞
X
r=µ
√
2πM
(2ω)
5
2 (r2 −1)
2ω −1
ω
,
(12)
where
En =
Z 1
0
Z 1
0
∞
X
ω=1
∞
X
r=0
cωrζωr(α)φω(β) −
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β)
2
d
Proof E2
n =
Z 1
0
Z 1
0
∞
X
ω=1
∞
X
r=0
cωrψωr(α)φω(β) −
2θ−1
X
ω=1
µ−1
X
r=0
cωrψωr(α)φω(β)
2
dαdβ,
E2
n =
Z 1
0
Z 1
0
∞
X
ω=2θ
∞
X
r=µ
c2
ωrψ2
ωr(α)φ2
ω(β)dαdβ,
⩽
∞
X
ω=2θ
∞
X
r=µ
2π2M2
(2ω)5 (r2 −1)2
2ω −1
ω
2
d h
d
l (12) where
En =
Z 1
0
Z 1
0
∞
X
ω=1
∞
X
r=0
cωrζωr(α)φω(β) −
2θ−1
X
ω=1
µ−1
X
r=0
cωrζωr(α)φω(β)
2
dαdβ
dαdβ
Proof E2
n =
Z 1
0
Z 1
0
∞
X
ω=1
∞
X
r=0
cωrψωr(α)φω(β) −
2θ−1
X
ω=1
µ−1
X
r=0
cωrψωr(α)φω(β)
2
dαdβ,
E2
n =
Z 1
0
Z 1
0
∞
X
ω=2θ
∞
X
r=µ
c2
ωrψ2
ωr(α)φ2
ω(β)dαdβ,
⩽
∞
X
ω=2θ
∞
X
r=µ
2π2M2
(2ω)5 (r2 −1)2
2ω −1
ω
2
H
d h
i
d
l Proof E2
n =
Z 1
0
Z 1
0
∞
X
ω=1
∞
X
r=0
cωrψωr(α)φω(β) −
2θ−1
X
ω=1
µ−1
X
r=0
cωrψωr(α)φω(β)
2
dαdβ,
E2
n =
Z 1 Z 1
∞
X
∞
X
c2
ωrψ2
ωr(α)φ2
ω(β)dαdβ, ω
2
Hence proved the required result. Hence proved the required result. □ □ 5 Convergence analysis (11)
fter simplification we obtain (11) After simplification we obtain |Cωr| ≤
M
r
√
25θ+1π
Z π
0
|(sin x)εr(x)| dx, Again using the value of εr(x) from Eqs. (10) in (11), we get |Cωr| =
M
r
√
25θ+1π
Z π
0
sin x
sin(r −1)x
r −1
−sin(r + 1)x
r + 1
dx,
≤
M
r
√
25θ+1π
Z π
0
sin x
sin(r −1)x
r −1
+
sin x
sin(r + 1)x
r + 1
dx, ≤
2rπM
r (r2 −1)
√
25θ+1π
=
√
2πM
2
5θ
2 (r2 −1)
. Since ω ≤2θ−1. Therefore Since ω ≤2θ−1. Therefore |Cωr| ⩽
√
2πM
(2ω)
5
2 (r2 −1)
, i.e. |cωr| ⩽
√
2πM(2ω −1)
(2ω)
5
2 (r2 −1)
Z 1
0
φω(β)dβ, 7 |cωr| ⩽
√
2πM
(2ω)
5
2 (r2 −1)
2ω −1
ω
. Therefore
∞
X ∞
X
cωr is absolutely convergent to u(α, β). Therefore ω=1 r=0
(Error estimation). 5.1 Problem 1 Let us consider the problem [28] of the form (1) for R = 0 along with the
initial condition as below,
uβ + uuα = 0,
u(α, 0) = α. (13)
uβ + uuα = 0,
u(α, 0) = α. (13) Eq.(13) has the exact solution u(α, β) =
α
β−1. Let. Eq.(13) has the exact solution u(α, β) =
α
β−1. Let. uβ + uuα = 0. (14) (14) uβ + uuα = 0. β
(
)
Integrate Eq.(14) w.r.to β limit from 0 to β so that,
u(α, β) +
R β
0 uuαdβ = −α. Now, let’s use the proposed chebyshev wavelet method (CWM) for µ = 4
and θ = 3, So, let’s choose an truncated solution of the form, u(α, β) =
4
X
ω−1
3
X
r−0
cωrζωr(α)φω(β) 8 Therefore, applying the procedure as proposed in section 4, we get a system of
equations the solving them using a proper method we get the desired wavelet
coefficients as, c10 = −0.0783; c11 = −0.0554; c12 = 0; c13 = 0;
c20 = −0.2350; c21 = −0.1662; c22 = 0; c23 −0
c30 = −0.3917; c31 = −0.2769; c32 = 0; c33 = 0;
c40 = −0.5483; c41 = −0.3877; c42 = 0; c43 = 0. Hence u(α, β) = (c10ζ10 + · · · + c13ζ13) + (c20ζ20 + · · · + c23ζ23) β+
(c30ζ30 + · · · + c33ζ33) β2 + (c40ζ40 + · · · + c43) β3
u(α, β) = −α + (−α)β + (−α)β2 + (−α)β3
u(α, β) = (−α)
1 + β + β2 + β3 The series with infinite terms is obtained for larger values of µ. u(α, β) = (−α)
1 + β + β2 + β3 + β4 + · · ·
= (−α)
(1 −β)−1
u(α, β) =
α
β −1 This is also the precise solution to this problem. This problem demonstrates
the efficacy and high accuracy of the suggested approach because it converges
far faster than any other wavelet. Table 1 compares numerical values of the
Chebyshev wavelet solution (CWS). Numerical values obtained by this method and exact values are compared
in Table 1. Table 1 : Comparison of CWS and the exact solution for the problem 1. 5.2 Problem 2 Let us consider the problem [28] of the form (1) for R = 0 along with the
initial condition as below,
u˙β −uα =
2α −α2
e−β, α > 0, β > 0,
u(α, 0) = α2, 0 < α ⩽1. (15) (15) (15) has the exact solution u(α, β) = α2e−β. We solve Eq. (15) by the proposed Chebyshev wavelet method by fixing val-
ues θ = 3;
µ = 4 and we have the Chebyshev wavelet solution of the above
problem as u(α, β) = α2
1 −β + β2
2 −β3
6
. Hence for larger values of µ, the solution is the exact solution. The precise
solution, with β values discretized by 0.2 units and α values discretized by
0.25 units, has been compared to the Chebyshev wavelet solution. The subse- (15) has the exact solution u(α, β) = α2e−β. We solve Eq. (15) by the proposed Chebyshev wavelet method by fixing val-
ues θ = 3;
µ = 4 and we have the Chebyshev wavelet solution of the above
problem as u(α, β) = α2
1 −β + β2
2 −β3
6
.
Hence for larger values of µ, the solution is the exact solution. The precise
solution, with β values discretized by 0.2 units and α values discretized by
0.25 units, has been compared to the Chebyshev wavelet solution. The subse-
quent Table 2 shows these outcomes. Table 2 : Comparison between CWS and the Exact solution for th
α
β
Exact Solution CWS at µ = 5 Absolute error
0.25 0.2 0.0512
0.0512
1.61e −07
0.4 0.0419
0.0419
4.9e −06
0.6 0.0343
0.0343
3.67e −05
0.8 0.0282
0.0281
1.50e −04
0.50 0.2 0.2047
0.2047
6.45e −07
0.4 0.1676
0.1676
1.99e −05
0.6 0.1373
0.1372
1.47e −04
0.8 0.1129
0.1123
1.09e −04
0.75 0.2 0.4605
0.4605
1.45e −06
0.4 0.3771
0.3771
4.49e −05
0.6 0.3090
0.3087
3.30e −04
0.8 0.2541
0.2527
1.35e −03 ble 2 : Comparison between CWS and the Exact solution for the problem 2 5.1 Problem 1 Table 1 : Comparison of CWS and the exact solution for the prob
α
β
Exact Solution CWS at µ = 8 Absolute error
0.25 0.2 −0.3125
−0.3124
8.00e −10
0.4 −0.4166
−0.4163
2.73e −04
0.6 −0.6250
−0.6145
1.049e −02
0.8 −1.2500
−1.0402
2.09e −01
0.50 0.2 −0.6250
−0.6249
1.06e −06
0.4 −0.8333
−0.8327
5.46e −04
0.6 −1.2500
−1.2290
2.09e −02
0.8 −2.5000
−2.0805
4.19e −01
0.75 0.2 −0.9375
−0.9374
2.40e −06
0.4 −1.2500
−1.2491
8.19e −04
0.6 −1.8750
−1.8435
3.14e −02
0.8 −3.7500
−3.1208
6.29e −01 9 5.4 Problem 4 Let us consider the problem of the form (1) for R ̸= 0 along with the initial
condition as below,
1 2 uβ + 1
2
u2
α = α,
u(α, 0) = 2. (17) (17) which has the exact solution u(α, β) = 2 sechβ+α tanhβ. 5.3 Problem 3 Let us consider the problem [28] of the form (1) for R ̸= 0 along with the
initial condition as below,
uβ + uuα = 2β + α + β3 + αβ2,
u(α, 0) = 0. (16) (16) u(α, β) = β2 + αβ is the exact solution of (16). u(α, β) = β2 + αβ is the exact solution of (16). u(α, β) = β2 + αβ is the exact solution of (16). 10 Integrating Eq. (16) with respect to ’ β ’ and applying the initial condition,
we get u(α, β) +
Z β
0
uuαdβ = β2 + αβ + β4
4 + αβ3
3 0
We solve Eq. (16) by Chebyshev wavelet method for θ = 3; µ = 4. There-
fore the approximate solution takes the form as u(α, β) =
4
X
ω=1
3
X
r=0
cωrζωr(α)φω(β) and after solving the above equation for coefficients we get, c10 = 0; c11 = 0; c12 = 0; c13 = 0;
c20 = 0.7050; c21 = 0.0554; c22 = 0 : c23 = 0
c30 = 0.6267; c31 = 0; c32 = 0; c33 = 0;
c40 = 0; c41 = 0; c42 = 0; c43 = 0. H Hence, u(α, β) = (c10ζ10 + · · · + c13ζ13) + (c20ζ20 + · · · + c23 ζ23) β
+ (c30ζ30 + · · · + c33ζ33) β2 + (c40ζ40 + · · · + c43ζ43) β3. 5.5 Problem 5 Let us consider the nonhomogeneous Advection the problem equation,
uβ −uα = −1,
u(α, 0) = α2,
0 < α ⩽1. (18) (18) which has the exact solution u(α, β) = (α + β)2 −(α + β) + α. which has the exact solution u(α, β) = (α + β)2 −(α + β) + α. which has the exact solution u(α, β) = (α + β)2 −(α + β) + α. 5.7 Problem 7 Let us consider the problem of the form (1) for R ̸= 0 along with the initial
condition as below, uβ + 1
2
u2
α = eα + β2e2α,
u(α, 0) = 0. (20) (20) which has the exact solution u(α, β) = βeα. which has the exact solution u(α, β) = βeα. 5.6 Problem 6 Let us consider the problem of the form (1) for R ̸= 0 along with the initial
condition as below,
uβ + uuα = 1 −e−α (β + e−α) ,
u(α, 0) = e−α. (19) (19) which has the exact solution u(α, β) = β + e−α. 11 Fig. 1 Graphical representation of the CWS solution for the problem 4. Fig. 1 Graphical representation of the CWS solution for the problem 4. Fig. 2 Surface representation of CWS for the problem 6. Fig. 2 Surface representation of CWS for the problem 6. 12 Fig. 3 Plot representation of CWS for the problem 6. Fig. 3 Plot representation of CWS for the problem 6. Fig. 3 Plot representation of CWS for the problem 6. 5.8 Problem 8 Let us consider the nonhomogeneous Advection the problem equation, uβ + 1
2
u2
α = α,
u(α, 0) = 2. (21) (21) which has the exact solution u(α, β) = 2 sechβ+α tanhβ. 13
Fig. 4 Plot representation of CWS for the problem 7. 13 13
Fig. 4 Plot representation of CWS for the problem 7. Fig. 4 Plot representation of CWS for the problem 7. Fig. 4 Plot representation of CWS for the problem 7. 5.9 Problem 9 Let us consider the nonhomogeneous Advection problem equation,
uβ −uα = −α2β, α > 0, β > 0
u(α, 0) = 2−sin α . (22) (22) which has the exact solution u(α, β) = 2−sin(α −β) + α4
12 −α3β
3
−(α−β)4
12
. which has the exact solution u(α, β) = 2−sin(α −β) + α4
12 −α3β
3
−(α−β)4
12
. which has the exact solution u(α, β) = 2−sin(α −β) + α4
12 −α3β
3
−(α−β)4
12
. 6 Conclusion It can be inferred from the research above that the Chebyshev wavelet-based
collocation method is significantly more effective than many other exist-
ing methods for numerically solving nonlinear Advection partial differential
equations in a variety of scientific and engineering disciplines. Increase the
number of collocation points to obtain more precise results. Declarations • Funding
Not Applicable • Conflict of interest/Competing interests All Authors declare that they have
no conflict of interest. • Conflict of interest/Competing interests All Authors declare that they have
no conflict of interest. • Ethics approval Not Applicable • Consent to participate Yes • Consent for publication Yes • Availability of data and materials Yes • Code availability Not Applicable • Authors’ contributions Yes 7 Acknowledgements The authors are thankful to the Director, Institute of Engineering and Tech-
nology, Lucknow for encouraging and providing the facilities for the research
work. 14
Fig. 5 Surface representation of CWS for the problem 7. 14 Fig. 5 Surface representation of CWS for the problem 7. Please refer to Journal-level guidance for any specific requirements. References [1] Saberi-NadjafiJ, Ghorbani A. He’s homotopy perturbation method:
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Fig. 8 Comparison of the Chebyshev wavelet solution with exact solution at β = 0.2 for
the problem 9. 17
Fig. 8 Comparison of the Chebyshev wavelet solution with exact solution at β = 0.2 for 17 Fig. 8 Comparison of the Chebyshev wavelet solution with exact solution at β = 0.2 for
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solving the initial and boundary value problems of Bratu-type equations. Applied Numerical Mathematics 2018;128;205-216. [25] Kajani MT, Vencheh AH, Ghasemi M. The Chebyshev wavelets opera-
tional matrix of integration and product operation matrix. International
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Hall/CRC; 2002 Sep 17. [27] Mason JC, Handscomb DC. Chebyshev polynomials. Chapman and
Hall/CRC; 2002 Sep 17. [28] Liu N, Lin EB. Legendre wavelet method for numerical solutions of
partial differential equations. [28] Liu N, Lin EB. Legendre wavelet method for numerical solutions of
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Equations: An International Journal. 2010 Jan;26(1):81-94. [27] Mason JC, Handscomb DC. Chebyshev polynomials. Chapman and
Hall/CRC; 2002 Sep 17. References Numerical Methods for Partial Differential
Equations: An International Journal. 2010 Jan;26(1):81-94. Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. ¦g.eps ¦g.eps
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https://openalex.org/W4205771740
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https://www.frontiersin.org/articles/10.3389/fpubh.2021.744601/pdf
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English
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Prevalence and Potential Risk Factors for Occupational Low Back Pain Among Male Military Pilots: A Study Based on Questionnaire and Physical Function Assessment
|
Frontiers in public health
| 2,022
|
cc-by
| 8,574
|
Prevalence and Potential Risk
Factors for Occupational Low Back
Pain Among Male Military Pilots: A
Study Based on Questionnaire and
Physical Function Assessment 1 Department of Rehabilitation Medicine, Air Force Medical Center, PLA, Beijing, China, 2 Department of Rehabilitation
Medicine and Physiotherapy, Anhui Medical University, Hefei, China Keywords: low back pain, military pilots, functional test, transversus abdominis, risk factor Objectives: Low back pain (LBP) has negative implications for the military’s combat
effectiveness. This study was conducted to determine the prevalence and risk factors of
LBP among pilots through a questionnaire and physical function assessments. Edited by:
Christopher Scheibler,
Harvard University, United States Methods: Data on the demographic and occupational characteristics, health habits,
physical activity, and musculoskeletal injuries of 217 male pilots (114 fighter, 48 helicopter,
and 55 transport pilots) were collected using a self-reported questionnaire and physical
function assessments. Reviewed by:
Wayne Albert,
University of New Brunswick
Fredericton, Canada
Aleksandra Truszczy ´nska-Baszak,
Józef Piłsudski University of Physical
Education in Warsaw, Poland
Manuel Monfort Pañego,
University of Valencia, Spain Results: LBP prevalence was 37.8% in the total cohort and 36.0, 45.8, and 34.5%
among fighter, helicopter, and transport pilots, respectively. Multivariate regression
analysis revealed that the risk factors significantly associated with LBP were neck
pain [odds ratio (OR): 3.559, 95% confidence interval (CI): 1.827–6.934], transversus
abdominis activation (OR: 0.346, 95% CI: 0.172–0.698), and hip external rotator strength
(OR: 0.001, 95% CI: 0.000–0.563) in the total cohort; neck pain (OR: 3.586, 95% CI:
1.365–9.418), transversus abdominis activation (OR: 0.268, 95% CI: 0.094–0.765), hip
external rotator strength (OR: 0.000, 95% CI: 0.000–0.949), and weekly flying hours (OR:
3.889, 95% CI: 1.490–10.149) in fighter pilots; irregular strength training (OR: 0.036, 95%
CI: 0.003–0.507) and hip external rotator strength (OR: 0.000, 95% CI: 0.000–0.042) in
helicopter pilots; and neck pain (OR: 6.417, 95% CI: 1.424–28.909) in transport pilots. *Correspondence:
Chaoqun Ye
yechaoqun37@163.com Specialty section:
This article was submitted to
Occupational Health and Safety,
a section of the journal
Frontiers in Public Health Specialty section:
This article was submitted to
Occupational Health and Safety,
a section of the journal
Frontiers in Public Health Received: 25 August 2021
Accepted: 13 December 2021
Published: 04 January 2022 Received: 25 August 2021
Accepted: 13 December 2021 Conclusions:
High volume flight schedules and weak core muscle functions have
significant negative effects on pilots’ back health. LBP is commonly associated with
high weekly flying hours, worsening neck pain, transversus abdominis insufficient
activation, and reduced hip extensor/rotator strength. Risk factors vary among pilots
of different aircraft. Thus, specific core muscle training would be especially important for
military pilots. ORIGINAL RESEARCH
published: 04 January 2022
doi: 10.3389/fpubh.2021.744601 Abbreviations: LBP, low back pain; PLA, People’s Liberation Army of China; BMI,
body mass index; TrA, transversus abdominis; SD, standard deviation; OR, odds
ratio; CI, confidence interval. Citation: Yang Y, Liu S, Ling M and Ye C (2022)
Prevalence and Potential Risk Factors
for Occupational Low Back Pain
Among Male Military Pilots: A Study
Based on Questionnaire and Physical
Function Assessment. Front. Public Health 9:744601. Front. Public Health 9:744601. doi: 10.3389/fpubh.2021.744601 January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org Yang et al. Back Pain Among Military Pilots Study Design This cross-sectional study was designed to investigate the
prevalence of LBP among military pilots. The participants
were recruited at the Airforce Medical Center and the data
collection was started from July 2019 to January 2021. Before
initiating this experiment, the study protocol was approved
by the ethics committee of the Air Force Medical Center of
People’s Liberation Army of China (PLA) (Process No. 2020-
150-PJ01). All participants were asked to complete the consent
form in which they were informed about study aim and
experimental procedures. p
Analyzing the relationship between pilots and LBP is
complicated, and different aircraft vary greatly. Different types
of aircraft have shown different prevalence of LBP (1) because
of different +Gz forces (flying loads), whole-body vibration,
and other properties. Specifically, fighter pilots have a high
+Gz exposure (6, 10) and helicopter pilots are more affected
by vibration during flight (11–14). Therefore, it is necessary to
discuss the LBP problem in different aircraft separately. Some
occupational factors, such as flight experience and flying hours
(11, 15) and the fact that the duration of occupational exposure of
pilots varies (1), may be other reasons for the occurrence of LBP. Though, compared with utility pilots, military pilots demand
better physical function such as stronger core muscle to tolerate
high load or prolonged confined sitting (1), but few studies in
the literature have discussed these issues. Furthermore, previous
studies investigating LBP within the last 12 months have reported
different rates of LBP in the same type of aircraft, as recent and
more severe pain may be remembered more clearly than earlier
episodes of pain (16). Therefore, evaluating the incidence of LBP
within the last 3 months may be more reliable. INTRODUCTION This study aimed to identify the factors related to the
development
of
occupational
LBP
in
military
pilots. It
was hypothesized that the demographic and occupational
characteristics, along with physical functions, were related to the
incidence of LBP among pilots. The incidence of low back pain (LBP) among flight personnel
has increased significantly due to the continuously increasing
intensity of flight training (1–5), and one out of three pilots
has reported LBP (6). LBP among pilots imposes significant
challenges to the strength of the military. Spinal-related disorders
such as LBP have become one of the highest causes of grounding
for service pilots in China (7). In addition, LBP may affect pilots’
attention and concentration (8), motor control (9), postural
stability (9), and ultimately operational safety. This non-combat
injury has become a major cause of troop attrition in modern
warfare, so effective strategies must be found to reduce the high
incidence of LBP in pilots. Participants Only male participants were recruited from the four Air Force
military units and they were certified pilots aged between
20 and 55 years old. Participants who belong to any of the
following criteria were excluded: (1) a current or past history of
known spinal trauma, signs of neurological deficit, osteoarthritis,
rheumatoid arthritis, or instrumental lesions, (2) suspended
from flying for more than four consecutive weeks in the last 6
months (i.e., holiday, study, etc.). In total, 249 male participants
who engaged in flying fighter, helicopter, or transport aircraft
volunteered to participate in this study, but only 217 of them
carried out the physical function assessment. The dropout data
were: (1) 28 pilots were temporarily dispatched to other places;
(2) two pilots withdrew because of injury during physical fitness
test; and (3) two pilots had intolerable back pain caused by
prolonged car driving. Previous studies (16–18) have found that patients with LBP
differ from healthy people in physical functional abilities such
as hip mobility (19), low back muscle strength and endurance
(16, 20, 21), and transversus abdominis activation (22, 23). Lifestyle factors, such as physical activities and other health
habits, were also associated with occurrence of LBP (24). These
predictive factors may be similar, but the impact of pilot-specific
occupational characteristics should receive more attention. The
physical functional abilities’ assessments can guide the targeted
training to prevent LBP. At present, the targeted training
mainly focuses on the exercise of abdominis and back muscle
strength, but ignores the core muscle functional training, such
as transversus abdominis (TrA) activation. These related issues
have not been mentioned in the recently published studies of the
pilot’s LBP, and therefore only through the analyses of personal
and occupational information of the pilots and the assessment
of physical function can the problematic parts of the current
training be identified and improved. Physical Function Assessments It was held for 5 s in each test, and the
average values of three replicates were used for data analysis. The maximum isometric strengths of the trunk included two
tests: participants sat on a backrest chair with arms across the
chest and feet offthe ground; then they were fixed the body
on the backrest by a strap and the hand-held dynamometer
on their trunk by another strap; by the dynamometer was
placed on the sternum stem/the height of the level with the
fourth or fifth thoracic vertebra to allow the participant to resist
bending forward/backward. The maximum isometric strengths
of hip included four tests: the extensor required participants to
maintain the prone position with hip extension 0◦and knee
flexion 90◦, and the dynamometer was placed on the midline of
the posterior thigh to allow the participant to resist extension;
the abductor required participants maintain the supine position
with hip extension 0◦, and the dynamometer was placed on the
lateral supra patella to allow the participant to resist abduction;
the rotator required participants maintain the prone position
with hip extension 0◦and knee flexion 90◦, and the dynamometer
was placed on the lateral malleolus/medial malleolus to allow
the participant to resist internal rotation/external rotation. The same tester (SL) performed all measurements to ensure
consistency, and muscle strength testing order was randomized
to minimize bias. The balance test was defined as standing on one foot without
shoes with eyes closed, and the participant got a 0 point if he
failed to remain balanced for <30 s, otherwise one point. When
the participant was unable to maintain balance, such as jumping,
raising the leg to the ground, or maintaining with external force,
the test ended. All tests were performed by the same test group (YY, ML, and
SL) and the same tester performed the same measurements to
ensure consistency (YY for ROM test, ML for PRONE test, and SL
for muscle strength tests). The intra-tester reliability of measures
was carried out before data collection, and intra-tester reliability
was good with associations of intraclass correlation coefficient
ranging from 0.80 to 0.92. No standard test protocol exists for
the physical function assessments of pilots. It took about 90–
120 min to complete the above tests in the same day. Physical Function Assessments Physical function assessments contain the range of motion
(ROM) of hip medial/lateral rotation, trunk muscle strength
(isometric strength of trunk flexor and extensor) (27, 28), trunk
muscle endurance [flexor endurance test, Sorensen test, and side-
bridge test (21)], isometric strengths of hip muscles [extensors,
abductors, and internal/external rotators (29)], and TrA muscle
activation and balance test. Detailed procedure of each outcome
was presented below. The ROM of hip was measured using the clinical inclinometer
as described by Eoghan et al. (30), which showed good test-
retest reliability [intraclass correlation coefficient (ICC), 0.86]. Participants were asked to maintain the prone position with the
measured hip placed in 0◦of abduction and knee flexed to 90◦. The inclinometer was placed in the distal tibia and adjusted to
0◦; then the hip was allowed to do internal or external rotation
until the contralateral pelvis lifted to stop. Repeat three times
in each direction. The ROM was measured in the dominate leg,
defined as the preferred leg for kicking a football in the lab, and
the maximum value was reported. TrA muscle activation (PRONE test) was assessed using a
pressure biofeedback unit (Stabilizer Pressure Biofeedback Unit,
Chattanooga Group Inc., USA) as described previously (31, 32),
which showed good test-retest reliability (ICC =0.81) (31). This
test was designed measure the ability of performing abdominal
hollowing by holding the contraction for 10 s within 60–66
mmHg (70 mmHg began), and the score was expressed as
contraction seconds (max value was 10). When the participant
had compensatory movements such as rectus abdominis curling,
pelvic forward tilt, hip flexion, or inability to breathe abnormally,
or the pressure drops more than 10 mmHg or <4 mmHg
during the 10s, the test ended. Previous studies have stated that
the results of the PRONE test >2 s indicated the transverse
abdominis can be activated (32), so participants were sub-
grouped by the results into TrA activation group (≥3 s) and TrA
inactivation group (<3 s). Unlike the other tests, each participant
could have three to five attempts before the PRONE test. p
The
maximum
isometric
strengths
of
trunk
and
hip
muscles were measured using a calibrated digital hand-held
dynamometer (MicroFET 2, Hoggan Health Industries, USA)
as described previously (27–29), which showed good test-retest
reliability on measuring trunk and hip muscle strength (ICC,
0.86–0.97) (27, 29). Frontiers in Public Health | www.frontiersin.org Questionnaire Demographic information including age, height and weight
(used to calculate body mass index (BMI) were collected. Participants were also asked to report additional data on
occupational characteristics: (1) total flying hours (divided into
four groups:<1000h, 1000–2000h, 2000–3000h and ≥3000h) and
weekly flying hours (6 h was used a cut-offpoint: ≥6 h per
week refers to high intensity while <6 h was defined as low
intensity) in the past 6 months; (2) health habits (alcohol and
smoking); (3) weekly strength training and core muscle training
of >3 times; and (4) musculoskeletal injuries (as measured by
the Nordic musculoskeletal questionnaire) (25). To minimize
recall bias (16), pain experience in the last 3 months was
measured, which was different from a previous study using
the 12-month reports. LBP in this study was defined as pain
symptoms that persisted for within the last 3 months, and the
pain or discomfort affected daily flight schedules and required
treatment (26). January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org 2 Back Pain Among Military Pilots Yang et al. prone with the lower body fixed to the bed and upper body
extended over the edge of the bed with the anterior superior
iliac spine parallel to the edge of the bed; a chair was placed
in front of the bed, and the arms were supported on the chair;
when the test started, the participant lifted his arms away from
the chair and crossed the chest, keeping the upper body on
the horizon; if the upper body had a downward trend, it was
allowed to remind once and return to the horizontal position,
and the test would end at another descent. Thirdly, the Side-
bridge test required participants to lie on their sides by left feet
and left elbow supported with lifting the hips offthe bed to
maintain a straight line; if the pelvis had a downward trend, it
was allowed to remind once and return to the straight position,
and the test would end at another descent. During all tests,
participants were reminded to maintain their position as long
as possible. Physical Function Assessments The order
of all the test items was the balance test, the ROM tests of hip,
the TrA muscle activation test, the maximum isometric strength
tests of the trunk and hip muscles, and trunk muscle endurance
tests. The maximum isometric strength tests and the trunk
muscle endurance tests were intervals by no <20 min. Besides,
these maximum isometric strength tests in each direction were
intervals by no <60 s, and the three trunk endurance tests by no
<5 min. Trunk muscle endurance was measured using three tests (21). Firstly, the Flexor endurance test was used in which participants
were asked to sit with the trunk risen to 60◦from the bed with
arms across the chest and the knees and hips flexed to 90◦; the
participant is asked to hold this position for as long as possible;
the test ends when the body leans backwards at an angle of
<60◦. Secondly, the Sorensen test required participants to lie January 2022 | Volume 9 | Article 744601 3 Back Pain Among Military Pilots Yang et al. TABLE 1 | Demographic and other characteristics of the study population. TABLE 1 | Demographic and other characteristics of the study population. TABLE 1 | Demographic and other characteristics of the study population. Physical Function Assessments Aircraft category
Total
Fighter pilots
Helicopter pilots
Transport pilots
p-value
(N = 217)
(n = 114)
(n = 48)
(n = 55)
Demographic data
Age (year)
36.7 (7.6)
36.6 (6.0)
37.2 (7.9)
36.6 (10.0)
0.901
Height (cm)
174.5 (7.8)
174.3 (10.0)
175.1 (4.2)
174.4 (4.2)
0.829
Weight (kg)
73.4 (6.9)
72.6 (5.6)
74.6 (8.0)
74.0 (8.0)
0.201
BMI (kg/m2)
24.2 (2.1)
24.3 (1.9)
24.0 (2.1)
24.2 (2.5)
0.826
<24 (n)
99 (45.6)
49 (43.0)
21 (43.8)
29 (52.7)
≥24 (n)
118 (54.4)
65 (57.0)
27 (56.3)
26 (47.3)
Smoking (n)
112 (51.6)
62 (54.4)
29 (60.4)
21 (38.2)
0.055
Alcohol (n)
69 (31.8)
33 (28.9)
18 (37.5)
18 (32.7)
0.557
Occupational data
Total flying hours (h)
2197.4 (1563.7)
2019.7 (1102.2)
2324.0 (1722.9)
2455.5 (2122.7)
0.194
<1000 (n)
47 (21.7)
17 (14.9)
11 (22.9)
19 (34.5)
≥1000, <2000 (n)
60 (27.6)
41 (36.0)
7 (14.6)
12 (21.8)
≥2000, <3000 (n)
49 (22.6)
30 (26.3)
15 (31.3)
4 (7.3)
≥3000 (n)
61 (28.1)
26 (22.8)
15 (31.3)
20 (36.4)
Week flying hour (h)
7.3 (7.1)
8.16 (7.9)
6.2 (6.4)
6.3 (5.4)
0.137
Low-weekly-hour (n)
126 (58.1)
71 (62.3)
19 (39.6)
36 (65.5)
High-weekly-hour (n)
91 (41.9)
43 (37.7)
29 (60.4)
19 (34.5)
Physical activity
Regular strength training (n)
156 (71.9)
84 (73.7)
38 (79.2)
34 (61.8)
0.122
Regular core muscle training (n)
85 (39.2)
49 (43.0)
21 (43.8)
15 (27.3)
0.112
Pain location
Neck (n)
54 (24.9)
34 (29.8)
10 (20.8)
10 (18.2)
0.199
Upper back (n)
20 (9.2)
12 (10.5)
4 (8.3)
4 (7.3)
0.768
Lower back (n)
82 (37.8)
41 (36.0)
22 (45.8)
19 (34.5)
0.421
Presented as mean (standard deviation) or as absolute values (percentages). Frontiers in Public Health | www.frontiersin.org Statistical Analysis The data obtained through the questionnaire and LBP assessment
were presented as mean with standard deviation (SD) and as
absolute values with percentages. Data normality was tested using
the Kolmogorov–Smirnov test. Normally distributed data were
evaluated using One way ANOVA, and a chi-square test was
used for non-normally distributed data. Then we conducted a
Bonferroni’s post-hoc analysis to identify changes among aircraft. A binary logistic regression of variables with p < 0.20 was used to
calculate the multivariate odds ratio (OR) and 95% confidence
interval (CI) according to the results of univariate regression
analysis. A stepwise backward elimination procedure was used
to determine the optimum regression equation. For a term
to be retained, the term should significantly contribute to the
prediction of y (p <0.05). Multivariate regression was adjusted
for age and total flying time. Using the logistic regression model,
the possibility of incidence of LBP (y) based on the independent
variable (x) was calculated depending on questionnaire data and
physical function data where A are regression coefficients and B
are dummy variables, and the full regression model was: Participant Characteristics Participant Characteristics Participant Characteristics
A total of 217 male pilots (age: 36.7 ± 7.6 years; BMI: 24.12
± 2.13 kg/m2), including 114 fighter pilots, 48 helicopter
pilots, and 55 transport pilots, volunteered to participate in
this study. The demographic data and other characteristics were
displayed in Table 1. No group differences were observed in the
demographic data, occupational features, and the pain location
of spine. Total flying hours was 2197.4 on average, and pilots
of different aircraft were distributed differently in hours groups. Fighter pilots (36.0%) were mainly distributed in 1,000–2,000 h,
but helicopters (62.6%) and transports (43.7%) above 2000 h. Weekly flying hours in the past 6 months was 7.3 and 60.4%
of helicopter pilots over 6 h. Physical functions were presented
in Table 2. Significant group differences were detected in fle/ext
strength (p = 0.040), Sorensen test (p < 0.001), side bridge
(p = 0.012), hip extensor strength (p < 0.001), hip abductor
strength (p = 0.035) and hip external rotator strength (p =
0.024). Results from further comparison indicated that the fighter
pilots showed significantly higher fle/ext strength (p = 0.035),
side-bridge score (p < 0.001), hip extensor strength (p < 0.001)
and hip external rotator strength (p = 0.042) than those of the
helicopter pilots. The fighter pilots had a significantly higher
Sorensen test score (p < 0.001), hip extensor strength (p <
0.001) and hip abductor strength (p = 0.039) than those of the
transport pilots. y = Ax + B
(1) (1) Collinearity was checked all the related independent variable
which a correlation coefficient <0.50. All analyses were
performed using SPSS version 17.0 (IBM Corporation, USA). A
p-value of <0.05 was considered to be statistically significant. January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org 4 Back Pain Among Military Pilots Yang et al. TABLE 2 | Physical functions in the study population. DISCUSSION Non-combat injuries are the leading cause of pilot attrition and
military discharge. Back pain, especially LBP (approximately 75%
of cases), is the most common complaint in military personnel
and appears to increase during training and combat deployments
(33). The present study tried to identify the risk factors of
occupational LBP among pilots by analyzing demographic,
occupational, and muscle function data. The main finding was
that LBP of pilots was positively related to neck pain history
and weekly flying hours, but negatively related to TrA muscle
activation and hip strength. Furthermore, these factors varied Epidemiology of LBP in Aircraft Pilots In this study, the prevalence of LBP was 37.8% in the total cohort
and 36.0%, 45.8%, and 34.5% in fighter, helicopter, and transport
pilots, respectively. The rate of back pain is high among military
pilots, from 32 to 89% (1, 2, 4, 5, 11, 15, 34). Besides, vibration
frequencies and +Gz gravity loads vary among aircraft types
and are the influential factors of LBP (6, 14). The prevalence of
LBP is remarkably higher among utility and attack helicopter
pilots (89.38% and 74.55%, respectively) than among fighter and
transport pilots (64.02% and 47.47%, respectively) (1). Lis et al. (35) found that helicopter pilots had the highest incidence of
LBP among all occupations. Compared with previous studies,
the LBP reporting rate in this study is relatively low, and the
results showed no significant differences in different aircraft. The
prevalence of LBP was lower in our cohort because the more
stringent definition of LBP increased the credibility of the results
to some extent. This finding is corroborated by another study in
which the 3-month prevalence of LBP is slightly lower than the
12-month prevalence (11). y1 = 1.269x1 −1.060x2 −7.131x3 + 1.692
(2)
y2 = 1.277x1 + 1.358x5 −1.316x2 −11.032x3 + 3.168 (3)
y3 = 20.386x4 −3.317x6 + 6.586
(4)
y4 = 1.859x1 −1.012
(5) y1 = 1.269x1 −1.060x2 −7.131x3 + 1.692
(2)
y2 = 1.277x1 + 1.358x5 −1.316x2 −11.032x3 + 3.168 (3)
y3 = 20.386x4 −3.317x6 + 6.586
(4)
y4 = 1.859x1 −1.012
(5) (2) (5) The risk factors for LBP were shown in Table 3. The risk factors for LBP were shown in Table 3. The unadjusted and adjusted analyses with linear regression
models for the LBP were reported in Table 3. The multivariate
regression analysis revealed that some factors significantly
predicted LBP. The full regression model of (1) the total cohort,
(2) the flight pilots, (3) the helicopter pilots, and (4) the transport
pilots were (y1: the total cohort, y2: the flight pilots, y3: the
helicopter pilots, y4: the transport pilots; x1: neck pain, x2:
TrA activation, x3: hip strength of external rotator, x4: hip
strength of internal rotator, x5: high-weekly-hour, x6: Strength
training irregularly): Participant Characteristics Total
Aircraft category
Fighter pilots
Helicopter pilots
Transport pilots
p-value
(N = 217)
(n = 114)
(n = 48)
(n = 55)
Hip ROM (◦)
Medial rotation
26.6 (7.4)
25.3 (8.2)
26.7 (5.8)
29.2 (6.3)
0.657
Lateral rotation
40.0 (11.0)
36.3 (11.8)
43.7 (9.2)
44.3 (7.5)
0.059
Fle/ext strength (%BW)
0.98 (0.37)
1.03 (0.34)
0.87 (0.36)*
0.99 (0.41)
0.040
Endurance (s)
Flexor endurance test
104.2 (50.3)
108.4 (48.4)
102.1 (53.3)
97.4 (51.6)
0.391
Sorensen test
88.0 (31.7)
95.1 (28.4)
85.8 (38.8)
75.1 (27.5)#
<0.001
Side bridge
65.5 (24.5)
70.1 (24.5)
58.9 (21.5)*
61.7 (25.4)
0.012
PRONE test (s)
2.6 (4.0)
2.9 (4.2)
2.3 (3.8)
2.4 (3.7)
0.641
TrA activation (n)
152 (70.0)
76 (66.7)
35 (72.9)
41 (74.5)
TrA inactivation (n)
65 (30.0)
38 (33.3)
13 (27.1)
14 (25.5)
Hip strength (%BW)
Extensor
0.35 (0.10)
0.38 (0.09)
0.31 (0.08)*
0.33(0.10)#
<0.001
Abductor
0.20 (0.05)
0.21 (0.05)
0.19 (0.06)
0.19 (0.04)
0.035
Internal rotator
0.16 (0.05)
0.16 (0.05)
0.14 (0.04)
0.16 (0.05)
0.100
External rotator
0.17 (0.04)
0.18 (0.05)
0.16 (0.04)*
0.16 (0.04)#
0.024
Balance test (n)
102 (47.0)
49 (43.0)
23 (47.9)
30 (54.5)
0.366
Presented as mean (standard deviation) or as absolute values (percentages). TABLE 2 | Physical functions in the study population. g
g
p
p
Indicated significant differences between flighter and Transport pilots. Frontiers in Public Health | www.frontiersin.org Factors Associated With LBP among pilots of different aircraft types. These findings may
provide new insights into the pilot training. Can Demographic Characteristics Predict
LBP in Pilots? As people age, inevitably increased BMI and its age-related
functional decline can contribute to the higher risk of developing
LBP (17), but the relationship between demographics and LBP
in pilots is inconclusive. A recent study showed that age is a risk
factor for LBP in pilots (4). By contrast, we found no correlation
between these factors which are consistent with other reports (5,
11). The mean BMI of pilots was higher than 24, which might be January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org 5 Back Pain Among Military Pilots Yang et al. TABLE 3 | Risk factors for low back pain in the study population. Univariate
Multivariatea
Pilot category
Variables
β
OR (95% CI)
p-value
β
OR (95% CI)
p-value
Total cohort
Irregular strength training
0.508
0.602 (0.319–1.137)
0.118
Neck pain
1.296
3.656 (1.925–6.943)
<0.001
1.269
3.559 (1.827–6.934)
<0.001
Upper back pain
1.237
3.445 (1.313–9.037)
0.012
Side bridge endurance
−0.010
0.990 (0.978–1.001)
0.080
TrA activation
−0.996
0.381 (0.197–0.736)
0.004
−1.060
0.346 (0.172–0.698)
0.003
Hip strength
Extensors
−2.402
0.090 (0.005–1.747)
0.112
Internal rotators
−7.343
0.001 (0.000–0.251)
0.016
External rotators
−7.477
0.001 (0.000–0.300)
0.019
−7.131
0.001 (0.000–0.563)
0.033
Fighter pilots
Age
0.247
1.050 (0.983–1.121)
0.149
BMI ≥24
0.575
1.776 (0.804–3.922)
0.155
Total flying hours
0.775
1.000 (1.000–1.001)
0.174
High–weekly-hour
1.053
2.867 (1.293–6.355)
0.009
1.358
3.889 (1.490–10.149)
0.006
Irregular core muscle training
0.523
0.593 (0.274–1.284)
0.185
Neck pain
1.390
4.014 (1.724–9.346)
0.001
1.277
3.586 (1.365–9.418)
0.010
Upper back pain
1.431
4.182 (1.175–14.889)
0.027
TrA activation
−1.057
0.347 (0.141–0.857)
0.022
−1.316
0.268 (0.094–0.765)
0.014
Hip strength
Extensors
−5.149
0.006 (0.000–0.613)
0.030
Internal rotators
−11.038
0.000 (0.000–0.081)
0.011
External rotators
−12.648
0.008 (0.000–0.038)
0.008
−11.032
0.000 (0.000–0.949)
0.049
Helicopter pilots
Height
−0.107
0.898 (0.779–1.037)
0.143
BMI ≥24
0.916
2.500 (0.766–8.160)
0.129
Irregular strength training
−2.409
0.090 (0.010–0.782)
0.029
−3.317
0.036 (0.003–0.507)
0.014
Flexor endurance test
−0.011
0.989 (0.976–1.002)
0.098
Hip internal rotator strength
12.110
0.000 (0.000–0.618)
0.125
20.386
0.000 (0.000–0.042)
0.020
Transport pilots
High-weekly-hour
−0.985
0.373 (0.103–1.352)
0.133
Neck pain
1.859
6.417 (1.424–28.909)
0.016
1.859
6.417 (1.424–28.909)
0.016
TrA activation
−0.853
0.426 (0.103–1.767)
0.240
aAdjusted for age and total flying time. Adjusted R2 of the total cohort = 0.412; adjusted R2 of fighter pilots = 0.319; adjusted R2 of helicopter pilots = 0.183; adjusted R2 of transport pilots = 0.194. TABLE 3 | Risk factors for low back pain in the study population. due to high muscle mass. Frontiers in Public Health | www.frontiersin.org Can Demographic Characteristics Predict
LBP in Pilots? In our study, the number of flying hours per week, but not total
flying time, was associated with LBP. The total number of flying
hours reflects the exposure time to a particular occupational
environment. Exposure time is related to LBP (37). Long-term
exposure associated with high-load pressure results in muscle
contraction and potentially leads to musculoskeletal disorders,
especially LBP and neck pain. Recent studies established that an
increased prevalence of LBP was mainly associated with pilots’
long flying time (1, 5, 33, 37). However, some studies reported
inconsistent findings (6). Flying more than 6 h a week increased
the risk of LBP in fighter pilots because of continuously high
+Gz exposure during flight and insufficient recovery time. We
found that the average total flying time of fighter pilots is the
least of the three aircraft, but their average weekly flying time
is indeed the longest. This finding also confirms the effect of
long weekly working hours on pilots. Pilots flying the same type
of aircraft may be exposed to different factors depending on
the military mission and flight schedule; therefore, discussing
total flying hours only is insufficient. The increased number
of flying hours per week in our cohort can reflect the level of
occupational exposure in the last 6 months and is therefore
a better indicator of chronic musculoskeletal pain. Vibration
is considered to be a characteristic of helicopters, but results
from different studies were contradictory (1, 5, 11, 37). The
whole-body vibration is transmitted to the entire body through
the seat, resulting in a reduction in muscle fatigue resistance,
then leading to LBP in helicopter pilots (38). In our study, the
prevalence of LBP was higher in helicopter pilots than the other
two types of aircraft; however, no relationship was found between
exposure (total-flying-hours and week-flying-hours) and LBP. So, the exposure time to vibration may be as an independent
indicator for helicopter pilots which is worth to be discussed in
future studies. muscles of transport aircraft pilots was significantly weaker,
and the performance in other tests were also weak. This may
be due to the fact that work as transport aircraft pilot is
less physically demanding than work as a fighter or helicopter
pilot (1). The TrA is an important respiratory muscle and deep
postural muscle and plays an important role in maintaining
core trunk stability (39). Can Demographic Characteristics Predict
LBP in Pilots? Moreover, contradictory to the findings
of previous investigations (5, 13), there was no significant
association between height and LBP in the present study. were more aware of the importance of exercise as evidenced by
the fact that 75% performed strength training regularly regardless
of the presence of LBP. Another reason might be that weak
deep postural muscles lead to superficial muscles overactivation,
so general strength training without kinetic control may be
counterproductive to the protection of the spine (36). g
y
Regular strength training is the foundation of good physical
performance and core muscle strengthening training can
decrease the risk of back pain (17). The findings showed a good
awareness of strength training among pilots of which a large
percentage of our cohort practice strength training regularly, and
this percentage was higher than that of a 2012 survey (50%) (10). However, the number of participants that perform core muscle
training was low. We also found that the concept of core muscle
training was unclear to the pilots, which may explain why the
results are contrary to the hypothesis. Most of them believed that
core muscle strengthening engaged the rectus abdominis (sit-
ups) and erector spinae (back extension). This misconception
may lead to inadequate training. A paradoxical finding was that
irregular strength training was a protective factor for LBP in
helicopter pilots, which might be due to the fact that these pilots Neck pain and upper back pain were independent risk factors
for LBP in the total cohort and in fighter pilots; neck pain was a
negative predictor of LBP in the multivariate regression analysis
and was the greatest predictor of LBP in transport pilots. This
finding indicates that neck pain, upper back pain, and LBP are
interconnected (33). The spine should be viewed as a whole. The fatigue of the spine muscles during flight may change the
sitting posture, forcing the back to bend more. To maintain trunk
balance, the pilot may need to forward his neck further. This
series of compensatory actions may make the pilot’s spine more
susceptible to pain. Therefore, we should be alert to the possibility
of LBP in a patient with neck pain and vice versa. January 2022 | Volume 9 | Article 744601 6 Back Pain Among Military Pilots Yang et al. Is LBP Associated With Flying Experience? Can Demographic Characteristics Predict
LBP in Pilots? Respiratory muscle fatigue affects
anti-G respiratory maneuver training; for this reason, core
muscle strengthening is crucial (40). We found that TrA muscle
activation was associated with LBP, and the risk of LBP was
significantly reduced in the pilots who had a better TrA
function. A study found no difference in the TrA thickness
between the patients with recurrent LBP and healthy controls
(22). However, Hagin et al. (23) reported that the respiratory
exertion of individuals with LBP was higher when lifting heavy
weights; this finding suggests that the relationship between
TrA and LBP may be due to TrA dysfunction. Based on
clinical findings, weakness of the deep postural muscles can
lead to overcompensation of the superficial large muscles,
which can further lead to impaired muscle motor control
(22, 41, 42). Therefore, low-load motor control training is
an integral part of the solution to chronic pain compared to
endurance and strength training. A study by Salmon et al. (36) stated that training of the deep postural muscles was
effective in improving neck pain in helicopter pilots. Similarly,
a randomized controlled study found that the prevalence of
LBP is lower among US Air Force pilots who performed
specific core muscle exercises regularly vs. a control group that
did not perform these exercises (43). The advantages of TrA
were more pronounced in our cohort. The results indicate
the importance of good TrA function for LBP prevention and
suggest that the TrA improves tolerance to +Gz loads. Therefore,
biofeedback training and strengthening exercise in core muscle
training are essential to improve the TrA function of pilots for
LBP prevention. Frontiers in Public Health | www.frontiersin.org ETHICS STATEMENT The studies involving human participants were reviewed and
approved by the People’s Liberation Army of China (PLA)
Air Force Medical Center (Process No. 2020-150-PJ01). The
patients/participants provided their written informed consent to
participate in this study. This study has limitations. First, our study had recruited over
200 pilots who fly different types of aircraft. The sample sizes
for helicopters and transport aircraft are relatively small and
should conduct large-sample study in the future. Second, the
loss of follow-up might lead to bias. Third, this study was a part
of a larger study and the use of a uniform questionnaire while
the participants were surveyed may have resulted some targeted
information being missed. Additionally, some details related to
LBP were absent, such as active lifestyle habits and flexibility
work, so the survey study should be enhanced in the next phase
of the intervention study. We also admitted that the physical
functional assessments related to LBP may be inadequate, and
it would be improved in the future research. Finally, the cross-
sectional nature of the study does not allow the determination of
causality because evidence of the temporal relationship between
the risk factors and outcomes is lacking. Therefore, long-term
follow-up studies are necessary to assess the causes of LBP
in pilots. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding author. ACKNOWLEDGMENTS We thank TopEdit (www.topeditsci.com) for its linguistic
assistance during the preparation of this manuscript. FUNDING Funding
support
was
provided
by
Air
Force
LSD,
China (2017-651). Strengths and Limitations The study population was representative because the participants
came from different air force units in China and flew different
aircraft types. Hence, the study population can provide a broad
perspective on LBP for PLA Air Force researchers. Moreover, the
physical function results demonstrated the presence of muscle
weakness and biomechanical problems in this population. The
entire body should function optimally to maximize performance. These findings can provide a basis for developing training
programs to prevent or reduce LBP. Does Improvement in Core Muscle
Strength Reduce the Incidence of LBP in
Pilots? Previous studies focused on the influence of hip extensors
and abductors on LBP (19). We found that the strength of
hip extensors and internal/external rotators were associated
with LBP in pilots and might be related to flying posture,
as the pilots remain in a seated position and maintain their
pelvis stable, which reduce the demand on hip abductors
and increase the demand for push–pull movements by the
upper limbs. Moreover, internal and external hip rotators
are involved in the force transfer along myofascial chains
(44). The hip rotators are part of the functional line and
spiral line, which transfer the power of the lower body to
the upper body and provide core stability (45). Therefore,
this reason can also explain hip muscle weakness associated
with LBP in the context of myofascial chains. Hip flexor
strength was an independent risk factor for LBP and might
be
related
to
tolerance
to
+Gz
acceleration. Therefore,
physical training should focus on improving hip strength. Factors associated with LBP, including trunk flexor/extensor
strength ratio (20), did not increase the risk of LBP in our
participants probably because of occupational and physical
function characteristics. Research support for designing targeted training programs is
lacking because literature data on the physical functions of
military pilots are limited. The present study evaluated physical
functions that might contribute to LBP in military pilots and
found that pilots of different aircraft had different functional
performance. We also found that TrA muscle function and
hip rotator strength significantly impacted LBP. Thus, the
results provide new ideas for the targeted physical training of
military pilots. Our study showed that the core muscle strength of pilots
showed differences in different aircraft. The fle/ext strength
test found that fighter pilots’ abdominal muscle strength was
significantly better than back muscle strength, and the side-
bridge test results were also better than other models. These
tests measured core stability and endurance (21, 22) which
indicated that fighter pilots showed better core muscle function
due to their anti-G acceleration requirements during flight
(6, 10). The Sorensen test found that the endurance of back January 2022 | Volume 9 | Article 744601 7 Back Pain Among Military Pilots Yang et al. CONCLUSIONS An effective approach to reduce the incidence of LBP
is making ergonomic changes to the aircraft, however the
usefulness
of
such
changes
is
controversial. Therefore,
LBP prevention should focus on physical training, which
is critical in the short term. Targeted training improves
core muscle strength, reduces the workload of chronically
fatigued muscles such as the erector spinae and quadratus
lumborum, and further reduces the risk of chronic LBP
(17). However, no evidence-based guidelines or consensus
for LBP prevention in Chinese pilots are currently available. Developing training programs to improve physical functions in
military pilots and assessing the effects of these interventions
are fundamental. Demographic indicators were not significantly related to LBP in
military pilots. Several strategies can be adopted to reduce LBP
in this population, such as establishing adequate flight schedules
to improve rest and avoid fatigue and strengthening hip rotators
and core muscles, in particularly, the transversus abdominis
function. In addition, the interaction between neck pain and LBP
should be the focus of future research, and a holistic view of spinal
protection is needed. AUTHOR CONTRIBUTIONS CY and YY wrote and revised the research proposal and statistical
analysis plan. YY, ML, and SL collected, managed, and analyzed
the data and drafted and revised the manuscript. CY is the
guarantor and manager. All authors contributed to the article and
approved the submitted version. j
8. Mao CP, Zhang QL, Bao FX, Liao X, Yang XL, Zhang M. Decreased
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the publisher, the editors and the reviewers. Any product that may be evaluated in
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training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
11. Core
muscle
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
Part two
Nordic Questionnaire REFERENCES The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms. 28. Moreland J, Finch E, Stratford P, Balsor B, Gill C. Interrater reliability of
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strength using a hand-held dynamometer is reliable. Scand J Med Sci Sports. (2010) 20:493–501. doi: 10.1111/j.1600-0838.2009.00958.x January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org 9 Back Pain Among Military Pilots Yang et al. APPENDIX
Questionnaire (English Version)
Part one
1. Sex: □male □female
2. Date: xx/xx/xxxx
3. Height: ___cm
4. Weight: ___kg
5. Smoking: □yes □no
6. Alcohol: □yes □no
7. Aircraft type: □fighter, ____; □helicopter, ____; □transport,
____; □other, _____
8. Total flying hours: ____h
9. In the past 6 months, week flying hour: ____h
10. Strength
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
11. Core
muscle
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
Part two
Nordic Questionnaire APPENDIX
Questionnaire (English Version)
Part one
1. Sex: □male □female
2. Date: xx/xx/xxxx
3. Height: ___cm
4. Weight: ___kg
5. Smoking: □yes □no
6. Alcohol: □yes □no
7. Aircraft type: □fighter, ____; □helicopter, ____; □transport,
____; □other, _____
8. Total flying hours: ____h
9. In the past 6 months, week flying hour: ____h
10. Strength
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
11. Core
muscle
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.)
Part two
Nordic Questionnaire APPENDIX
Questionnaire (English Version)
Part one
1. Sex: □male □female
2. Date: xx/xx/xxxx
3. Height: ___cm
4. Weight: ___kg
5. Smoking: □yes □no
6. Alcohol: □yes □no
7. Aircraft type: □fighter, ____; □helicopter, ____; □transport,
____; □other, _____
8. Total flying hours: ____h
9. In the past 6 months, week flying hour: ____h
10. APPENDIX y
g
9. In the past 6 months, week flying hour: ____h 10. Strength
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.) 11. Core
muscle
training
habits:
□yes
□no
(defined that training at least three times a week and
each time not <30 min.) January 2022 | Volume 9 | Article 744601 Frontiers in Public Health | www.frontiersin.org 10
|
https://openalex.org/W4396517350
|
https://nhess.copernicus.org/articles/24/1501/2024/nhess-24-1501-2024.pdf
|
English
| null |
A satellite view of the exceptionally warm summer of 2022 over Europe
|
Natural hazards and earth system sciences
| 2,024
|
cc-by
| 14,982
|
Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024
https://doi.org/10.5194/nhess-24-1501-2024
© Author(s) 2024. This work is distributed under
the Creative Commons Attribution 4.0 License. Correspondence: João P. A. Martins (joao.martins@ecmwf.int) Correspondence: João P. A. Martins (joao.martins@ecmwf.int) Received: 16 September 2023 – Discussion started: 16 October 2023
Revised: 12 February 2024 – Accepted: 21 March 2024 – Published: 30 April 2024 Received: 16 September 2023 – Discussion started: 16 October 2023
Revised: 12 February 2024 – Accepted: 21 March 2024 – Published: 30 April 2024 Abstract. Summer heatwaves are becoming increasingly
dangerous over Europe, and their close monitoring is essen-
tial for human activities. Typically, they are monitored using
the 2 m temperature from meteorological weather stations or
reanalysis datasets. In this study, the 2022 extremely warm
summer over Europe is analysed using satellite land surface
temperature (LST), specifically the LSA SAF (Land Surface
Analysis Satellite Application Facility) all-sky LST product
(available from 2004 onwards). Since climate applications
of LST are still poorly explored, heatwave diagnostics de-
rived from satellite observations are compared with those de-
rived using ERA5/ERA5-Land reanalysis data. Results high-
light the exceptionality of 2022 in different metrics such as
the mean LST anomaly, area under extreme heat conditions,
number of hot days and heatwave magnitude index. In all
metrics, 2022 ranked first when compared with the remain-
ing years. Compared to 2018 (next in all rankings), 2022 ex-
ceeded its LST anomaly by 0.7 °C and each pixel had on av-
erage 7 more hot days. Satellite LST complements reanal-
ysis diagnostics, as higher LST anomalies occur over areas
under severe drought, indicating a higher control and am-
plification of the heatwave by surface processes and vegeta-
tion stress. These cross-cutting diagnostics increase the con-
fidence across satellite data records and reanalyses, fostering
their usage in climate applications. A satellite view of the exceptionally warm
summer of 2022 over Europe João P. A. Martins1,2, Sara Caetano1,3, Carlos Pereira1, Emanuel Dutra1,4, and Rita M. Cardoso4
1Instituto Português do Mar e da Atmosfera, 1749-049 Lisbon, Portugal
2European Centre for Medium-Range Weather Forecasts, Robert-Schuman-Platz 3, 53175 Bonn, Germany
3Direção-Geral do Território, 1099-052 Lisbon, Portugal
4 4Instituto Dom Luiz, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisbon, Portugal J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe longer than 30 years), (2) the absence of reliable all-
sky LST datasets, (3) the lack of intercomparison between
different LST time series and intercomparison among differ-
ent climate indicators, and finally (4) the lack of validation
over heterogeneous sites. However, the potential of the stan-
dard LSA SAF clear-sky LST dataset for monitoring heat-
waves has already been highlighted by Gouveia et al. (2022),
who derived heatwave diagnostics similar to those obtained
using well-established products such as MODIS LST (Wan,
2014) or ERA5/ERA5-Land (Hersbach et al., 2020; Muñoz-
Sabater et al., 2021), which have been more frequently used
to study these phenomena (Zaitchik et al., 2006; Galanaki
et al., 2022; Sutanto et al., 2020; Hundhausen et al., 2023;
Morlot et al., 2023; Agathangelidis et al., 2022). Good et
al. (2022) also demonstrated the usefulness of LST for cli-
mate monitoring and found good agreement between LST
anomalies derived from the ESA Climate Change Initiative
LST (Pérez-Planells et al., 2023) datasets and in situ 2 m
temperature anomalies derived from ECA&D E-OBS (Eu-
ropean Climate Assessment & Dataset observational dataset
over Europe) (Cornes et al., 2018). Nevertheless, the asso-
ciation between LST and 2 m temperature anomalies under
heatwave conditions is not always straightforward (Agath-
angelidis et al., 2022; Mildrexler et al., 2011), as they can ,
;
,
)
On top of these dynamic aspects, heatwave intensities over
Europe are strongly modulated by thermodynamic effects
involved in land surface–atmosphere interactions (Sousa et
al., 2020; Suarez-Gutierrez et al., 2020; Miralles et al.,
2019, 2014). The abovementioned typical synoptic situations
favour subsidence heating, reduced cloudiness and there-
fore increased net surface radiation. The excess surface en-
ergy is released as surface (longwave) radiative and turbu-
lent fluxes, both over land and over ocean, particularly over
the Mediterranean (Suarez-Gutierrez et al., 2020; Juza et al.,
2022). Soil moisture availability controls the partitioning of
surface turbulent fluxes, since in the case of water scarcity,
all the excess energy at the surface is re-emitted mostly as
longwave radiative flux and sensible heat flux, both acting
to increase near-surface temperatures. Compound events of
drought and extreme heat conditions are among the riski-
est climate-related events for Europe, especially considering
their effects on mortality, crop and forest productivity, and
wildfire risk (Zscheischler et al., 2018, 2020; Manning et al.,
2018). J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe (2022),
who derived heatwave diagnostics similar to those obtained
using well-established products such as MODIS LST (Wan,
2014) or ERA5/ERA5-Land (Hersbach et al., 2020; Muñoz-
Sabater et al., 2021), which have been more frequently used
to study these phenomena (Zaitchik et al., 2006; Galanaki
et al., 2022; Sutanto et al., 2020; Hundhausen et al., 2023;
Morlot et al., 2023; Agathangelidis et al., 2022). Good et
al. (2022) also demonstrated the usefulness of LST for cli-
mate monitoring and found good agreement between LST
anomalies derived from the ESA Climate Change Initiative
LST (Pérez-Planells et al., 2023) datasets and in situ 2 m
temperature anomalies derived from ECA&D E-OBS (Eu- management. Furthermore, when mega drought and heat-
wave events happen, there is a sense of urgency by the pub-
lic and the media, particularly concerning their connection
and attribution to climate change (Schiermeier, 2018). While
such diagnostics may take weeks to perform, remote sensing
provides the means for a real-time overview of the magni-
tude of a given event, from minutes to just a few hours after
the relevant measurements were acquired. Satellite agencies,
particularly the European Organisation for the Exploitation
of Meteorological Satellites (EUMETSAT), European Space
Agency (ESA), Copernicus and the National Aeronautics and
Space Administration (NASA), are making efforts to main-
tain stable, accurate and long-term data records of essential
climate variables (ECVs; Bojinski et al., 2014), as well as
access to digested information from those datasets. The EU-
METSAT Land Surface Analysis Satellite Application Fa-
cility (LSA SAF; Trigo et al., 2011) provides a collection
of data records related to the monitoring of land surface en-
ergy balance (including land surface temperature, LST) and
vegetation indicators from 2004 onwards. Most products are
based on measurements made by the Spinning Enhanced Vis-
ible and Infrared Imager (SEVIRI) on board the Meteosat
Second Generation (MSG) series, and their production will
be ensured in the next generation of geostationary meteoro-
logical satellites. Despite the relatively good temporal and
spatial coverage of LST products over areas with significant
heatwaves in the past decades (such as south-central Europe),
Reiners et al. (2023) showed that these products have not
been much used to study these phenomena. According to
these authors, this is due to (1) the lack of long time series
(i.e. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1502 often compromise air quality over the affected regions due to
their high dust aerosol loads (Sousa et al., 2019; Miralles et
al., 2014; Díaz et al., 2017). management. Furthermore, when mega drought and heat-
wave events happen, there is a sense of urgency by the pub-
lic and the media, particularly concerning their connection
and attribution to climate change (Schiermeier, 2018). While
such diagnostics may take weeks to perform, remote sensing
provides the means for a real-time overview of the magni-
tude of a given event, from minutes to just a few hours after
the relevant measurements were acquired. Satellite agencies,
particularly the European Organisation for the Exploitation
of Meteorological Satellites (EUMETSAT), European Space
Agency (ESA), Copernicus and the National Aeronautics and
Space Administration (NASA), are making efforts to main-
tain stable, accurate and long-term data records of essential
climate variables (ECVs; Bojinski et al., 2014), as well as
access to digested information from those datasets. The EU-
METSAT Land Surface Analysis Satellite Application Fa-
cility (LSA SAF; Trigo et al., 2011) provides a collection
of data records related to the monitoring of land surface en-
ergy balance (including land surface temperature, LST) and
vegetation indicators from 2004 onwards. Most products are
based on measurements made by the Spinning Enhanced Vis-
ible and Infrared Imager (SEVIRI) on board the Meteosat
Second Generation (MSG) series, and their production will
be ensured in the next generation of geostationary meteoro-
logical satellites. Despite the relatively good temporal and
spatial coverage of LST products over areas with significant
heatwaves in the past decades (such as south-central Europe),
Reiners et al. (2023) showed that these products have not
been much used to study these phenomena. According to
these authors, this is due to (1) the lack of long time series
(i.e. longer than 30 years), (2) the absence of reliable all-
sky LST datasets, (3) the lack of intercomparison between
different LST time series and intercomparison among differ-
ent climate indicators, and finally (4) the lack of validation
over heterogeneous sites. However, the potential of the stan-
dard LSA SAF clear-sky LST dataset for monitoring heat-
waves has already been highlighted by Gouveia et al. 1
Introduction In the last couple of decades, long and extremely hot sum-
mers have become more frequent over Europe (Hoy et al.,
2020), with increased heatwaves risk (Morlot et al., 2023). Recently, Rousi et al. (2022) reported that their frequency
is increasing 3 to 4 times faster than in other midlatitude
regions. Future climate projections have consistently indi-
cated the increase in both mean and extreme temperatures,
with extreme heat conditions becoming more likely when
more severe greenhouse gas emission scenarios are consid-
ered (Christidis et al., 2015; Lhotka et al., 2018; Amengual et
al., 2014; Zhang et al., 2020; Seneviratne et al., 2021; Hund-
hausen et al., 2023). Although the increased radiative forc-
ing due to increasing greenhouse gas concentrations in the
atmosphere is the root cause of the general shift in global
temperature distributions, its spatial patterns are not homo-
geneous. Over Europe, warm extremes have been linked to
the presence of stationary anticyclonic systems and to atmo-
spheric blocking events (Schaller et al., 2018; Bieli et al.,
2015; Brunner et al., 2018; Garcia-Herrera et al., 2010; Chan
et al., 2022). The increasing frequency of this blocking pat-
tern has been linked to the increasing persistence of double
jet stream structures, particularly because the region between
the sub-tropical and polar-front jets (i.e. latitudes between 45
and 65° N) is characterized by negative wind anomalies and
positive surface temperature anomalies (Rousi et al., 2022;
Kornhuber et al., 2017). Furthermore, the atmospheric circu-
lation induced by these synoptic configurations favours the
advection of warm Saharan air masses into Europe, which d by Copernicus Publications on behalf of the European Geosciences Union. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 2.1
LSA SAF LST The goal of this article is to illustrate the exceptionality
of summer 2022 in Europe, mainly using the datasets pro-
vided by the LSA SAF. Clouds have been identified as a
caveat to the use of this kind of dataset for monitoring heat
extremes, as they introduce spatial and temporal disconti-
nuities (Reiners et al., 2023; Gouveia et al., 2022). In par-
ticular, these discontinuities hamper a correct count of the
number of hot days (especially the number of consecutive
hot days, which is relevant for the determination of heat-
wave conditions) or a correct assessment of the spatial ex-
tent of extreme heat conditions. In this work, the new all-
sky LST product (Martins et al., 2019) is used instead. This
product is based on thermal geostationary observations by
SEVIRI, is available since 2004 and complements the infor-
mation provided by the clear-sky LST product (Trigo et al.,
2021), even over areas with significant cloud coverage. LSTs
under cloudy conditions are estimated using a surface energy
balance scheme (Barrios et al., 2024), which is used at the
LSA SAF to estimate evapotranspiration and surface turbu-
lent fluxes along with the cloudy-sky LST. The scheme uses
radiation flux and vegetation products from the LSA SAF,
H SAF (Support to Operational Hydrology and Water Man-
agement Satellite Application Facility) soil moisture and a
few screen-level variables from ECMWF as the main inputs. Thus, it overcomes the main limitations of the standard LST
products, especially of those that have been recently used for
climate monitoring (Agathangelidis et al., 2022; Mildrexler
et al., 2011; Gouveia et al., 2022). By using a product that
fills in those gaps using a physically based algorithm (i.e. es-
timates a land surface temperature value taking into account
the changes in radiative fluxes under clouds, as well as the
vegetation state and soil moisture conditions), interpolations
that are unphysical many times are avoided. Although clear-
sky conditions are typically the norm for heatwave condi-
tions, clouds are nonetheless frequent and ubiquitous. Com-
parisons of the derived information with corresponding in-
formation derived using ERA5 data are performed, not only
to provide confidence to the obtained diagnostics but also to
explore the physical differences between the 2 m temperature
(which is the standard variable used in extreme heat monitor-
ing studies) and skin surface temperature (which is what is
observable by satellite). 2.1
LSA SAF LST Furthermore, a cross-cutting analy-
sis of temperature, vegetation and soil moisture anomaly pat-
terns, all obtained using different measurement principles, is
also useful for a robust assessment of their physical consis-
tency, which, if demonstrated, improves users’ trust in those
datasets and further fosters their usage for climate applica-
ti LST corresponds to the radiative temperature of the surface
“skin”, i.e. the ground or the surface of the canopy over veg-
etated areas (Hulley and Ghent, 2019; Li et al., 2013). The
LSA SAF (Trigo et al., 2011) has been providing near-real-
time (NRT) LST estimates over Europe, Africa and part of
South America since 2004, based on infrared observations
from the Spinning Enhanced Visible and Infrared Imager
(SEVIRI) on board the four Meteosat Second Generation
(MSG) satellites. This dataset has been extensively validated,
using a set of ground stations covering a wide range of land
surface conditions (Göttsche et al., 2016; Trigo et al., 2021),
ensuring compliance of the product with its requirements in
terms of accuracy, uncertainty and temporal stability. How-
ever, a major limitation of the product is the fact that it is
not available in cloudy situations. With this in mind, a new
all-sky LST product was developed and is now distributed
in NRT (Martins et al., 2019), the MLST-ASv2 (MSG LST,
all-sky product; available at https://datalsasaf.lsasvcs.ipma. pt/PRODUCTS/MSG/MLST-ASv2/, last access: 16 Septem-
ber 2023). The new product fills in the blanks in the clear-sky
retrievals due to clouds by solving a surface energy balance
model (Barrios et al., 2024) whose main inputs are satellite
retrievals of longwave and shortwave downwelling radiative
fluxes, as well as albedo and the leaf area index (LAI), all
produced at the LSA SAF. Other inputs include soil moisture
from the EUMETSAT H SAF and screen-level meteorologi-
cal variables (including 2 m temperature, 2 m dew point tem-
perature, 10 m wind and surface pressure) from the European
Centre for Medium-Range Weather Forecasts (ECMWF). The model uses an iterative method to determine four un-
knowns: sensible heat flux, latent heat flux, skin temperature
and friction velocity. This skin temperature is used to fill in
the gaps in the clear-sky LST product, while the latent heat
flux is distributed as a product per se. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe The following section presents the data and methods fol-
lowed by the results and the main conclusions of this study. pecially towards higher temperatures. An additional limita-
tion on the analysis of LST daily time series based on polar-
orbiting satellites (Agathangelidis et al., 2022; Good et al.,
2022) is that measurements over a particular area are ob-
tained at slightly different times of day and with different
viewing and illumination geometries. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe The mechanisms involved in feedbacks between these
processes are still under debate, not only due to the remaining
uncertainties within the theoretical framework but also due
to the lack of suitable observations to support it (Miralles et
al., 2014, 2019; Seneviratne et al., 2021; Barriopedro et al.,
2023). This means that there is a significant spread in the
heatwave metrics that are provided by different global and
regional models, even at higher spatial resolutions (Petrovic
et al., 2024; Lin et al., 2022; Furusho-Percot et al., 2022;
Molina et al., 2020; Hundhausen et al., 2023). According
to
the
Copernicus
Climate
Change
Service
(C3S),
the
summer
of
2022
over
Europe
was
by
far
the
warmest
on
record
to
date,
with
an excess of 0.4 °C with respect to 2021, the pre-
vious
warmest
year
(https://climate.copernicus.eu/
2022-saw-record-temperatures-europe-and-across-world,
last access: 31 August 2023). The World Health Orga-
nization (WHO) revealed at least 15 000 deaths were
due to the extreme heat conditions, particularly over
Spain,
Portugal,
the
United
Kingdom
and
Germany
(https://www.who.int/europe/news/item/07-11-2022-
statement—climate-change-is-already-killing-us–but-
strong-action-now-can-prevent-more-deaths, last access: 31
August 2023). The combined effects of drought and extreme
heat also led to a wide range of economic impacts, namely
an overall crop loss (particularly cereal) of 9 % with respect
to the previous years’ 5-year average production (FAO,
2022), causing a generalized increase in food and grocery
prices. According
to
the
Copernicus
Climate
Change
Service
(C3S),
the
summer
of
2022
over
Europe
was
by
far
the
warmest
on
record
to
date,
with
an excess of 0.4 °C with respect to 2021, the pre-
vious
warmest
year
(https://climate.copernicus.eu/
2022-saw-record-temperatures-europe-and-across-world, last access: 31 August 2023). The World Health Orga-
nization (WHO) revealed at least 15 000 deaths were
due to the extreme heat conditions, particularly over
Spain,
Portugal,
the
United
Kingdom
and
Germany
(https://www.who.int/europe/news/item/07-11-2022- g
y
g
strong-action-now-can-prevent-more-deaths, last access: 31
August 2023). The combined effects of drought and extreme
heat also led to a wide range of economic impacts, namely
an overall crop loss (particularly cereal) of 9 % with respect
to the previous years’ 5-year average production (FAO,
2022), causing a generalized increase in food and grocery
prices. Near-real-time (NRT) monitoring of these extreme heat
events as well as of the wide range of their associated vari-
ables is therefore key to timely actions from stakeholders,
namely those involved in civil protection and agricultural https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 1503 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 2.4
ERA5/ERA5-Land reanalyses This product only assimilates scatterometer
data from ASCAT (A/B/C) and uses a stand-alone sur-
face analysis derived from the 9 km operational anal-
ysis. Although H141/H142 and H26 are not identi-
cal, a comparison during an overlap period (in 2020)
showed that the differences over Europe were reduced
(not shown). Soil moisture products from the H SAF are available
through their web portal (https://hsaf.meteoam.it/Products/
ProductsList?type=soil_moisture, last access: 16 September
2023). In the case of the selected dataset, soil moisture is lin-
early rescaled between the wilting point (0) and field capacity
(1), defining the soil wetness index (SWI). In this work, an
SWI average of the first three layers (i.e. down to 1 m below
the surface) from the daily data is used to compute monthly
means, from which the 2004–2021 climatology and anoma-
lies are derived. Soil moisture products from the H SAF are available
through their web portal (https://hsaf.meteoam.it/Products/
ProductsList?type=soil_moisture, last access: 16 September
2023). In the case of the selected dataset, soil moisture is lin-
early rescaled between the wilting point (0) and field capacity
(1), defining the soil wetness index (SWI). In this work, an
SWI average of the first three layers (i.e. down to 1 m below
the surface) from the daily data is used to compute monthly
means, from which the 2004–2021 climatology and anoma-
lies are derived. 2.4
ERA5/ERA5-Land reanalyses To provide a synoptic context for the summer 2022 con-
ditions (see Sect. 3.1) the ECMWF ERA5 climate reanal-
ysis (Hersbach et al., 2020) was analysed. ERA5 provides
hourly data on several atmospheric, land surface and ocean
parameters together with their respective uncertainties, on
a 0.25° × 0.25° grid, and can be downloaded from the
Copernicus Climate Change Climate Data Store (https://
cds.climate.copernicus.eu/, last access: 16 September 2023). Variables used here include the 850 hPa temperature (T850),
the 500 hPa geopotential height (Z500), wind speed (v) and
precipitation (precip), for the period 1980–2022 (43 years). a. The H141 data record (Fairbairn and de Rosnay, 2020)
was used from 2004–2018 and complemented with the
data record extension H142 for 2019–2020. The prod-
uct is based on a land data assimilation system which
assimilates scatterometer data (including ERS SCAT –
European Remote Sensing satellite scatterometer – and
Metop ASCAT-A/B – Meteorological Operational satel-
lite Advanced Scatterometer) and screen-level variables
(2 m temperature and relative humidity). The HTESSEL
(revised land surface Hydrology Tiled ECMWF Scheme
for Surface Exchanges over Land) land surface model is
used to propagate soil moisture information through the
soil down to the root zone. ERA5-Land is a reanalysis that provides a more detailed
description of the variables characterizing the continental
surfaces, with a higher spatial resolution (0.1°), and is pro-
duced with atmospheric forcing from ERA5 (Muñoz-Sabater
et al., 2021), relying on the ECMWF land surface model. It
is therefore more accurate since surface fields such as orog-
raphy or land cover are more detailed. The ERA5-Land vari-
ables used here are the 2 m temperature (T2m) and the skin
temperature (SKT), which is comparable in terms of physi-
cal meaning to satellite LST. Although ERA5 is by far the
most widely used dataset in heatwave studies, in this study
T2m estimates from ERA5-Land are used, as this choice al-
lows for focusing on the physical differences between T2m
and SKT, without having to consider differences introduced
by comparing different modelling systems and spatial reso-
lutions. Both SKT and T2m are used to derive “reference”
heatwave indicators, to which the ones derived by satellite
are compared. b. Since H141/H142 was not available from 2021 on-
wards, the H26 product (Fairbairn and de Rosnay, 2021)
was used. 2.3
LSA SAF vegetation products slots from 06:00 to 15:00 UTC, when at least half the data
over that period are available. This way, chances of getting an
unphysical maximum are reduced. The all-sky LST, despite
being much more spatially complete than the correspond-
ing clear-sky product, still has missing data. The product’s
ATBD (Algorithm Theoretical Basis Document) (Martins et
al., 2018) mentions that the surface energy balance model
used for cloudy scenes is not able to provide reliable data
over inland waters, which are excluded from the analysis. There are a few situations where the model does not reach
convergence after the upper limit of iterations, in which case
the model also returns a missing value. Vegetation plays an important role in the exchange of en-
ergy between the soil and the atmosphere (Katul et al., 2012;
Hoek van Dijke et al., 2020), by efficiently promoting the
water exchange between the surface and the atmosphere. The LSA SAF provides several satellite-derived vegetation
parameters. Here, the fraction of vegetation cover (FVC;
García-Haro et al., 2019) obtained from MSG observations
is used to study the vegetation state as a response to the high
temperatures experienced in the summer of 2022. FVC is the
horizontal fraction of soil covered by green vegetation, rang-
ing from 0 to 1. It is available since 2004 as a daily prod-
uct (in https://datalsasaf.lsasvcs.ipma.pt/PRODUCTS/MSG/
MTFVC/, last access: 16 September 2023) and is also an in-
dicator of drought conditions, exhibiting pronounced nega-
tive anomalies over areas under significant drought. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1504 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 2.2
H SAF soil moisture The H SAF produces, among other variables, several soil
moisture datasets. However, a soil moisture data record that
is fully compliant with the existing NRT products does not
exist yet. Therefore, to reprocess the surface energy balance
model back to 2004 and to analyse soil moisture anomalies
in Sect. 2.4, a combination of two products was used. 2.1
LSA SAF LST The results in the prod-
uct validation report (Martins and Dutra, 2022) showed an
overall accuracy (bias) of 0.0 K and a root mean squared dif-
ference of 2.9 K, when product time series are compared to
measurements from 33 in situ stations over a range of land
cover types and climate zones. The product is available ev-
ery 30 min in NRT, with a 3 km spatial resolution at nadir
(and about 4–5 km over Europe), and is then reprojected onto
a 0.05° regular grid. It was reprocessed from 2004 onwards
using the satellite data records available at the LSA SAF, me-
teorological variables from ERA5 reanalysis and a combina-
tion of soil moisture products from the H SAF. In this study, the daily maximum LST (LSTMax) is derived
from the 30 min data by taking the maximum over all time https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 3.1
Synoptic context Before the analysis of the satellite data characterizing the
exceptional summer 2022 over Europe, the synoptic con-
text is provided in this section for completeness. ERA5 data
were used for this purpose. Figure 1 shows this synoptic
context for the March–April–May period (meteorological
spring, MAM, left panels) and for June–July–August (me-
teorological summer, JJA, right panels). The different pan-
els illustrate the anomalies of temperature at 850 hPa (T850)
and 500 hPa geopotential height (Z500), as well as normal-
ized anomalies of accumulated precipitation (i.e. where the
1981–2022 seasonal mean was subtracted from the 2022 pre-
cipitation and then divided by the seasonal standard devia-
tion of the whole period). As may be seen in Fig. 1a and c,
there was an extended area of positive Z500 anomalies over
the North Sea region, covering the British Isles, the Scan-
dinavian Peninsula and central Europe. The Z500 anomalies
in the centre of this system were above the 95th percentile
of the distribution of the seasonal anomalies for this period
(not shown). This blocking pattern, characterized by strong
subsidence warming and relatively lower humidity, inhib-
ited cloud formation and, consequently, induced large areas
of negative anomalies of precipitation. Moreover, this pat-
tern was associated with an anomalous easterly/northeast-
erly wind regime, bringing drier continental air into central
Europe. Transient lows from the North Atlantic were de-
flected from these regions by anticyclonic blocking. There-
fore, above-normal T850 values (Fig. 1a) and below-normal
precipitation (Fig. 1c) were observed in northern and cen-
tral Europe, with areas in southeastern France showing some
of the warmest and driest anomalies in the whole reference
period (highlighted as dotted areas). The JJA synoptic con-
figuration (Fig. 1b) shows that the atmospheric blocking pat-
tern over central Europe persisted and even aggravated across
the summer, with the centroid of the anomaly located more
towards central Europe (when compared to the MAM con-
figuration). Once again, it is characterized by exceptionally
high positive Z500 anomalies and associated with a promi-
nent anomalous easterly wind towards central/western Euro- To facilitate comparison across different periods and re-
gions, the magnitude of a heatwave is estimated through a
standardization of the daily maximum temperature Tx. The
daily magnitude Md proposed by Russo et al. (2015) is used: Md =
Tx−P25
P75−P25 , if Tx > P25
0, if Tx ≤P25. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe for all the heatwave days in that period: for all the heatwave days in that period: taking the median of that monthly mean across the whole ref-
erence period. For JJA anomalies, the June, July and August
means are averaged for each year and then the median over
all years is calculated to obtain the reference climate for that
case. When computing monthly means, it was ensured that
at least 85 % of the days in each month were available. This
prevents spurious values in the disc edge contaminating the
monthly value. HWMI =
d=d2
X
d=d1
Md,
(2) (2) where d1 and d2 are the Julian days between which the sum is
computed. By considering the duration and intensity of heat-
waves, HWMI allows for the quantification of the magnitude
of heatwaves in different periods and regions of the world
(Russo et al., 2015). In this work, HWMI is always computed
for the whole JJA period. y
Heatwaves are commonly characterized as a consecutive
number of days when the temperature is excessively higher
than normal (Sutanto et al., 2020; Xu et al., 2016; Meehl and
Tebaldi, 2004). However, several authors use different defi-
nitions, which has a significant influence on the assessment
of the impact of climate change on this phenomenon. In this
study, heatwaves are defined as a period of 3 or more consec-
utive days with a daily maximum temperature (Tx) above the
90th percentile (P90) of the reference period of 2004–2021. These days where P90 is exceeded are hereafter referred to as
hot days. The percentile is calculated for each day of the Ju-
lian calendar considering a 31 d window around the Julian
day, for all years in the reference period, based on Russo
et al. (2015). P90 and the multi-year medians used in this
study were calculated relying on a bootstrapping technique
as defined by Zhang et al. (2005). This technique consists
of replacing the year for which the percentile is calculated
with the next year in the time series, except for the last year,
where a mean of the previous years’ estimates is used. This
kind of procedure avoids the possible effects of heterogene-
ity between the distributions of values in the reference period
and the year where the percentile is evaluated. 3.1
Synoptic context (1) (1) Here, Tx may be obtained using the LSA SAF LST or either
SKT or T2m from ERA5. As in Cardoso et al. (2019), a slight
difference from the standard index relies on the fact that the
25th and 75th percentiles (P25 and P75) are calculated con-
sidering all Tx values in the reference period, whereas in
Russo et al. (2015) these were obtained from annual max-
ima time series. This means that there is one percentile value
per pixel, which implies that the same temperature anomaly
causes a larger daily magnitude Md over areas with less tem-
perature variability within the reference period. In the case of
P50 and P90, the annual variability is still represented to en-
sure that anomalies/exceedances are evaluated against their
expected values for a given time of year. Finally, also following Russo et al. (2015), an adapted
version of the heatwave magnitude index (HWMI) is used,
which is simply the sum of the daily magnitudes Md over a
given period (e.g. a single heatwave, a month or a full year), https://doi.org/10.5194/nhess-24-1501-2024 2.5
Heatwave definition and metrics To derive monthly and seasonal anomalies, the reference
“climate” is calculated by first estimating Tx monthly means
for all months in the reference period of 2004–2021 and then https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 1505 https://doi.org/10.5194/nhess-24-1501-2024 3.2
LST anomalies and comparison with ERA5 We start the analysis by focusing on the summer LST cli-
matology and anomalies for the years 2018, 2019, 2021 and
2022 (Fig. 2). These were among the recent years with the
largest JJA anomalies over Europe. The 2004–2021 climatol-
ogy is represented in Fig. 2a and shows that higher LSTMax
values are observed around the Mediterranean, except for
coastal areas that are more prone to the occurrence of low
clouds (such as western Iberia). Figure 2b, c, d and e show the
JJA anomalies for 2018, 2019, 2021 and 2022, respectively. The 2022 LSTMax JJA anomaly was much stronger when
compared to other years, both in terms of the anomaly mag-
nitude and its spatial extent. Seasonal anomalies of 3–5 °C
were observed over most of central Europe, in an area ex-
tending from northern Spain to the British Isles and to eastern
Germany. Large areas of France exhibited seasonal anoma-
lies of up to 7–8 °C. The area with the largest anomalies was
over Hungary, where LSTs were 9.5 °C above normal. Over
the considered domain, 2022 showed an area-averaged JJA
anomaly of 2.2 °C (where the anomaly was weighted by the
area of each pixel), while the remaining years with the high-
est area-averaged JJA anomalies in the data record show val-
ues of 1.1 °C (2018 and 2019) and 0.8 °C (2012). For instance, temperature anomalies over burned areas are
generally higher when they are determined based on LST ob-
servations than when they are based in SKT, since the rele-
vant information in the physiographic fields is not included
in ERA5-Land (e.g. surface emissivity, albedo, vegetation
cover). Some of these fire scars are visible in Fig. 3 (e.g. over
the Iberian Peninsula). Close inspection of pixels roughly
corresponding to burned areas associated with fires that oc-
curred in July 2022, namely in northwestern (Castilla and
Leon) and southern (Andalusia) Spain, reveals LST −SKT
mean differences of up to 14 °C in the August maps. LST-
based anomalies are generally comparable to SKT-based
anomalies in June. A more pronounced negative difference
is observable over the Alps, and more pronounced (3–5 °C
or more) positive differences were observed over western in-
land Iberia, Hungary, Romania and Ukraine. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1506 pean countries, such as France, Germany and Italy. Persis-
tent warm and dry advection of continental air masses from
eastern Europe contributed to the exceptionally high T850
anomalies over France, Italy, and parts of Spain and Ger-
many. A lack of precipitation was also observed over the
Iberian Peninsula, Germany and the British Isles (Fig. 1d),
with many areas where it was below the 10th percentile
(shown as dotted areas). This configuration of higher-than-
normal temperatures in spring and summer and overall lack
of rainfall, especially during springtime, led to the intensifi-
cation of the widespread drought event that started in early
spring and lasted throughout the entire summer. anomalies extended into eastern Europe, where the monthly
anomaly ranged around 2–5 °C. Southern Balkan countries
and Ukraine were left out of the general pattern of very high-
temperature anomalies in August. p
g
Although there are a few recent climate assessments made
using remotely sensed LST (Wang et al., 2022; Good et al.,
2022; Gouveia et al., 2022), this data type is not typically
used to derive this kind of climate monitoring information,
and therefore a comparison with more standard datasets is
relevant in the context of this work. In Fig. 4, a comparison
between the anomalies shown in Fig. 3 and the correspond-
ing anomalies using ERA5-Land SKT and T2m is shown. All anomalies used the same reference period and calculation
methodology. While LST and SKT are highly comparable in
terms of their physical meaning, T2m results mostly from
assimilated surface meteorological observations, while SKT
has a stronger model weight. LST for clear-sky situations
(which typically prevail in heatwave conditions) is derived
from thermal infrared brightness temperatures. For cloudy
skies, a surface energy balance model is employed, which
mostly is based on optical and infrared satellite information
but also relies on screen-level data from ERA5 for the esti-
mations of surface fluxes. Similarly, ERA5-Land SKT is also
derived from a surface energy balance driven by ERA5 fluxes
and screen-level data, modulated by the land surface charac-
teristics (e.g. vegetation cover) and conditions (available soil
moisture). Therefore, it is influenced by ERA5 errors, such
as errors in the cloud fraction, or errors in the representa-
tion of the physiographic fields such as vegetation cover/LAI
(which are static) or sub-surface conditions (soil moisture). https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 3.2
LST anomalies and comparison with ERA5 Throughout the
summer, these positive difference patterns intensified, and
positive differences rose all over central and Mediterranean
Europe, with August being the month where the overall dif-
ferences were higher, reaching 3 to 5 °C or more. Figure 3 shows the evolution of monthly anomalies of the
3 summer months (JJA). In June, a general pattern of pos-
itive temperature anomalies is evident throughout Europe,
with values ranging around 3–4 °C, and in Hungary, northern
Spain and Italy there were anomalies of up to 8 °C. North-
western Iberia, the British Isles and countries bordering the
Aegean Sea exhibited slightly colder-than-normal tempera-
tures. In July, the anomalies over Hungary and Romania suf-
fered a very strong increase, with values around 7–10 °C,
while in central/western Europe there were anomalies rang-
ing between 3–6 °C. In northeastern Europe and Türkiye,
the anomalies were negative, with temperatures about 1–3 °C
lower than normal. In August, the anomaly was the most
intense of the 3 summer months, with an average value of
around 3 °C above the climatological reference. The areas
most affected by anomalously warm conditions were cen-
tral Europe and Hungary, with the monthly anomaly for
these areas ranging from 8–10 °C. The pattern of positive Regarding the differences between the LST-based and the
T2m-based anomalies in June, the spatial patterns are similar
to those of the LST difference with SKT (Fig. 4a, b). How-
ever, there some differences worth noting: (i) the absence
of a pronounced negative difference over the Alps, (ii) a https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1507 Figure 1. Anomalies of the synoptic atmospheric configuration over Europe for two seasons in 2022, as given by ERA5: (a, c) MAM and
(b, d) JJA. (a, c) T850 anomalies (in colour) and Z500 (black contours). Dotted areas denote areas where T850 was above its 95th percentile. (b, d) Normalized anomaly of accumulated precipitation (in colour) and v anomalies (black arrows). Dotted areas denote areas where the
precipitation anomaly was below the 10th percentile. All anomalies were computed with respect to the 1981–2022 reference period. Arrows
are spaced 2° × 2° for the sake of readability. Figure 1. 3.2
LST anomalies and comparison with ERA5 Anomalies of the synoptic atmospheric configuration over Europe for two seasons in 2022, as given by ERA5: (a, c) MAM and
(b, d) JJA. (a, c) T850 anomalies (in colour) and Z500 (black contours). Dotted areas denote areas where T850 was above its 95th percentile. (b, d) Normalized anomaly of accumulated precipitation (in colour) and v anomalies (black arrows). Dotted areas denote areas where the
precipitation anomaly was below the 10th percentile. All anomalies were computed with respect to the 1981–2022 reference period. Arrows
are spaced 2° × 2° for the sake of readability. tent clouds, which, if undetected, could introduce a neg-
ative bias in LST (Martins and Dutra, 2022; Trigo et al.,
2021; Martins et al., 2019). These situations are however
relatively infrequent. For mid-range LSTMax values, differ-
ences are generally positive, with larger LSTMax −T2mMax
values, especially in July when they reach around 2 °C. For
LSTMax around 45–55 °C, temperature differences are rela-
tively lower, but they increase again for very high LSTMax
values. more consistent positive difference over central Europe and
(iii) larger differences over the regions where the pronounced
positive differences were identified in the SKT map. For July
and August, the differences became much greater, exceeding
5 °C over Hungary, central Europe, northern Italy and areas
around the northern Black Sea. However, in August, a pat-
tern of negative differences of up to −3 °C became apparent
in the easternmost parts of the domain (Fig. 4f). Regarding SKTMax −T2mMax, one should note that these
are entirely produced by reanalysis alone. The comparison
reveals that thermal contrasts between SKTMax and T2mMax
are much smoother than those between LSTMax and T2mMax. Since the surface sensible heat flux is proportional to this
difference, this suggests that sensible heat fluxes are weaker
in ERA5-Land under extreme heat conditions compared to
observations (i.e. LSTMax −T2mMax differences), although
other model parameters might play a role in the sensible flux
modulation (e.g. surface roughness). Therefore, despite the good correlations between LST and
T2m found by Good et al. (2022), these results show that
there is a wide range of situations where these temperatures
may be very different. 3.3
Number of hot days and HWMI Following the previous results focusing on the seasonal
and monthly anomalies, we now assess several aspects of
heatwave-related metrics. Figure 6a presents the number of
hot days in JJA, using LSTMax. The most striking result is
that only a few regions have less than 10 hot days, which in-
clude eastern Iberia and the southern Balkans. In contrast,
large areas over the whole west-central Europe area had
more than 40 hot days (and even more than 50). The heat- In Fig. 5, the temperature differences are further analysed
as a function of the absolute LSTMax. Their behaviour is
consistent across the absolute LSTMax range. For instance,
for lower LSTMax values, both differences are small and
negative. A large part of this can be explained by persis- https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1508 igure 2. (a) JJA median of LSTMax for the period 2004–2021. (b, c, d, e) Seasonal LSTMax anomalies for 2012, 2018, 2019 and 2022. Figure 2. (a) JJA median of LSTMax for the period 2004–2021. (b, c, d, e) Seasonal LSTMax anomalies for 2012, 2018, 2019 and 2022. Figure 2. (a) JJA median of LSTMax for the period 2004–2021. (b, c, d, e) Seasonal LSTMax anomalies ax for the period 2004–2021. (b, c, d, e) Seasonal LSTMax anomalies for 2012, 2018, 2019 and 2022. the physical differences between these three variables nec-
essarily impact these heatwave diagnostics. In central Iberia,
LSTMax reveals a pattern with up to 40 fewer hot days when
compared to SKT and T2m, consistent with negative differ-
ences in thermal anomalies with respect to the ERA5 vari-
ables (see Fig. 4). In central Europe, using LSTMax, up to
20 more hot days were detected and increases around 20–
40 in HWMI were observed. The largest differences are over
northern Italy, Hungary and eastern Romania, where there
are up to 40 more hot days and differences of up to 60 in
HWMI, with respect to both the ERA5-Land variables. wave magnitude index (HWMI) for JJA shown in Fig. 6b
provides a cumulative view of the extreme heat conditions
for each pixel. The differences between Fig. 6a and b stem
from the requirement of at least 3 consecutive hot days for
HWMI to be positive. Heatwave conditions were particularly
severe (i.e. with HWMI values of up to 120) in northeast-
ern Portugal and Spain, southeastern France, Hungary and
Slovakia, parts of Romania, and to a lesser extent (i.e. with
HWMI values between 60 and 100) northwestern France and
Luxembourg. Regions such as southeastern Spain, Scotland,
Austria, Czech Republic and the southern Balkans were not
severely affected by damaging heat conditions in summer
2022. In Fig. 7, a time series view of the mean behaviour of
the heatwave diagnostics within the box in Fig. 6a is shown
to illustrate some methodological aspects. Large day-to-day
LSTMax variability is observed over this region, with tem-
perature drops of more than 10 °C over a couple of days
(e.g. https://doi.org/10.5194/nhess-24-1501-2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J
p
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Figure 3. LSTMax monthly anomalies for (a) June, (b) July and (c) August 2022 over Europe. Figure 3. LSTMax monthly anomalies for (a) June, (b) July and (c) August 2022 over Europe. VIRI on MSG. This is the same instrument used to produce
the clear-sky LSTs (also providing the main inputs for its
cloudy scenes), although LST relies on infrared information
rather than visible and near-infrared data like in the case of
FVC. FVC quantifies the fraction of each pixel that is oc-
cupied by green vegetation and responds to soil moisture
and surface net radiation anomalies with a delay related to
plant physiology. SWI is obtained through scatterometry data
(i.e. radar) obtained by polar-orbiting instruments (such as
ASCAT on Metop) and consists of an index quantifying how
close the root-zone soil moisture is to the soil field capac-
ity (SWI = 1) or to the plant wilting point (SWI = 0). SWI
is used as input to the surface energy balance model that is
used to derive cloudy-sky LST. However, it can be inferred
that most of the retrievals under heatwave periods are made
for clear skies. Therefore, it can be assumed that LST, FVC
and SWI are mostly independent from each other. and advection of colder air masses into the region. Even so,
40 hot days were observed, 33 of which belonged to heat-
wave periods (red circles). These counts are of course very
sensitive to P90, shown as a dashed line in Fig. 7a. Both
the number of days in the rolling window and the number
of years used to determine the percentile influence the heat-
wave diagnostics because on one hand a larger moving win-
dow tends to lower the summer P90 and on the other hand
including more earlier years (which are colder due to climate
change) would also lead to a decrease in these P90 values. Therefore, if any of these options would have been consid-
ered, the observable heatwaves at the end of June and the be-
ginning and middle of August could last for slightly longer
and the last 2 hot days observed in August could have been
part of a heatwave; the overall HWMI also would be slightly
higher for the region. In Fig. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 8 the monthly FVC anomalies and the monthly
anomalies of the SWI index for June, July and August 2022
are shown. In June, most of central Europe shows small pos-
itive anomalies of FVC. Major exceptions with strong neg-
ative anomalies are France and northwestern Iberia, eastern
Hungary, and Italy (with smaller values). The SWI anomaly
patterns are not necessarily similar but broadly correspond
to the anomalies in accumulated precipitation (cf. Fig. 1),
with some exceptions. Over Switzerland, the MAM precip- 3.4
Vegetation anomalies and soil moisture anomalies In this section, independent remote sensing datasets are ex-
plored for the study period with a twofold motivation: (i) to
assess the extreme heat conditions in the context of the
drought conditions (see Fig. 1) and (ii) to identify potential
causes for the differences between LSTMax and ERA5-Land
diagnostics observed in the previous sections. FVC measure-
ments are obtained through optical imagery, made by SE- https://doi.org/10.5194/nhess-24-1501-2024 from 29 to 31 July), mostly caused by cloudy conditions The impact of using SKT or T2m instead of LSTMax to de-
rive the heatwave diagnostics is assessed in Fig. 6c–f, show-
ing the differences in the indices. In general, the patterns are
similar to those observed in Fig. 4, translating the fact that https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ 1509 https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1510 re 4. Comparison between LSTMax monthly anomalies and the corresponding anomalies using reanalysis SKT (left) and T2m
t). Comparisons are made for (a, b) June, (c, d) July and (e, f) August. Figure 4. Comparison between LSTMax monthly anomalies and the corresponding anomalies using reanalysis SKT (left) and T2m data
(right). Comparisons are made for (a, b) June, (c, d) July and (e, f) August. Figure 5. Mean differences between LSTMax and SKTMax (orange, diamonds) anomalies and between LSTMax and T2mMax (blue, circles)
anomalies as a function of mean LSTMax, for (a) June, (b) July and (c) August. On top, the number of pixels used in the calculation. Whiskers
represent the standard deviation over each interval. Figure 5. Mean differences between LSTMax and SKTMax (orange, diamonds) anomalies and between LSTMax and T2mMax (blue, circles)
anomalies as a function of mean LSTMax, for (a) June, (b) July and (c) August. On top, the number of pixels used in the calculation. Whiskers
represent the standard deviation over each interval. https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1511 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe Figure 6. (a) Number of JJA hot days detected using LSTMax (i.e. days when LSTMax > P90). (b) Total JJA HWMI derived with LSTMax. (c, e) Differences between the number of hot days obtained with LSTMax and with SKT and T2m, respectively. (d, f) Difference between the
SKT-based HWMI and T2m-based HWMI, respectively. The blue square in (a) denotes the area used for the extraction of time series data
which are analysed below. Figure 6. (a) Number of JJA hot days detected using LSTMax (i.e. days when LSTMax > P90). (b) Total JJA HWMI derived with LSTMax. (c, e) Differences between the number of hot days obtained with LSTMax and with SKT and T2m, respectively. (d, f) Difference between the
SKT-based HWMI and T2m-based HWMI, respectively. The blue square in (a) denotes the area used for the extraction of time series data
which are analysed below. as surface temperature) can be questioned when these strong
vegetation anomalies are in place (Johannsen et al., 2019;
Nogueira et al., 2020, 2021; Duveiller et al., 2022). If dy-
namic vegetation was prescribed in ERA5-Land, a negative
anomaly in FVC would imply (a) a decrease in latent heat re-
lease, since the surface cannot evaporate water so efficiently,
and (b) a reduced sensible heat flux, since roughness is re-
duced when vegetation cover decreases. Both of these effects
would act to increase SKT (especially the surface rough-
ness increase), considering that in that situation soil mois-
ture is assimilated (thus already implying reduced evapo-
transpiration). Under these circumstances, the only effective
way to compensate the excess surface net radiation is to in-
crease thermal longwave emission (i.e. increasing skin tem-
perature). itation anomaly was among the highest over the reference
period, while the SWI anomaly is negligible. Over Germany
and Poland, negative SWI anomalies are observed, which are
associated with the very low precipitation over the region, but
their expression in FVC only becomes evident from July on-
wards. Eastern countries exhibit strong positive FVC anoma-
lies and positive SWI anomalies. The arc-like feature covering Hungary, Serbia, Romania
and Moldova is consistent among precipitation, LSTMax,
SWI and FVC. This is also the region where differences be-
tween LSTMax-based heatwave diagnostics and those derived
from SKTMax (and in a lesser extent, T2mMax) are larger. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe The
positive vegetation anomaly in the eastern parts of the do-
main is also consistent with the negative LSTMax −T2mMax
differences over that area. Thermal anomalies observed from satellites and those ob-
tained from ERA5-Land SKT would then be more similar
(i.e. differences in Figs. 4 and 5 would be much lower). These
results not only highlight the added value of using LST for
heatwave monitoring instead of more standard datasets such This consistency among different remote sensing (and re-
analysed) products, obtained via different instruments and
measurement principles, suggests problems in the repre-
sentation of these variables in ERA5-Land. In fact, since
vegetation dynamics is prescribed as static information in
ERA5-Land, the reliability of some surface variables (such https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ 1512 Figure 7. (a) Evolution of LSTMax (green curve) and the respective P90 (dashed curve). Hot days are marked as a yellow circle at the top;
if they belong to a heatwave (set of 3 or more consecutive days), they are marked as a red circle. (b) Explicit differences between LSTMax
and P90. (c) Daily heatwave magnitude Md is in blue and the accumulated HWMI is in red, with values on the right axis. All data are area
averages from the blue box in Fig. 6. Figure 7. (a) Evolution of LSTMax (green curve) and the respective P90 (dashed curve). Hot days are marked as a yellow circle at the top;
if they belong to a heatwave (set of 3 or more consecutive days), they are marked as a red circle. (b) Explicit differences between LSTMax
and P90. (c) Daily heatwave magnitude Md is in blue and the accumulated HWMI is in red, with values on the right axis. All data are area
averages from the blue box in Fig. 6. as ERA5 but also may contribute to identifying ways of im-
proving ERA5, especially its surface scheme. (1) the spatial average of the seasonal LST anomaly, (2) the
average area under extreme heat conditions (i.e. Md > 2),
(3) the mean number of hot days and (4) the spatial aver-
age of the summer HWMI. There are considerable dispari-
ties between the four rankings, reflecting the way different
characteristics under analysis impacted Europe in each year. Nevertheless, our results show that 2022 was remarkably ex-
ceptional, independently of the ranking criterion, followed
by 2018. As already discussed in Sect. 2.2, in terms of mean
LSTMax anomalies, 2022 ranks in first with a mean anomaly
of 2.2 °C, followed by 2019 and 2018 with a mean anomaly
of 1.1 °C. The coldest year was 2004, with a mean anomaly
of −1.2 °C. Even in the context of a general increase in these
mean anomalies over Europe, 2022 stands out as truly excep-
tional. Regarding the mean area occupied by extreme heat
conditions (i.e. the time average of the curves in Fig. 9), a
very strong increase has been observed since 2018, with the
top 3 years being 2022, 2018 and 2020. https://doi.org/10.5194/nhess-24-1501-2024 In 2022, more than 3.5
Exceptionality of the 2022 heatwave To conclude the results, we show evidence of the exception-
ality of the 2022 heatwave magnitude and spatial extent. Fig-
ure 9 shows a time series of the proportion of European land
area affected by daily magnitudes greater than 2, from 1 June
to 1 September, together with the corresponding data from
the individual years from 2004 to 2023. For 22 % of the sum-
mer days in 2022, the proportion of land area occupied by
Md > 2 was the largest among all years. These days occurred
mainly in the middle of July and for 7 d in a row in mid-
August. Other years like 2018, 2019 and 2015 also had large
periods where a significant percentage of European land area
was under extreme heat stress. In Fig. 10, all the summers in the LST data record (2004–
2023) are ranked in terms of four different heatwave metrics: https://doi.org/10.5194/nhess-24-1501-2024 P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1513 Figure 8. (a, c, e) FVC monthly anomalies and (b, d, f) SWI, for June (a, b), July (c, d) and August (e, f). Reference period is 2004–2021. Figure 8. (a, c, e) FVC monthly anomalies and (b, d, f) SWI, for June (a, b), July (c, d) and August (e, f) Figure 8. (a, c, e) FVC monthly anomalies and (b, d, f) SWI, for June (a, b), July (c, d) and August (e, f). Reference period is 2004–2021. Figure 9. Time series of the percentage of land area affected by Md > 2, from 1 June to 31 August. The bold red curve represents 2022 data,
while other colours represent the same variable for the other years in the data record. Stars mark the days in the area where Md > 2 in 2022
was the greatest over all years. Figure 9. Time series of the percentage of land area affected by Md > 2, from 1 June to 31 August. The bold red curve represents 2022 data,
while other colours represent the same variable for the other years in the data record. Stars mark the days in the area where Md > 2 in 2022
was the greatest over all years. Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1514 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe
0. Ranking of summers over the study period according to (a) their mean LSTMax anomaly, (b) the average fraction of area
y extreme heat conditions (Md > 2), (c) the average number of hot days and (d) the area-averaged HWMI. Colours are mainly for
e purposes, where each year was classified according to the severity associated with each parameter (from less severe in blue to
severe in dark red). Figure 10. Ranking of summers over the study period according to (a) their mean LSTMax anomaly, (b) the average fraction of area
covered by extreme heat conditions (Md > 2), (c) the average number of hot days and (d) the area-averaged HWMI. Colours are mainly for
illustrative purposes, where each year was classified according to the severity associated with each parameter (from less severe in blue to
extremely severe in dark red). Another way of inspecting the exceptionality of the 2022
extreme heat conditions is to look for areas where new tem-
perature records were set, which is illustrated in Fig. 11. In
the JJA anomaly, new maxima were set for large areas of
northern Iberia, France, southern Germany, Italy and Hun-
gary (in bright cyan). These areas have strong signals over
the individual monthly maps as well. The 2018 (dark grey)
heatwave still holds the record for large parts of north-central
Europe (Hoy et al., 2020), while the 2010 (red) heatwave
set the overall record over Russia (Barriopedro et al., 2011). In the June map, the 2019 (light grey) heatwave introduced
records for this month over a large part of north-central Eu-
rope, while 2023 (light blue) set new records for northern
France and the Benelux area. In July, 2022 (bright cyan) set
new monthly records over south-central Europe and 2006
(orange) set the record for large areas from France to the
Baltic countries, while in north-central Europe the record
was set by the 2018 (dark grey) heatwave. In August, the 2 % of Europe was under extreme heat conditions, on aver-
age, and a similar picture was seen in 2018. In 2020 that value
was just over 1 %, although with an overall lower mean tem-
perature anomaly (ranking in sixth in the left panel). J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe The year
with the smaller average area covered by extreme heat con-
ditions was 2008, with only 0.09 %. As for the mean number
of hot days, again 2022 stands out in first place with nearly
21 hot days, for the JJA average for every land pixel over
Europe. This is a very large difference towards the second
in the ranking, since in 2012 around 14 hot days on aver-
age were observed. An average of 3.7 hot days were ob-
served in 2004, the last in the ranking. Finally in terms of
area-averaged HWMI, a mix between the information in the
previous rankings is observed but with an evident similarity
to the ranking of the number of hot days. This reinforces the
idea of HWMI being an index combining all the relevant in-
formation on heatwaves, namely their magnitude and their
temporal and spatial extent. 2 % of Europe was under extreme heat conditions, on aver-
age, and a similar picture was seen in 2018. In 2020 that value
was just over 1 %, although with an overall lower mean tem-
perature anomaly (ranking in sixth in the left panel). The year
with the smaller average area covered by extreme heat con-
ditions was 2008, with only 0.09 %. As for the mean number
of hot days, again 2022 stands out in first place with nearly
21 hot days, for the JJA average for every land pixel over
Europe. This is a very large difference towards the second
in the ranking, since in 2012 around 14 hot days on aver-
age were observed. An average of 3.7 hot days were ob-
served in 2004, the last in the ranking. Finally in terms of
area-averaged HWMI, a mix between the information in the
previous rankings is observed but with an evident similarity
to the ranking of the number of hot days. This reinforces the
idea of HWMI being an index combining all the relevant in-
formation on heatwaves, namely their magnitude and their
temporal and spatial extent. https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe 1515 Figure 11. Year where the record maximum average LSTMax occurred for the periods (a) JJA, (b) June, (c) July and (d) August. Figure 11. Year where the record maximum average LSTMax occurred for the periods (a) JJA, (b) June, (c) July and (d) August. role of the surface–atmosphere coupling in the modulation of
these events. The combination of these ingredients has been
key in setting the magnitude of the 2022 heatwave as well as
that of other recent mega heatwaves. year 2022 set new records for large areas from northwest-
ern Portugal to France and the British Isles and up to the
Baltic countries. The 2015 (light brown) heatwave still holds
the record for August over areas such as Poland, Belarus and
western Ukraine. It is worth noting that 2003 set JJA tem-
perature records over large areas of western Europe (Sousa
et al., 2020; Lhotka et al., 2018), but since that year is not
within the period covered by the LSA SAF LST dataset, it
does not show up in Fig. 11. This can be regarded as a caveat
to this dataset, since due to its relatively short length, it does
not allow for a full picture of the most relevant extreme heat
events over Europe for the past couple of decades. The analysis presented in this study showed that the sum-
mer extreme heat conditions was unprecedented, considering
the relatively short reference period. In particular, the analy-
sis showed that this year exhibited the largest summer tem-
perature anomaly (about 2.2 °C above the reference period
median), the largest average area under extreme heat condi-
tions (just above 2.5 % of Europe was on average under ex-
treme heat conditions, Md > 2) and the largest mean number
of hot days (average of 21 d). The combined effect of these
factors is accounted for by HWMI, which also translates the
exceptionality of summer 2022 over Europe, with an aver-
age value of 22, which was followed in the ranking by 2012
and 2018 with a summer HWMI of 11.9 and 11.7, respec-
tively. Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 This study also showed that despite some areas in June
and August having their highest monthly anomalies in 2023,
summer 2022 as a whole remains exceptionally warm, rank-
ing first in all considered metrics. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ Original draft preparation: JPAM,
RMC and SC. Review and editing: all authors. Author contributions. Conceptualization and methodology: JPAM,
RMC and SC. Data curation: ED and JPAM. Formal analy-
sis: JPAM, SC, CP, RMC and ED. Original draft preparation: JPAM,
RMC and SC. Review and editing: all authors. Competing interests. The contact author has declared that none of
the authors has any competing interests. Given the physical similarities between them, the compar-
isons of LSTMax- and SKTMax-based metrics further rein-
forced the confidence of the former, not only because they
compare relatively well in general but also because when
they do not, there are plausible reasons for it. ERA5-Land
does not rely on dynamic vegetation information, and there-
fore strong negative anomalies such as those reported here
over central Europe and over the Hungary–Romania region
are not well represented by the model. This means that sur-
face roughness and evapotranspiration efficiency are too high
in the model, leading to colder SKTs when compared to the
observed LSTs. On the other hand, the cross-cutting analysis
comparing LST, FVC and SWI anomalies shows a remark-
able physical consistency between the observed patterns,
thus reinforcing confidence in these datasets. This kind of
analysis is key to fostering their usage for all sorts of climate
applications, thus increasing user uptake of these datasets. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims made in the text, pub-
lished maps, institutional affiliations, or any other geographical rep-
resentation in this paper. While Copernicus Publications makes ev-
ery effort to include appropriate place names, the final responsibility
lies with the authors. Special issue statement. This article is part of the special issue
“Methodological innovations for the analysis and management of
compound risk and multi-risk, including climate-related and geo-
physical hazards (NHESS/ESD/ESSD/GC/HESS inter-journal SI)”. It is not associated with a conference. Acknowledgements. This work was performed within the frame-
work of the LSA SAF (https://lsa-saf.eumetsat.int, last access:
26 April 2024) project, funded by EUMETSAT. Data from ERA5
and ERA5-Land were generated using Copernicus Climate Change
Service information (2022). Carlos Pereira acknowledges the sup-
port from the project FRESAN (grant no. FED/2017/389-710),
financed by the European Union and managed by Camões I.P. Rita M. Cardoso would like to acknowledge the financial support
from the Fundação para a Ciência e a Tecnologia, I.P./MCTES,
through national funds (PIDDAC) to the Instituto Dom Luiz
(grant no. J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ 1516 (point data) which may then be interpolated into gridded
datasets (such as E-OBS) or assimilated into and earth sys-
tem models or reanalyses (such as ERA5). These methods in-
troduce interpolation and model errors into the spatially con-
tinuous T2m fields. For satellite observations, spatial cover-
age is much higher, and therefore spatial interpolation errors
are mostly absent (although there are relatively small uncer-
tainties associated with geolocation and regridding from the
original satellite observations to regular grids such as those
used in this study). Furthermore, LST is more directly linked
to the surface energy balance and provides extra informa-
tion when compared to T2m. In this work, it was shown
that when there are strong drought conditions linked with
vegetation anomalies (i.e. low SWI and low FVC), differ-
ences between LSTMax and T2m anomalies are amplified,
and therefore these results highlight that these observed dif-
ferences may be used as proxies for surface–atmosphere cou-
pling metrics. It should be noted however that SWI is used as
input to the cloudy-sky LST retrieval. However, most of the
retrievals under heatwave conditions are made under clear
skies, so most of the LST signal comes from the infrared re-
trieval and not from the surface energy balance scheme used
for cloudy-sky retrievals. from the EUMETSAT LSA SAF Data Service (2024a, b; https:
//datalsasaf.lsasvcs.ipma.pt/PRODUCTS/MSG/MLST-ASv2
and
https://datalsasaf.lsasvcs.ipma.pt/PRODUCTS/MSG/MTFVC/, re-
spectively). Soil moisture data is a combination of two datasets: The
H141 (https://doi.org/10.15770/EUM_SAF_H_0008, H SAF, 2000)
and the H26 (https://hsaf.meteoam.it/Products/Detail?prod=H26,
EUMETSAT,
2024c). The
ERA5
data
were
retrieved
from
the
Copernicus
Climate
Change
Climate
Data
Store
(https://doi.org/10.24381/cds.bd0915c6,
Hersbach
et
al.,
2023). The
ERA5-Land
data
were
retrieved
from
the
Copernicus
Climate
Change
Climate
Data
Store
(https://doi.org/10.24381/cds.e2161bac,
Muñoz-Sabater
et
al.,
2019). All datasets require a simple registration before they can be
downloaded. from the EUMETSAT LSA SAF Data Service (2024a, b; https:
//datalsasaf.lsasvcs.ipma.pt/PRODUCTS/MSG/MLST-ASv2
and
https://datalsasaf.lsasvcs.ipma.pt/PRODUCTS/MSG/MTFVC/, re-
spectively). Soil moisture data is a combination of two datasets: The
H141 (https://doi.org/10.15770/EUM_SAF_H_0008, H SAF, 2000)
and the H26 (https://hsaf.meteoam.it/Products/Detail?prod=H26,
EUMETSAT,
2024c). The
ERA5
data
were
retrieved
from
the
Copernicus
Climate
Change
Climate
Data
Store
(https://doi.org/10.24381/cds.bd0915c6,
Hersbach
et
al.,
2023). The
ERA5-Land
data
were
retrieved
from
the
Copernicus
Climate
Change
Climate
Data
Store
(https://doi.org/10.24381/cds.e2161bac,
Muñoz-Sabater
et
al.,
2019). All datasets require a simple registration before they can be
downloaded. Author contributions. Conceptualization and methodology: JPAM,
RMC and SC. Data curation: ED and JPAM. Formal analy-
sis: JPAM, SC, CP, RMC and ED. 4
Conclusions The year 2022 was exceptional for Europe in terms of spatial
and temporal extent and in terms of the magnitude of the heat
extremes that affected the continent. The dominant synop-
tic configuration caused unprecedented blocking, subsidence
heating and warm advection into the continent, as well as
a prolonged record drought from spring onwards. Although
some uncertainties persist, a theoretical framework is already
able to broadly explain the broad mechanisms involved in the This study also highlights the importance of looking into
LST as a complement to the information provided by T2m. The main source of T2m is surface meteorological stations https://doi.org/10.5194/nhess-24-1501-2024 Nat. Hazards Earth Syst. Sci., 24, 1501–1520, 2024 J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europe J. P. A. Martins et al.: A satellite view of the exceptionally warm summer of 2022 over Europ UIDB/50019/2020) and the project LEADING (grant
no. PTDC/CTA-MET/28914/2017). New perspectives on climate monitoring are supported
by the increasing availability of high-quality satellite data
records. Although the MSG-based LST data record is not still
at the stage of being used to derive fully compliant climate
normals (which typically use 30 years of data), it already
provides useful perspectives to complement the study and
monitoring of decadal surface temperature variability. These
include the study of surface–atmosphere coupling within
extreme heat events based on observations and diagnosing
caveats in more standard datasets used for the monitoring
those events. Financial support. This research has been supported by the Euro-
pean Organisation for the Exploitation of Meteorological Satellites
(LSA SAF grant); the European Commission Directorate-General Data availability. All the datasets used in this study are openly
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A multi-perspective study of Perceived Inclusive Education for students with Neurodevelopmental Disorders
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Journal of autism and developmental disorders
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Journal of Autism and Developmental Disorders (2024) 54:1611–1617
https://doi.org/10.1007/s10803-022-05643-7 Journal of Autism and Developmental Disorders (2024) 54:1611–1617
https://doi.org/10.1007/s10803-022-05643-7 BRIEF REPORT BRIEF REPORT A multi-perspective study of Perceived Inclusive Education for
students with Neurodevelopmental Disorders Emma Leifler1,2
· Anna Borg1 · Sven Bölte1,3,4 Accepted: 6 June 2022 / Published online: 4 July 2022
© The Author(s) 2022 Abstract Consensus is often a prerequisite for communities to develop initiatives to improve practice and create a future together. We investigated the consensus around the perceived educational inclusion of autistic and other neurodivergent students,
their caregivers, and their teachers. Seventeen triads of informants plus two single students from mainstream secondary
and high schools in Sweden underwent the standardized INCLUSIO interview operationalizing educational inclusion
domains. Inclusive practice was reported across groups to be poorly to sufficiently developed for different domains and
measures. Discrepancies were extensive between informants and most pronounced for students and parents versus teach
ers. The findings highlight limited consensus about inclusive education in practice and suggest enhanced participation of
neurodivergent students and their parents to improve inclusive education implementation. Keywords Inclusive education · Autism · ADHD · Consensus · Assessment · Teachers · Parents Keywords Inclusive education · Autism · ADHD · Consensus · Assessment · Teachers · Parents (Hardy & Woodcock, 2015). The coming into effect of
these policies has been paralleled by an increasing number
of children diagnosed with autism, attention-deficit hyper
activity disorder (ADHD), and other neurodevelopmental
disorders (NDDs) (Danielson et al., 2016; Maenner et al.,
2021). Thus, considerably more students diagnosed with
NDDs currently attend mainstream schools (Florian, 2014). Guidance for school professionals regarding how to imple
ment inclusive education and the provision of resources to
facilitate inclusion vary between countries and schools and
are often limited (Gitschthaler et al., 2021; Schwab, 2020). Furthermore, dissent exists about what defines inclusive
education, how it should be translated into practice, what
level of inclusion is meaningful, and what has been accom
plished (Nilholm, 2021; Nilholm & Göransson, 2017). Inclusive education is a core component of human rights,
fostering equal learning opportunities and social justice
(Armstrong, 2005). International declarations agree that
inclusive education should entail the accommodation of
all students’ needs, no matter the prerequisites, and avoid
stigma (European Agency for Special Needs and Inclusive
Education, 2018; UN, 2016; UNESCO, 1994, 2005). Many
high- and middle-income countries have introduced inclu
sive educational policies embracing these statements, aim
ing to reduce segregated education of divergent students Inclusive education is a core component of human rights,
fostering equal learning opportunities and social justice
(Armstrong, 2005). International declarations agree that
inclusive education should entail the accommodation of
all students’ needs, no matter the prerequisites, and avoid
stigma (European Agency for Special Needs and Inclusive
Education, 2018; UN, 2016; UNESCO, 1994, 2005). Abstract Many
high- and middle-income countries have introduced inclu
sive educational policies embracing these statements, aim
ing to reduce segregated education of divergent students Emma Leifler
emma.leifler@ki.se
Sven Bölte
sven.bolte@ki.se
1
Karolinska Institutet Center of Neurodevelopmental
Disorders (KIND), Centre for Psychiatry Research,
Department of Women’s and Children’s Health, Karolinska
Institutet, & Stockholm Health Care Services, Region
Stockholm, Stockholm, Sweden
2
Department of Pedagogical, Curricular and Professional
Studies, University of Gothenburg, Göteborg, Sweden
3
Child and Adolescent Psychiatry, Stockholm Health Care
Services, Region Stockholm, Stockholm, Sweden
4
Curtin Autism Research Group, School of Allied Health,
Curtin University, Perth, Western, Australia Sven Bölte
sven.bolte@ki.se Despite increasing evidence for and against certain
approaches to facilitating the educational inclusion of stu
dents with NDDs (Crosland & Dunlap, 2012; Leifler et
al., 2022; Lovett & Nelson, 2021), continuous challenges
to achieve educational inclusion have been reported, par
ticularly for students with NDDs (Pellicano et al., 2018). In
a previous study, we examined inclusive practices for stu
dents with NDDs in Sweden, as reported by a large sample
of school staff across various professional backgrounds
and school types (Bölte et al., 2021). 1
Karolinska Institutet Center of Neurodevelopmental
Disorders (KIND), Centre for Psychiatry Research,
Department of Women’s and Children’s Health, Karolinska
Institutet, & Stockholm Health Care Services, Region
Stockholm, Stockholm, Sweden
2
Department of Pedagogical, Curricular and Professional
Studies, University of Gothenburg, Göteborg, Sweden
3
Child and Adolescent Psychiatry, Stockholm Health Care
Services, Region Stockholm, Stockholm, Sweden
4
Curtin Autism Research Group, School of Allied Health,
Curtin University, Perth, Western, Australia Participants measures, particularly the absence of mentor systems and
coordination with outside school services for implement
ing support. Importantly, there were considerable differ
ences between the reports by principals, teachers, special
educators, youth workers, and school health team members. Substantial variation in perceived inclusion by neurotypical
and neurodivergent people and their families has also been
found in other studies (Jones et al., 2022). The sample consisted of groups of neurodivergent students,
their parents, and their teachers recruited in the form of 17
complete triads. Specifically, included parents and teach
ers were the caregivers and educators of the specific target
students. In addition, two solitary students were included. Hence, the study had 53 participants: 19 students (4 female,
15 male), 17 of their parents (13 mothers, 4 fathers), and 17
of their teachers. The students’ ages ranged from 15 to 20
years (M = 17.0, SD = 1.6). The average professional experi
ence among the teachers was 14.7 years (SD = 8.0, range = 2
to 32 years). The participants originated from seven main
stream secondary and high schools across an urban munici
pality on the west coast of Sweden. Participants were
recruited via requests sent to the schools and forwarded
to students, parents, and teachers by the principals. Three
of the neurodivergent participants had a self- and parent-
reported primary clinical diagnosis of autism spectrum dis
order (ASD), seven reported a double diagnosis of ASD and
ADHD, and nine reported an ADHD diagnosis. There were
additional co-existing neurodevelopmental and psychiatric
difficulties reported in the neurodivergent group: dyslexia
(3), obsessive compulsive disorder (1), and post-traumatic
stress disorder (1). However, consensus is crucial for making decisions that
are in the best interest of a community (Dressler, 2006). Hence, discovering and addressing incongruent views is
key, as they are likely to cause miscommunication, false
expectancies, or the blocking of decisions leading to fruit
ful community development. In addition, as the attitudes of
some stakeholders could be implicitly or explicitly priori
tized over those of others by decision makers, for example,
those of professionals over those of individuals with NDDs
and their caregivers, the needs of the target groups that
policies claim to serve might be neglected. Therefore, this
study’s objective was to investigate educational inclusion
by assessing the implementation of inclusive measures from
the perspective of students with NDDs, their caregivers, and
their teachers. Abstract Participants reported
poorly or modestly implemented educational inclusion 1
Karolinska Institutet Center of Neurodevelopmental
Disorders (KIND), Centre for Psychiatry Research,
Department of Women’s and Children’s Health, Karolinska
Institutet, & Stockholm Health Care Services, Region
Stockholm, Stockholm, Sweden
2
Department of Pedagogical, Curricular and Professional
Studies, University of Gothenburg, Göteborg, Sweden
3
Child and Adolescent Psychiatry, Stockholm Health Care
Services, Region Stockholm, Stockholm, Sweden
4
Curtin Autism Research Group, School of Allied Health,
Curtin University, Perth, Western, Australia 1
Karolinska Institutet Center of Neurodevelopmental
Disorders (KIND), Centre for Psychiatry Research,
Department of Women’s and Children’s Health, Karolinska
Institutet, & Stockholm Health Care Services, Region
Stockholm, Stockholm, Sweden 1
Karolinska Institutet Center of Neurodevelopmental
Disorders (KIND), Centre for Psychiatry Research,
Department of Women’s and Children’s Health, Karolinska
Institutet, & Stockholm Health Care Services, Region
Stockholm, Stockholm, Sweden
2
Department of Pedagogical, Curricular and Professional
Studies, University of Gothenburg, Göteborg, Sweden
3
Child and Adolescent Psychiatry, Stockholm Health Care
Services, Region Stockholm, Stockholm, Sweden
4
Curtin Autism Research Group, School of Allied Health,
Curtin University, Perth, Western, Australia 2
Department of Pedagogical, Curricular and Professional
Studies, University of Gothenburg, Göteborg, Sweden 3
Child and Adolescent Psychiatry, Stockholm Health Care
Services, Region Stockholm, Stockholm, Sweden 4
Curtin Autism Research Group, School of Allied Health,
Curtin University, Perth, Western, Australia 1 3 Journal of Autism and Developmental Disorders (2024) 54:1611–1617 1612 Participants Based on our experience and previous find
ings, we hypothesized that limited consensus would exist
among groups; specifically, the opinions of neurodivergent
students and their parents about implemented inclusive mea
sures would be less favorable than those of their teachers. Methods Participants completed a 21-item standardized interview
version derived from the INCLUSIO scale (Bölte et al.,
2021). The original INCLUSIO is a school staff report ques
tionnaire operationalizing inclusive educational measures
for neurodivergent students in mainstream schools. It opera
tionalizes inclusive actions on eight domains (see Table 1). The content validity of the INCLUSIO scale was established
based on a cross-professional Delphi process in a group of
NDD, education, and inclusion professionals and experts. Reliability in terms of internal consistency (Cronbach’s
alpha) for all 61 questionnaire items in a large sample of
4,778 school staff was α = 0.87, and between α = 0.70 to 0.89
for its domains (Bölte et al., 2021). For the current study,
INCLUSIO was transformed to an interview format using
a Delphi process to ensure a comparable comprehension of
item meanings across students, teachers, and parents, and
was reduced to a selection of 21 items (see Table 1) that
would be possible to evaluate across informants. For the
generated interview version, internal consistency in the
given sample was α = 0.89. INCLUSIO items are Likert-
scaled and scored from 0 to 3. Items can be answered with
“yes” (score 3, indicating inclusive practice), “rather yes”
(score 2, indicating moderate inclusive practice), “rather Data Analysis Interview data collected from students, parents, and teachers
were analyzed in SPSS/Win. 27. Descriptive statistics (M, Table 1 INCLUSIO Domains and Items: Comparisons Between Ratings by Students, Parents, and Teachers
Students
(S)
M (SD)
Parents
(P)
M (SD)
Teachers
(T)
M (SD)
F
P
Eta2
Post-hoc
Total score
32.5
(9.3)
33.3
(14.6)
42.5
(10.2)
5.2
0.001
0.23
T > S = P
Assessment of support needs (max. 9)
4.8 (1.7)
5.5 (2.6)
5.8 (2.5)
2.2
0.49
0.11
Recommendations from clinical services are used for support planning
1.8 (1.2)
1.1 (1.2)
1.8 (0.7)
1.9
0.078
0.07
There is a specific and accessible support plan document for the students
1.5 (0.9)
2.2 (1.2)
2.1 (1.2)
2.6
0.043
0.09
P = T > S
Staff involved in support plans meet regularly and support plans are
evaluated
2.0 (1.1)
2.1 (1.2)
1.8 (1.1)
0.3
0.371
0.01
Use of individualized support (max. 12)
6.4 (3.2)
7.0 (3.4)
9.5 (1.8)
5.1
0.002
0.23
T > S = P
Students are offered alternative options to demonstrate knowledgea*
1.9 (0.9)
1.9 (0.8)
2.8 (0.4)
9.0
0.0005
0.26
T > S = P
School rules are adapted to students’ needsb
1.3 (1.2) 1.94 (1.0)
2.3 (1.0)
4.3
0.09
0.15
Students receive the individual special education support needed
1.5 (1.1)
1.6 (1.2)
2.1 (1.1)
1.2
0.148
0.05
Everyday individual adaptations in the classroom and schedule are provided
1.7 (1.1)
1.6 (1.1)
2.3 (0.6)
2.7
0.036
0.10
T > S = P
Implementation of a structured learning environment (max. 9)
4.1 (2.0)
4.1 (2.6)
4.8 (2.4)
1.4
0.127
0.07
The school uses visualization of schedules and timec*
2.2 (1.0)
1.2 (1.1)
1.7 (1.1)
3.6
0.017
0.12
S > P = T
Students are offered organizational aidd
1.0 (0.8)
1.0 (1.0)
1.9 (1.1)
5.7
0.003
0.18
T > S = P
Changes to procedures are communicated to NDD* students as early as
possible
0.9 (1.0)
1.8 (1.0)
1.1 (1.1)
3.8
0.014
0.13
P > S = T
Individual changes applied to schedule/teaching (max. 6)
3.1 (1.9)
2.2 (1.5)
3.6 (1.4)
3.5
0.011
0.17
T > P
Students’ interests are integrated in teaching
1.1 (1.0)
1.4 (1.0)
1.7 (1.1)
1.2
0.149
0.05
Strategies for handling stressful situations are provided
2.0 (1.2)
0.8 (0.7)
1.9 (1.0)
7.4
0.001
0.23
S = T > P
Functional response to behavioral characteristics
(max. Design This study was approved by the Swedish Ethical Review
Authority. Informed consent was signed prior to participa
tion. We conducted an ex-post facto quasi-experimental
and correlational study. Inclusive educational practices, as
reported by three groups (neurodivergent students, their
parents, their teachers), were examined using standardized
interview findings. Group differences and convergence of
reported experienced educational inclusion were analyzed. Data collection started in the fall of 2019 and was finalized
in the fall of 2021. Due to the social distancing regulations
during periods of the COVID-19 pandemic, data collection
was performed during both face-to-face meetings and in
remote form using Zoom video conferencing, Google Meet,
or telephone. 1 3 1 3 Journal of Autism and Developmental Disorders (2024) 54:1611–1617 1613 SD) for all 21 INCLUSIO interview items, the 8 domains,
and the total score are provided for the 3 groups. Inference
statistics included three steps. First, groups were compared
for reported inclusive practices, applying general linear
modeling (MANOVA) across INCLUSIO total, domain,
and item scores, followed by post hoc Tukey tests for
single group comparisons. Then, Kappas were computed
using crosstabs within the total sample across groups for
all INCLUSIO items to determine the degree of agree
ment between groups for single inclusive measures. Finally, no” (score 1, indicating doubtful inclusive practice), “no”
(score 0, indicating no inclusive practice), and “don’t know”
(scored 9 [and converted to 0], indicating no knowledge
of inclusive practice). Item scores are summed to domain
scores and a total score. Data Analysis 6)
3.3 (1.9)
3.2 (1.6)
4.6 (1.6)
4.2
0.005
0.19
T > P = S
The staff gets time to discuss NDD* students’ behavior and support plans
1.5 (1.3)
1.3 (1.1)
2.4 (0.9)
4.9
0.005
0.16
T > S = P
The school offers space for rest and withdrawal
1.8 (1.1)
1.9 (1.0)
2.2 (1.1)
0.5
0.315
0.02
Cooperation with parents (max. 9)
6.1 (2.0)
5.3 (2.8)
6.4 (2.3)
2.2
0.495
0.11
There is a mutual exchange of knowledge about students with NDDs between
home and
school
2.1 (1.2)
1.8 (1.1)
2.5 (0.9)
1.6
0.107
0.06
The school uses caregivers’ knowledge to optimize support
1.5 (1.1)
1.8 (1.2)
2.1 (1.0)
1.1
0.173
0.04
There are regular exchanges between caregivers and responsible staff around
students with NDDs
2.4 (0.8)
1.8 (1.2)
1.9 (1.0)
2.1
0.067
0.08
Consideration of peer-relations (max. 6)
2.0 (1.5)
3.2 (2.5)
4.1 (1.2)
5.2
0.001
0.23
T > S
In group work, the composition of the group takes into account knowledge of
students with NDDs
1.3 (0.9)
1.8 (1.4)
2.5 (1.0)
4.6
0.007
0.16
T > S = P
NDD students are prepared and trained for unstructured social situations
0.7 (1.1)
1.4 (1.2)
1.6 (1.2)
3.2
0.023
0.11
T > S = P
Staff education/professionalism (max. 6)
2.8 (1.6)
2.8 (1.5)
3.8 (1.3)
1.9
0.069
0.10
The school staff has good knowledge of NDDs
1.0 (0.9)
1.0 (0.7)
1.4 (1.0)
1.9
0.143
0.05
The staff understands that individualized support might be necessary for a
student with NDD
1.8 (0.9)
1.8 (1.0)
2.4 (0.7)
2.6
0.04
0.10
T > S = P
Note. Items have been translated from Swedish and shortened for the reader’s ease and summary presentation; a e.g., allowed to present orally
instead of in written form, or vice versa; b e.g., can spend breaks in the classroom; c e.g., provide time-tables visualized schemes; d e.g., check
lists planning aids; *Neurodevelopmental disorder Use of individualized support (max. 12) 1 1 3 3 Journal of Autism and Developmental Disorders (2024) 54:1611–1617 1614 Table 2 Informant Agreement (Kappas) for INCLUSIO Items
Domains / items
Kappa
(p)
Assessment of support needs
Recommendations from clinical services are used for sup
port planning
− 0.21
(0.002)
There is a specific and accessible support plan document
(IEP) and support plans are followed-up and evaluated
0.00
(0.99)
Staff involved in support plans meet regularly
− 0.19
(0.01)
Use of individualized support
Students are offered alternative options to demonstrate
knowledge a
0.20
(0.03)
School rules are adapted to students’ needs b
0.19
(0.01)
Students receive the individual special education support
needed
0.01
(0.87)
Everyday individual adaptations in the classroom and
schedule are provided
0.11
(0.21)
Implementation of a structured learning environment
The school uses visualization of schedules and time c
− 0.11
(0.16)
Students are offered organizational aids d
0.04
(0.57)
Changes to procedures are communicated to NDD* stu
dents as early as possible
0.14
(0.03)
Individual changes applied to teaching
Students’ interests are integrated in teaching
0.06
(0.38)
Strategies for handling stressful situations are provided
− 0.18
(0.02)
Functional response to behavioral characteristics
The staff gets time to discuss NDD students’ behavior and
support plans
0.05
(0.50)
The school offers space for rest and withdrawal
0.09
(0.26)
Cooperation with parents
There is a mutual exchange of knowledge about students
with NDDs between home and school
0.07
(0.39)
The school uses caregivers’ knowledge to optimize support − 0.02
(0.82)
There are regular exchanges between caregivers and
responsible staff around students with NDDs
− 0.09
(0.22)
Consideration of peer-relations
In group work, the composition of the group takes into
account the knowledge of students with NDDs
0.10
(0.16)
NDD students are prepared for unstructured social situa
tions and trained in social interactions
0.11
(0.07)
Staff education/professionalism
The school staff has good knowledge of NDDs
0.28
(0.001)
The staff understands that individualized support might be
necessary for a student with NDD
0.03
(0.75)
Note. Use of individualized support (max. 12) Items have been translated from Swedish and shortened for the
reader’s ease and summary presentation; a e.g., allowed to present
orally instead of in written form, or vice versa; b e.g., can spend breaks
in the classroom; c e.g., provide time-tables, visualized schemes; d Pearson intercorrelations were computed between item and
domain scores, on the one hand, and total scores, on the
other, in the whole sample to investigate relations between
single inclusive measures and domains and rated overall
educational inclusion. An alpha level of 5% was adopted
for all statistics, and one-tailed tests were conducted for
directional hypotheses. Given the sample size and alpha, the
power (1-beta) to detect differences and associations in this
study was good to high (71 to 0.99) for large effects, low
to good (0.33 to 0.73) for medium effects, and low (0.10 to
0.17) for small effects (G*Power). 1 3 Results Parents and teachers scored higher than students
on the item There is a specific and accessible support plan
document, an individual educational plan (IEP) for the stu
dents (p = .04), and students scored higher than teachers and
parents for The school uses visualization of schedules and
time” (p = .002). Students and teachers scored higher than
parents on the item Strategies for handling stressful situ
ations are provided (p = .001). Parents scored higher than
students and teachers on the item Changes to procedures
are communicated to NDD students as early as possible
(p = .01). to adequate implementation ratings while teachers rated
inclusion significantly higher, even though their evalua
tions suggested there was room for improvement. Students
stated that they were not prepared for transitions and expe
rienced limited transparency in case of changes during the
school day. Transitions and limited predictability are known
challenges for neurodivergent individuals (Mandy et al.,
2016), and there are strategies to guide teachers in support
ing students to master change (Borg et al., 2021). Students
also experienced some practices as well-developed, such
as visual supports in accordance with TEACCH principles
(Virues-Ortega et al., 2013). Students and parents found that
there was a culture of exchange with responsible school staff
to discuss students’ situations. Parents agreed with teachers
that there was adequate access to students’ support plans,
but students did not share this view. Educational legislation often states that all students
have the right to be provided with a learning environment
that enables them to reach their educational goals in a safe
environment. Despite this, schools struggle to provide such
environments for multiple reasons, such as due to a lack of
best practice guidelines on inclusive education (Nilholm &
Göransson, 2017; Nilholm, 2021) and limited knowledge
about the needs of neurodivergent students (De Boer et al.,
2011; Hume et al., 2021; Långh et al., 2017; Toye et al.,
2019). Similarly, informants in our study agreed that the
school staff’s knowledge was low, indicating that the edu
cation of school staff, especially teachers, is of paramount
importance. However, in Sweden, for example, in teacher
education at universities, only recently have a small num
ber of higher education credits in NDDs been introduced as
mandatory, and there are no specific requirements to focus
on educational inclusion. Results See Table 1 for INCLUSIO item, domain, and the total
scores across groups and complete inference statistics for
group comparisons. The average student item score was
1.5. Student ratings were highest for the items, There are
regular exchanges between caregivers and responsible staff
around students with NDD (score 2.4, domain: Cooperation
with parents) and The school uses visualization of schedules
and time (score 2.2, domain: Implementation of a struc
tured learning environment), and lowest for NDD students
are prepared and trained for unstructured social situations
(score 0.7, domain: Consideration of peer-relations) and
Changes to procedures are communicated to NDD students
as early as possible (score 0.9, domain: Implementation of a
structured learning environment). The average parent item score was 1.6. Parents scored the
items There is a specific and accessible support plan docu
ment for the students (score 2.2) and Staff involved in sup
port plans meet regularly and support plans are evaluated
(both domain: Assessment of support needs) the highest. Low parent scores were found for Strategies for handling
stressful situations are provided (score 0.8, domain: Individ
ual changes applied to schedule/teaching) and The school
staff has good knowledge of NDD (score 1.0, domain: Staff
education/professionalism). The average teacher item score was 2.0. Teachers rated
the following items the highest: Students are offered alterna
tive options to demonstrate knowledge (score 2.8, domain:
Use of individualized support) and In case of group work,
the composition of the group takes into account knowledge
of students with NDD (score 2.5, domain: Consideration of
peer-relations) and the following items the lowest: Changes
to procedures are communicated to NDD students as early
as possible (score 1.1) and The school staff has good knowl
edge of NDD (score 1.4). In line with our hypothesis, the total score of all INCLU
SIO interview items was higher in teachers than in both
students and parents (p = .001). The same was true for the 1 3 Journal of Autism and Developmental Disorders (2024) 54:1611–1617 1615 domains Use of individualized support (p = .002) and Func
tional response to behavioral characteristics (p = .005), and
7 of the 21 items (p < .04). In addition, teacher scores were
higher than student scores for the domain Consideration of
peer-relations (p = .001) and higher than parent scores for
the domain Individual changes applied to schedule/teaching
(p = .01). Results Additionally, despite evidence that
professional development with regard to inclusive action for
neurodivergent students has beneficial effects (Petersson-
Bloom & Bölte, 2022), this is not systematically provided.i Kappa values for agreement between groups for all
INCLUSIO items are shown in Table 2. In total, 8 items
yielded negative to no agreement (r = − .21 to .00), 12
yielded none to slight agreement (r = .01-.20), and 1 yielded
fair agreement (r = .28, p = .001) [The school staff has good
knowledge of NDD]. In the whole group, all INCLUSIO
domains correlated highly (≥ r = .64) with the total score,
and the domain Use of individualized support demonstrated
the highest association (r = .85, p < .0001) with the overall
inclusion score. All items correlated moderately to highly
with the total score (r ≥ .33, p ≤ .006), and the items The
school uses the caregiver’s knowledge to optimize support
(r = .73, p ≤ .0001) and The staff gets time to discuss NDD
students’ behavior and support plans (r = .72, p ≤ .0001)
showed the closest association with the total score. Discussion The comprehensive educational inclusion of autistic and
other neurodivergent students is still far from being a reality
in high- and middle-income countries, despite statements
and legislation. A barrier to the implementation of educa
tional inclusion is a lack of consensus about what inclusion
should entail and the degree of inclusion accomplished. We
investigated educational inclusion from the perspective of
neurodivergent students, their caregivers, and teachers to
generate awareness of possible disagreement as a basis to
improve and guide better implementation practices. Con
sistent with previous findings (Bölte et al., 2021; Jones et
al., 2022), the experience of implemented educational prac
tice varied substantially between informants. There was
disagreement between all informants, although mostly, but
not exclusively, between students and parents versus teach
ers. Students and parents gave educational inclusion poor )
y
y p
Our findings imply that the most promising, essential and
non-bureaucratic avenue to achieve consensus and progress
for the educational inclusion of neurodivergent students in
different schools and learning environments is the extended
participation of students and parents in shaping practice and
solutions. First, the domain Use of individualized support
had the highest association with overall experience of inclu
sion, and students and parents are likely the best sources
of information in this regard. Second, and corroborating
the latter, the single measure that had the highest associa
tion with overall inclusion ratings was the school taking
advantage of the caregiver’s knowledge to optimize sup
port. Third, there were several indications from all infor
mants that the schools had already established some culture
of exchange between them, students, and parents. Enhanc
ing the participation of neurodivergent students and their 1 3 1616 Journal of Autism and Developmental Disorders (2024) 54:1611–1617 Bölte, S., Leifler, E., Berggren, S., & Borg, A. (2021). Inclusive prac
tice for students with neurodevelopmental disorders in Sweden. Scandinavian Journal of Child and Adolescent Psychiatry and
Psychology, 9, 9–15. https://doi.org/10.21307/sjcapp-2021-002f parents to achieve educational inclusion has been suggested
by Florian (2014), Fletcher-Watson et al., (2019), and Lord
et al., (2022) as a strategic research goal in the field. Crosland, K., & Dunlap, G. (2012). Effective strategies for the
inclusion of children with autism in general education class
rooms. Behaviour Modification, 36, 251–269. https://doi. org/10.1177/0145445512442682 Our results should be treated with caution because of
methodological limitations inhibiting generalizability. Declaration Conflict of interest All authors declare no conflict of interest in rela
tion to this article. Sven Bölte discloses that he has in the last 3 years
acted as an author, consultant, or lecturer for Medice and Roche. He
receives royalties for textbooks, and diagnostic and intervention tools
from Hogrefe, Kohlhammer, Liber, and UTB. Bölte is a shareholder in
SB Education/Psychological Consulting AB and NeuroSupportSolu
tions International AB. Anna Borg receives royalties from Liber and
Studentlitteratur. Emma Leifler receives royalties from Liber. Hume, K., Steinbrenner, J. R., Odom, S. L., Morin, K., Nowell, S.,
Tomaszewski, B. … Savage, M. N. (2021). Evidence-based prac
tices for children, youth, and young adults with autism: Third gen
eration review. Journal of Autism and Developmental Disorders,
51, 4013–4032. https://doi.org/10.1007/s10803-020-04844-2 Jones, S. C., Gordon, C. S., Akram, M., Murphy, N., & Sharkie, F. (2022). Inclusion, exclusion and isolation of autistic people:
Community attitudes and autistic people’s experiences. Journal
of Autism and Developmental Disorders, 52, 1131–1142. https://
doi.org/10.1007/s10803-021-04998-7l Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format,
as long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Leifler, E., Coco, C., Fridell, A., Borg, A., & Bölte, S. (2022). Social
skills group training for students with neurodevelopmental dis
abilities in senior high school – A qualitative multi-perspec
tives of social validity. International Journal of Environmental
Research and Public Health, 19, 1487. https://doi.org/10.3390/
ijerph19031487 Lord, C., Charman, T., Havdahl, A., Carbone, P., Anagnostou, E.,
Boyd, B. … McCauley, J. B. (2022). The Lancet Commission
on the future care and clinical research in autism. Lancet, 399,
271–334. https://doi.org/10.1016/S0140-6736(21)01541-5 Lovett, B. J., & Nelson, J. M. (2021). Systematic review: Educational
accommodations for children and adolescents with attention
deficit/hyperactivity disorder. Discussion A
larger sample including a more diverse group of schools
from other urban and rural areas would have been more
desirable. We only investigated schools in Sweden, a high-
income country with a strong social welfare infrastructure,
although Sweden scores below the OECD average on the
Programme for International Student Assessment. Data were
collected during the COVID-19 pandemic, which might
have affected the informants’ perceptions of educational
inclusion. Other pivotal stakeholder groups, e.g., school
leaders and policymakers of paramount importance for edu
cational inclusion were omitted, but the study focused on a
micro-environmental perspective, where informants should
have access to comparable information. De Boer, A., Pijl, S. J., & Minnaert, A. (2011). Regular primary school
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high-commitment decisions. Berrett-Koehler Publishers Evidence of the link between inclusive education and social inclusion:
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and Inclusive Education, & Symeonidou (2018). Ed.). Denmark Fletcher-Watson, S., Adams, J., Brook, K., Charman, T., Crane, L.,
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National Research School Special Education for Teachers Educators,
funded by the Swedish Research Council (grant no. 2017–06039). We
are grateful to all participating adolescents, parents, and teachers for
making this research possible. Gitschthaler, M., Kast, J., Corazza, R., & Schwab, S. (2021). Resources
for inclusive education in Austria: An insight into the perception
of teachers. In J. Goldan, J. Lambrecht, & T. Loreman (Eds.),
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Applying the functional abnormality ontology pattern to anatomical functions
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Aberystwyth University Applying the functional abnormality ontology pattern to anatomical functions. Hoehndorf, Robert; Ngonga Ngomo, Axel-Cyrille; Kelso, Janet Applying the functional abnormality ontology pattern to anatomical functions. Hoehndorf, Robert; Ngonga Ngomo, Axel-Cyrille; Kelso, Janet Published in:
Journal of Biomedical Semantics
DOI:
10.1186/2041-1480-1-4
Publication date:
2010 Citation for published version (APA):
Hoehndorf, R., Ngonga Ngomo, A.-C., & Kelso, J. (2010). Applying the functional abnormality ontology pattern to
anatomical functions. Journal of Biomedical Semantics, 1(1), Article 4. https://doi.org/10.1186/2041-1480-1-4 General rights General rights
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leechuck@leechuck.de
1 Institute for Medical
Informatics, Statistics and
Epidemiology, University of
Leipzig, Leipzig, Germany
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mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited. Research
Applying the functional abnormality ontology
pattern to anatomical functions Robert Hoehndorf*1,2,3,4, Axel-Cyrille Ngonga Ngomo2 and Janet Kelso3 * Correspondence:
leechuck@leechuck.de
1 Institute for Medical
Informatics, Statistics and
Epidemiology, University of
Leipzig, Leipzig, Germany
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email: is@aber.ac.uk Download date: 24. Oct. 2024 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Abstract Background: Several biomedical ontologies cover the domain of biological functions,
including molecular and cellular functions. However, there is currently no publicly available
ontology of anatomical functions. Consequently, no explicit relation between anatomical structures and their functions is
expressed in the anatomy ontologies that are available for various species. Such an
explicit relation between anatomical structures and their functions would be useful both
for defining the classes of the anatomy and the phenotype ontologies accurately. Results: We provide an ontological analysis of functions and functional abnormalities. From this analysis, we derive an approach to the automatic extraction of anatomical
functions from existing ontologies which uses a combination of natural language
processing, graph-based analysis of the ontologies and formal inferences. Additionally, we
introduce a new relation to link material objects to processes that realize the function of
these objects. This relation is introduced to avoid a needless duplication of processes
already covered by the Gene Ontology in a new ontology of anatomical functions. Conclusions: Ontological considerations on the nature of functional abnormalities and
their representation in current phenotype ontologies show that we can extract a skeleton
for an ontology of anatomical functions by using a combination of process, phenotype and
anatomy ontologies automatically. We identify several limitations of the current ontologies
that still need to be addressed to ensure a consistent and complete representation of
anatomical functions and their abnormalities. Availability: The source code and results of our analysis are available at http://bioonto.de. Background The notion of function is important throughout biology. It is used to characterize biological
sequences [1], cell types [2], anatomical structures [3] and to annotate gene products [4]. Functions are also used in the description of phenotypes of functionings, i.e., observable
phenomena regarding the functioning or malfunctioning of biological entities. These phe-
notypes play an important role in the discovery of gene functions and in the description of
abnormalities, diseases, signs and symptoms. Phenotype ontologies We define a phenotype as any observable characteristic of an organism, part of an organism
or process in which an organism or one of its parts is involved. Phenotypes may include
both structural and behavioral properties. Functional phenotypes are either observable
characteristics of a process that realizes a function of an organism or a part of the organism, Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Page 2 of 15 or properties of an organism that involve its functions (such as having a function or lack-
ing a function). Phenotype ontologies for mouse and human phenotypes were developed to annotate
research databases of mouse and human phenotypes. The Mammalian Phenotype
Ontology (MPO) focuses on mutant mouse phenotypes [5] and the Human Phenotype
Ontology (HPO) focuses on Mendelian diseases in man [6]. They make an explicit refer-
ence to anatomy ontologies in their cross-product definitions [7], and implicit reference
to the anatomy ontologies in the naming of their categories. The HPO uses the Foundational Model of Anatomy (FMA) [3] to refer to anatomical
entities in humans, and the MPO uses the Adult Mouse Anatomy Ontology (MA) [8]. These anatomy ontologies describe anatomical entities by using, among others, part-
whole relations, i.e., they focus on the anatomical structure. Although the phenotype ontologies describe both structurally and functionally abnor-
mal phenotypes, the anatomy ontologies do not include an elaborate description of the
anatomical functions. As a consequence, although the classification of structural abnor-
malities in the phenotype ontologies follows well-defined principles, the classification of
phenotypes of functionings is often unprincipled and sometimes ambiguous. To address
the issue of representing functional phenotypes, we provide an ontology design pattern
[9] for functional abnormalities. This design pattern is applicable in phenotype ontolo-
gies, especially in the MPO and HPO. We discuss the benefits of the application of the
design pattern and relate the design pattern to the composite names of the categories in
the phenotype ontologies. Based on the category names, we apply a pattern-based
approach to extract a skeleton for an ontology of anatomical functions from a combina-
tion of the anatomy and phenotype ontologies together with the Biological Process
ontology of the Gene Ontology (GO) [4]. Biological functions There is an ongoing discussion in the philosophy of biology and theoretical biology as to
the exact nature of a biological function. While functions of artifacts come into being
due to the intentions of a designer, biological entities have evolved over time, and biolog-
ical functions are not dependent on intentions in the same way as artifacts are. Philosophical theories of biological functions range from reductions to causality over
social accounts of functions to the denial of the existence of biological functions. The
first two are of major importance, i.e., the causal view of biological functions and the
social view of biological functions. The major proponents of causal explanations of func-
tionality are Wright [10] and Millikan [11], while the social view is defended by Searle
[12]. Wright gives the following definition of function [10]: Wright gives the following definition of function [10]:
Definition 1. "The function of X is Z" means
1. X is there because it does Z,
2. Z is a consequence (or result) of X's being there. Definition 1. "The function of X is Z" means Definition 1. "The function of X is Z" means 1. X is there because it does Z, 2. Z is a consequence (or result) of X's being there. In the definition, X is a category of structures and Z is a process category, and instances
of X are involved in instances of Z. In its definition, Wright does not distinguish between
functions and the processes which realize the function. Furthermore, the definition
assumes that an entity has only one function. However, as discussed by Wright [10], the
definition can be restated for entities having multiple functions by replacing "the func-
tion of X is Z" with "a function of X is Z". Page 3 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 In the social view, functions are ascribed to brute facts by a conscious observer [12]. A
detailed analysis is provided by Hartmann [13] and is illustrated in Figure 1. Hartmann
distinguishs three elements to the ascription of a function: the setting of a goal in the
future, the planning of how to achieve the goal, resulting in a structure that is capable of
achieving the goal through causal means. Biological functions Figure 1 shows how some entity obtains a sin-
gle function, according to Hartmann [13]. For an entity to have multiple functions, the
same three steps are performed, yet the goal and the initial situation may change. Ontology of functions We do not choose a particular definition of biological function, and we do not add
another definition to the literature. The method we present is compatible with most
major views of function. An analysis of how to represent functions has been provided by the Ontology of Func-
tions (OF) [14,15]. A function in the OF is described in terms of a requirement situation
type, a goal situation type and a processual role. The requirement situation type serves as
precondition for any function realization, the goal situation type is the postcondition,
and the processual role [16] is used to describe how a function bearer brings about the
goal from the requirements. One major advantage of the treatment of functions in the
OF is the explicit inclusion of preconditions for the function realizations, which serve to
model the contexts in which a function can be realized. Function realizations Functions can be realized multiple times. Each realization of a function is a process, and
in each realization of a function the function bearer achieves the goal of the function,
starting at a situation satisfying the preconditions of the function. While a function is an entity that is similar to a property in that it inheres in its bearer,
a functioning is a process that is a realization of a function. For example, while the func-
tion of the heart "to pump blood" is a property that the heart has in virtue of being a
heart and in virtue of the evolutionary history of hearts, a functioning is the actual pro-
cess of pumping blood which realizes the function of the heart [17]. In particular, the Figure 1 Three steps for function ascription. The figure shows the three conditions for the ascription of a
single function to an entity. First, the goal of the function is established in the future. Second, the means for
achieving the goal are selected or created. Finally, the goal can be realized by causal means, i.e., without the
need for accessing or anticipating future states of the world. Figure 1 Three steps for function ascription. The figure shows the three conditions for the ascription of a
single function to an entity. First, the goal of the function is established in the future. Second, the means for
achieving the goal are selected or created. Finally, the goal can be realized by causal means, i.e., without the
need for accessing or anticipating future states of the world. Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Page 4 of 15 function of the heart is "to pump blood" even when the heart is not functioning. This
could be the case during a heart transplantation or during a malfunctioning of the heart. Furthermore, the function of the heart is "to pump blood" even when the heart cannot
realize this function. Function realizations always require a disposition to realize the
function in the function bearer, while the function itself can exist without such a disposi-
tion. For our present work, we use the simple conditional analysis of dispositions [18]:
"something x is disposed at time t to give response r to stimulus s, iff, if x were to undergo
s at t, x would r". Abnormal functionings Abnormal functionings are processes which are similar to a functioning, but which are
impaired in some way. We distinguish between abnormal functionings and malfunction-
ings: in the case of a malfunctioning, the function bearer cannot cause the goal of its
function although the preconditions for a function realization are given. An entity e has
the property of being malfunctional (with respect to the function f), if e has a function f,
but not a disposition d to realize the function f . Functions and dispositions are disjoint
categories (i.e., neither is a subcategory of the other), yet they are related in a particular
way [19]. While abnormal functionings are processes, being malfunctional is a property
of the function bearer; in the case of a malfunctional entity, no process of functioning
can occur. There are various kinds of abnormal functionings: functionings may be more or less
effective, have unwanted side-effects or similar. We focus on the malfunctional property
here. A classification of kinds of abnormal functionings is out of the scope of this paper
and will be subject to future work. Function realizations There are other possible causes for a heart to not function, e.g., an abnormality in the
nervous system. This is not a malfunctioning of the heart. We define a function by its
preconditions and postconditions. In the case that the nervous system fails, a precondi-
tion of the heart's function is not satisfied. If this precondition was satisfied, the heart
would in fact pump blood (assuming the heart is functional). Therefore, it is not the heart's being malfunctional but rather a non-satisfied precondi-
tion that causes the heart to not function. Within a wider context, i.e., the whole body,
this may appear to be a malfunctioning of the heart, but the original cause was elsewhere
- in the nervous system. The heart is functional, the nervous system abnormally func-
tioning. A repair or treatment of such a condition should treat the nervous system and
not the heart. Function and Structure There is an important relationship between function and structure. Biological functions
are usually realized through causal processes (cf. [13] and Figure 1) and the function
bearer has developed through evolution to play a particular role in processes of a certain
kind (e.g., the role of the heart as a pump in its function to pump blood). Therefore, if the
heart - the function bearer - becomes unable to play this role in the function realization,
while everything else remains unchanged, this loss of disposition is due to a change in
the heart's structure. In general, the loss of a disposition in the case of malfunctional
entities must go along with a change in the structure of the bearer of the disposition and
function. As a result, if e has a biological function f, and e is malfunctional with respect to f, then
e is abnormal. This pattern is already implied in the taxonomic backbones of the pheno- Page 5 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 type ontologies and reflected in the naming and the definitions of the phenotype ontolo-
gies' categories. The functional abnormality pattern The functional abnormality pattern The functional abnormality pattern is an ontology design pattern [9] for ontologies that
classify both abnormal structural and functional phenotypes, such as both the Human
and Mammalian Phenotype Ontology. According to the functional abnormality pattern, an abnormality of functioning (a
property of a process) implies an abnormality of the function bearer (provided that
external circumstances are normal). If multiple types of entities have the same kind of
function, then an abnormality of the functioning implies a disjunction of the abnormali-
ties of each possible kind of function bearer. On the other hand, being malfunctional (a
property of the function bearer) is a sub-category of (is-a) an abnormality of the function
bearer, and if multiple types of entities have the same function, then being malfunctional
is a sub-category of a disjunction of the abnormalities of each possible kind of function
bearer. For example, an abnormality in HearingP processes (we use HearingF to refer to the
function, and HearingP to refer to the process realizing the function; HearingP processes
are functionings of the HearingF function) implies an abnormality of the ears, if the
function of the ears is HearingF (and only the ears have the function HearingF). If the
function of both the left ear and the right ear was HearingF, then an abnormality of
HearingP implies an abnormality of the left ear or an abnormality of the right ear. In this
case, the category "abnormality of the left ear or abnormality of the right ear" should be
named "ear abnormality" and defined as a disjunction of the two categories "abnormality
of the left ear" and "abnormality of the right ear", which are both sub-categories of "ear
abnormality". On the other hand, the ears' being malfunctional with respect to their HearingF func-
tion is a property of the ears, and should be classified as a sub-category of Ear abnormal-
ity. The ears' being malfunctional is defined as the absence of a disposition which would
normally be present (due to the ears' having a function whose realization requires the
disposition), and the loss of a disposition entails a structural modification according to
the theory of dispositions [18]. Therefore, a loss of a disposition is a special kind of struc-
tural change of the disposition's bearer. Naming patterns in the phenotype ontologies g p
p
yp
g
Our goal is to represent functional phenotypes formally. While there is no ontology of
anatomical functions yet, such an anatomical function ontology is implied in the pheno-
type ontologies. These ontologies classify abnormal phenotypes, and in these phenotype
ontologies, abnormal functionings are usually classified as a sub-category of abnormal
structures which bear the function that is impaired. Therefore, the phenotype ontologies
can serve as a seed for the construction of an ontology of anatomical functions. However, as the phenotype ontologies rarely define abnormal functionings formally,
the challenge is to extract the information about anatomical structures and their func-
tions from the current ontology structure, category names and definitions of the pheno-
type ontologies. Such an approach will be insufficient to create an exhaustive ontology of
anatomical functions, because only few functions are addressed in the phenotype ontol-
ogies, nor will this approach provide a high-quality ontology that is suitable for use in
applications. Instead, our goal is to extract functions that can be used as the backbone of
an ontology of formally defined function categories after a manual review process. Page 6 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 The second major challenge in the extraction of anatomical functions is to provide an
analysis and formal representation of the relations between anatomical functions, their
bearers and the processes that realize the functions. Formal representation of anatomical functions In our formal analysis, we use the definition of the category Deafness in both the Mam-
malian and Human Phenotype Ontology as an example. The definition in the cross-
products of both ontologies is the following statement in the OBO Flatfile Format [20]: In the OBO Flatfile Format, the definition of an ontological category is started with a
[Term] statement, followed by a unique identifier of the category. Everything following
an exclamation mark is considered to be a comment. The GO category GO:0007605 is named "sensory perception of sound" and has a
synonym "hearing". The definition of Deafness in the two phenotype ontologies we use
in our analysis claims that Deafness is a process of HearingP in which the quality Absent
inheres. Inherence is a dependence relation between an instance of a quality and the
bearer of the quality [21]. There are several problems with the analysis of Deafness in the phenotype ontologies. Naming patterns in the phenotype ontologies The first problem is that, according to the definition, Deafness is a process of HearingP. Deafness seems to be something different from a process, and certainly different from a
HearingP process. An absence of hearing means that there is no HearingP process what
ever properties such a process might have. In particular, Absent cannot inhere in a non-
existing process and is arguably not a quality at all. The second problem is that there can be an absence of hearing without there being a
case of Deafness. In a completely silent environment, both a human or a mouse will
experience an absence of HearingP even when their disposition to hear is present. More
precicely, according to the definition of Deafness, an absence of sound would also entail
Deafness. Therefore, to represent the phenotype Deafness formally, we are faced with two chal-
lenges: there is an absence of HearingP processes and there is also an absence of the dis-
position to hear. Using our ontological framework for representing malfunctionality, we can represent
Deafness as the ears' being malfunctional with respect to their HearingF function. How-
ever, a vital point is missing to apply our framework: an ontology of anatomical func-
tions. The absence of such an ontology is one reason for the phenotype ontologies to
model abnormal functionings by using processes from the GO. While the anatomical functions are not yet covered in an ontology, the processes that
realize the anatomical functions are present in GO's Biological Process ontology. There-
fore, we define a new relation that we call the CC-has-function-realized-by (hf rb) rela-
tion. This relation is based on the relations CC-has-function and CC-realized-by. The
prefix CC indicates that the relation takes two ontological categories as arguments. The
relations between categories are defined using relations between individuals (II-rela- Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Page 7 of 15 tions), following the pattern of defining CC-relations from the OBO Relationship Ontol-
ogy [5]. Naming patterns in the phenotype ontologies The definition of the relation CC-has-function is given in formula 1, where E
denotes a category of Presentials (in GFO [21]), Continuants (in BFO [22]) or Endurants
(in DOLCE [23]): CC-has-function
II-has-function
( , )
(
( , )
(
E F
x instanceOf x E
y
⇔∀
→
∃
( , )
( , )))
x y
instanceOf y F
∧
(1) (1) According to this definition, the category E has the function (CC-relation) F if and only
if for every instance x of E there is an instance y of F such that x has the function (II-rela-
tion) y. While the relation CC-has-function follows the standard pattern for defining relations
between categories [20], the relation CC-realized-by cannot follow the same pattern. Applying the same pattern would require that, for every function, there is a process that
realized the function. Yet, not every function instance is realized, and, according to our
considerations about malfunctionality, not every function can be realized. Therefore, we
have to employ a different definition for the CC-realized-by relation given in formula 2. In the formulation of the definition of the CC-realized-by relation, we assume that func-
tions are not necessarily realized, but when they are realized, then always by processes of
a certain kind. We recognize that this claim is controversial. There may be functions that can be real-
ized by different kinds of processes. However, we assume that it is possible to find a
super-category for these kinds of processes that include all and only those process cate-
gories that can realize the function. For example, a TransportF function will always be
realized by TransportP processes (yet, arguably, not every TransportP process is a real-
ization of a TransportF function), and these TransportP processes can be of many differ-
ent kinds, all of which are sub-categories of the TransportP process category. CC-realized-by
II-realized-by
( , )
, (
( , )
( ,
F P
x y instanceOf x F
x
⇔∀
∧
y
instanceceOf y P
)
( , ))
→
(2) (2) According to this definition, the function category F is realized by (CC-relation) the
process category P if and only if whenever an instance x of F is realized by some y, then y
is an instance of P . Naming patterns in the phenotype ontologies With these definitions of the two relations CC-has-function and CC-realized-by, we
can give a definition for the relation CC-has-function-realized-by: CC-hfrb
CC-hasfunction
CC-realizedBy
( , )
(
( , )
( , ))
E P
F
E F
F P
⇔∃
∧
(3) (3) This relation is a connection of the two previously defined relations with an implicit
function as argument. The relation CC-has-function-realized-by holds between the cat-
egory E and the category P if and only if E has the function F and F is realized by P . Page 8 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 The relation CC-has-function-realized-by is a relation between two categories. The
relation can be defined in OWL2 as a connection between the two CC-relations by using
a property chain: CC-hfrb
CC-has-function
CC-realized-by
=
(4) (4) Such a definition can be used in an OWL ontology in which ontological categories are
in the domain of discourse (cf. [21,24,25]), i.e., in which there are OWL classes which
have ontological categories as their instances. A similar connection between the two relations II-has-function and II-realized-by is
very different from the relation between the categories: it is a relation between an entity
with a function that is in fact (and currently) realized by a process: II-hfrb
II-hasFunction
( , )
(
( , )
e p
z
e z
⇔∃
∧ II-hfrb
II-hasFunction
II-realizedBy
( , )
(
( , )
( , ))
e p
z
e z
z p
⇔∃
∧
(5) II-hfrb
II-hasFunction
( , )
(
( , )
e p
z
e z
⇔∃
∧ (5) II-realizedBy( , ))
z p Application to anatomy and phenotype ontologies We apply the framework for representing functional abnormalities to the automated
extraction of anatomical functions from the HPO and MPO. For this purpose, we exploit
the naming of the categories in the phenotype ontologies. We make use of three types of ontologies in our approach: 1. the phenotype ontology that contains abnormal functional phenotypes, either the
HPO or the MPO, 2. an anatomy ontology that contains the structures affected by the malfunctionings
represented in the phenotype ontology, either the Adult Mouse Anatomy Ontology 3. a process ontology, which contains the processes that realize an anatomical func-
tion. Since functional abnormalities are already classified as subclasses of structural abnor-
malities in the phenotype ontologies that we consider, we search for a pattern in the phe-
notype ontologies where 1. a category C in the phenotype ontology has a name name(C); e.g., Hearing abnor-
mality (HP:0000364), 2. in name(C), the name or synonym name(D) of a GO Biological Process category D
occurs as a substring and name(D) is delimited by whitespaces in name(C); e.g.,
Hearing (GO:0007605), 3. the category C is a sub-category of a category E with a name name(E); e.g., Abnor-
mality of the ears (HP:0000598), 4. the name name(E) contains the name or synonym name(F) of a category F from
the anatomy ontology and name(F) is delimited by whitespaces in name(E); e.g., Ear
(FMA:52780). As a consequence, we find abnormalities of GO processes that are classified as sub-cat-
egories of abnormalities of anatomical structures in the phenotype ontologies. As a consequence, we find abnormalities of GO processes that are classified as sub-cat-
egories of abnormalities of anatomical structures in the phenotype ontologies. To exclude categories that are named after diseases or do not describe abnormalities,
we only consider the categories of the phenotype ontologies which contain "abnormal",
"impaired", "decreased" or "increased" in their name or synonyms and exclude the rest Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Page 9 of 15 from our analysis. Furthermore, we excluded the GO categories GO:0032502 (develop-
mental process), GO:0043473 (pigmentation) and GO:0001503 (ossification) from
our analysis (see Discussion section). Figure 2 shows an overview of our extraction pipeline. To match the names of the cate-
gories, we stemmed all category labels and synonyms in the input ontologies by using the
PlingStemmer [26]. Application to anatomy and phenotype ontologies The PlingStemmer generates the singular forms of English words. Furthermore, all category labels were reduced to their lower case form before the match-
ing was carried out. Results Using the HPO and the FMA, we could extract 25 structure-process pairs. These pairs
and their evaluation are available at our project page. Using the MPO and the MA ontol-
ogies, we extracted 331 structure-process pairs. A selection of the pairs which we
extracted and which do stand in the CC-has-function-realized-by relation is shown in
Table 1. In Table 2 we show pairs that do not stand in the CC-has-function-realized-by
relation. A manual evaluation of our results with respect to the phenotype ontologies
showed that we reach a precision of 75% for the HPO and FMA and a precision of
77.27% for the MPO and MA. However, these precision values are only valid within the context of the reference
ontologies. For example, the CC-has-function-realized-by relation holds between Ear
(FMAID:52780) and Hearing (GO:0007605) according to the HPO, although ears only
partially contribute to Hearing. The quality of the extraction results could be improved
by ameliorating the background data upon which the extraction is carried out. Functions of parts Although we successfully applied our proposed ontology pattern to harvest a basic
ontology of anatomical functions from the phenotype ontologies by using naming pat-
terns in the ontologies, there are cases in which our pattern yields incorrect results. In
particular, the relation between functions and parts of structures remains a topic for fur-
ther research. We have argued that an abnormality of a function should be a sub-category of an
abnormality of the bearer of the function. However, there may be cases where the bearer
of the function is not included in the anatomy ontology or the abnormality of the func-
tion bearer is not included in the phenotype ontology. Instead, a structure of which the
function bearer is a part or an abnormality of such a structure is included. The functional
abnormality pattern is valid if we assume that the abnormality of the part is an abnor-
mality of the whole. This assumption is supported by the phenotype ontologies. Never-
theless, to achieve completeness of both the anatomy ontologies and the phenotype Figure 2 Processing sequence of the input categories. Figure 2 Processing sequence of the input categories. Page 10 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Table 1: Selection of true positive matches
Structure
Process
cardiovascular system
anatomical structure morphogenesis
uterus
angiogenesis
blood vessel
cell migration
blood
coagulation
female reproductive system
diestrus
reproductive system
fertilization
pancreas
glucagon secretion
mammary gland
lactation
The true positive matches were extracted using the Adult Mouse Anatomy Ontology and the
Mammalian Phenotype Ontology. Table 1: Selection of true positive matches angiogenesis The true positive matches were extracted using the Adult Mouse Anatomy Ontology and the
Mammalian Phenotype Ontology. ontologies, and to provide a principled way for building the phenotype ontologies, it is
beneficial to include the abnormality of the function bearer whenever an abnormality of
a function is included in the phenotype ontologies. Text and naming problems While processing the phenotype ontologies, we discovered several naming problems. First, plural forms are apparently randomly mixed with singular forms of the same term. For example, the label of MP:0003677 is "abnormal ear lobe", while the label of its sub-
category MP:0003678 is "absent ear lobes" (plural). The same holds for HP:0000598
(abnormality of the ears) and HP:0000370 (abnormality of the middle ear). We suggest
to use the plural form only in the case of explicitly disjunctively defined categories. For
example, a category that is defined as the disjunction of the categories "abnormality of
the left ear" and "abnormality of the right ear" may be called "abnormality of the ears". Another difficulty is the mixture of structural and functional abnormalities as category
labels. For example, the category HP:0000251 is labeled "abnormality of tear glands
OR tear production". This name mixes structural and functional abnormalities: tear
glands are an anatomical structure, while tear production is a process that realizes a
function (the function "to produce tears"). To improve the usability and the possibilities
for automatic processing of the phenotype ontologies, we suggest a separation of func-
tion and structure-based abnormalities. For example, the category HP:0000251 should
be split into two distinct categories, one labelled "abnormality of tear glands", the other
"abnormality of tear production". Table 2: Selection of false positive matches extracted from mouse ontologies
Structure
Process
blood
morphogenesis of a branching structure
immune system
t-cell apoptosis
trunk
biological regulation
pancreas
cell differentiation
blood vessel
endothelial cell differentiation
Table 1: The false positive matches were extracted using the Adult Mouse Anatomy Ontology and the
Mammalian Phenotype Ontology. Table 2: Selection of false positive matches extracted from mouse ontologies Page 11 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 The third issue we found in the phenotype ontologies is the inconsistent use of cate-
gory labels. The MPO contains the categories "abnormal hearing physiology"
(MP:0001963), "hearing disability" and "hearing impairment" (exact synonyms for
MP:0001965), "deafness" (MP:0001967) and "impaired hearing" (MP:0006325). The
development of a naming convention would not only serve automatic processing of the
ontologies, but also help to improve the clarity of the phenotype ontologies. Text and naming problems The current use of polysemous words is one of the main drawback we face when trying
to extract functions out of the phenotype ontology. Ossification, for example, can be
understood both as the process of creating bone tissue and as a property of a bone (the
outcome of the process). Thus, while bone ossification relates to the ossification process,
skull ossification (HP:0002703) relates to the state of the skull, i.e., the result of the
ossification process of the skull. Similarly, pigmentation is used widely as a property and
not as the process of pigmentation. Finally, a major problem for the phenotype ontologies is the use of "absent" as a prop-
erty. In English, "absent" is used as an attributive adjective, and this is one reason why
"absent" is present in some ontologies of qualities, in particular the phenotypic quality
ontology PATO (PATO:0000462). In most ontologies, such as DOLCE [23], GFO [21]
or BFO [22], qualities are dependent on a bearer, an entity of which they are a quality. The meaning of "absent", however, entails that there is no such bearer. When "absent" is
used in "absent appendix", "absent nipple" or "absent hearing", it does not correspond to
an ontological quality [24]. While this fact is increasingly being taken into consideration
by the phenotype ontologies in the definition of categories pertaining to the absence of
structures, "absent" is still used as a quality in the definition of categories of absent pro-
cesses (or functions). These categories should be carefully examined and their definition
made clear. They can be defined formally by using the functional abnormality pattern
[19], which uses a form of the lacks relation [27] together with an ontological analysis of
functions and dispositions. The problem of "absent" is not a problem of the phenotype ontologies alone. The
PATO ontology also includes "absent" as a quality, and it should be removed from the
PATO. Ontology problems Our analysis is hindered by the lack of categories or synonyms for category names in
GO's Biological Process Ontology. For example, tear production, cardiac conduction,
hair pigmentation or taste sensation are not in the GO, yet their existence is indicated by
reference to these processes in the phenotype ontologies. An extension of the GO
together with a consistent naming of the categories in the phenotype ontologies could
improve our analysis and the clarity of the phenotype ontologies. The need for an ontology of anatomical functions One major problem in our analysis is the lack of an anatomical functions ontology. The
phenotype ontologies imply that HearingF would be a function of the ears, by stating
that an abnormality in HearingF is a sub-category of Abnormality of the ears. However,
the ears can be normal and be functioning normally and still there may be an absence of
HearingP. In particular, Deafness may be the result of an abnormality of the ears, or it
may be the result of an abnormality in the nervous system. For example, an abnormality
in the brain can impair HearingF just as well as an abnormality in the ears can. The ears
only partially contribute to HearingP, and not every abnormality of HearingF is an
abnormality of the ears. Therefore, the ears and HearingP do not stand in the CC-has- Page 12 of 15 Page 12 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 function-realized-by relation according to our definition: the ears have some function
which, if realized, is realized by processes that may be part of HearingP processes, but
are not necessarily HearingP processes themselves, nor are they always part of HearingP
processes. Instead, the function of the ears is realized by processes of the kind Detection
of mechanical stimulus involved in sensory perception of sound (GO:0050910), which
are a part-of Hearing in the GO. function-realized-by relation according to our definition: the ears have some function
which, if realized, is realized by processes that may be part of HearingP processes, but
are not necessarily HearingP processes themselves, nor are they always part of HearingP
processes. Instead, the function of the ears is realized by processes of the kind Detection
of mechanical stimulus involved in sensory perception of sound (GO:0050910), which
are a part-of Hearing in the GO. To prevent this kind of erroneous naming or definition of categories, an explicit rela-
tion between anatomical structures and their functions is needed, on which the category
definitions in the phenotype ontologies should be based. Such an explicit relation
between structures and their functions can be achieved by the introduction of an ontol-
ogy of anatomical functions and the use of the CC-has-function relation, or without the
introduction of an ontology of anatomical functions and the use of the CC-has-func-
tion-realized-by relation. The need for an ontology of anatomical functions One advantage of our introduction of the relation CC-has-function-realized-by is
that a needless duplication of the processes in the GO is avoided. In particular, many
functions do not need to be named explicitly because the processes in the GO are
defined as processes that realize a given function. A difficulty in hiding the function by
using the CC-has-function-realized-by relation occurs when one kind of function can
be realized by multiple kinds of processes. In this case, a new super-category for all the
kinds of processes that may realize the function must be introduced and used as the
argument in the CC-has-function-realized-by relation. This new category would be
defined as the category of all processes that realize a given function - a common form of
defining processes. However, a separate ontology of anatomical functions may provide benefits over indi-
rectly relating structures and processes by using CC-has-function-realized-by. With
the availability of an ontology of anatomical functions, the inner structure of functions
can be represented [14], relations between functions themselves can be established (such
as functions that support or prevent other functions [15]) and properties can be
assigned to functions. Suggestions for future development of phenotype ontologies Overall, our analysis of means for extracting and representing functions led to the dis-
covery of several shortcomings of current ontologies. The following list epitomizes these
shortcomings and presents suggestions for the future modelling of biomedical ontolo-
gies in general and phenotype ontologies in particular. • Naming conventions: plural and singular form seem to be used inconsistenly in cat-
egories labels. For example, MPO contains the categories labeled "abnormal ear lobe"
(MP:0003677) and "absent ear lobes" (MP:0003678). We suggest to use the plural
form exclusively for naming categories defined disjunctively. • Mixture of functional and structural abnormalities: several categories are labeled
with terms that denote a mix of structural and functional abnormalities. An example
for such a mixture is the category label "abnormality of tear glands OR tear produc-
tion" (HP:0000251). To provide a clear classification founded in ontological princi-
ples and to enable automatic processing, we suggest to split such classes into distinct
classes: Abnormality of tear glands and Abnormality of tear production. • Use of "Absent" as property: previous work has pointed out that Absent is not an
ontological property (see e.g., [24,27]). We propose the use of a variant of the lacks • Use of "Absent" as property: previous work has pointed out that Absent is not an
ontological property (see e.g., [24,27]). We propose the use of a variant of the lacks Page 13 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 relation instead of Absent to improve the clarity of the phenotype ontologies and to
enable consistent reasoning on them. • Missing categories: Some categories implied in the phenotype ontologies are absent
in the GO. These categories include Hair pigmentation and Taste sensation. Adding
these categories to the GO would improve the automatic extraction of cross-prod-
ucts to link phenotype ontologies with the GO, and the use of the CC-has-function-
realized-by relation in the anatomy ontologies provides a method to discover the
missing categories in the GO. • Ambiguous category names: Some terms have been used in literature to denote
both processes and states. An example for such a term is "ossification", which can
refer to the process of Ossification (GO:0001503) or a property of a bone which is
the outcome of such a process. Suggestions for future development of phenotype ontologies To ensure that terms in the phenotype ontology are
monosemous, we would suggest the addition of more specific terms to class labels. For example, we would suggest altering the label "abnormal bone ossification"
(MP:0008271) into "abnormal bone ossification process" or "abnormal bone ossifi-
cation state" as required. • Mapping of parts to functions of whole: Some of the functions of anatomical struc-
tures implied by the phenotype ontologies are mappings from a part of a complex
anatomical structure to the function realized by the whole of the complex structure. For example, Hearing abnormality (HP:0000364) being a subclass of Abnormality
of the ears (HP:0000598) implies that a function of the ears is realized by Hearing
processes. Rather, a function of the ears is realized by a part of Hearing processes,
namely by Detection of mechanical stimulus involved in sensory perception of sound
(GO:0050910). The use of the CC-has-function-realized-by relation in the anat-
omy ontologies can help to prevent these errors. Author Details 1Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany, 2Department of
Computer Science, University of Leipzig, Leipzig, Germany, 3Department for Evolutionary Genetics, Max Planck Institute
for Evolutionary Anthropology, Leipzig, Germany and 4European Bioinformatics Institute, Wellcome Trust Genome
Campus, Hinxton, Cambridge, CB10 1SD, UK Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. References Hayamizu TF, Mangan M, Corradi JP, Kadin JA, Ringwald M: The Adult Mouse Anatomical Dictionary: a tool for
annotating and integrating data. Genome Biology 2005, 6(3):. g
g
g
gy
9. Aranguren ME, Antezana E, Kuiper M, Stevens R: Ontology Design Patterns for bio-ontologies: a case study on the
Cell Cycle Ontology. BMC Bioinformatics 2008, 9(Suppl 5):S1+. g
g
g
gy
9. Aranguren ME, Antezana E, Kuiper M, Stevens R: Ontology Design Patterns for bio-ontologies: a case study on the
Cell Cycle Ontology. BMC Bioinformatics 2008, 9(Suppl 5):S1+. y
y
10. Wright L: Functions. Philosophical Review 1973 g
p
11. Millikan RG: Language, Thought, and Other Biological Categories: New Foundations for Realism MIT Press; 1988. 11. Millikan RG: Language, Thought, and Other Biological Categories: New Foundations for Realism MIT Press; 1 11. Millikan RG: Language, Thought, and Other Biolog 12. Searle JR: The Construction of Social Reality Free Press; 1997. 12. Searle JR: The Construction of Social Reality Free Press; 1997. 13. Hartmann N: Teleologisches Denken Walter de Gruyter; 1966. g
y
14. Burek P, Hoehndorf R, Loebe F, Visagie J, Herre H, Kelso J: A top-level ontology of functions and its application in
the Open Biomedical Ontologies. Bioinformatics 2006, 22(14):. 14. Burek P, Hoehndorf R, Loebe F, Visagie J, Herre H, Kelso J: A to 5. Burek P: Ontology of Functions. In PhD thesis University of Leip 16. Loebe F: Abstract vs. social roles - Towards a general theoretical account of roles. Applied Ontology 2007, 2(2 16. Loebe F: Abstract vs. social roles - Towards a general theoretical account o 17. Johansson I: Functions, Function Concepts and Scales. The Monist 2004. D: Finkish Dispositions. The Philosophical Quarterly 1997, 47(187): 19. Hoehndorf R, Kelso J, Herre H: Contributions to the formal ontology of functions and dispositions: an application
of non monotonic reasoning Proceedings of the 11th Bio Ontology Workshop 2009 19. Hoehndorf R, Kelso J, Herre H: Contributions to the formal ontology of functions
of non-monotonic reasoning. Proceedings of the 11th Bio-Ontology Workshop 2009 20. Golbreich C, Horrocks I: The OBO to OWL mapping, GO to OWL 1.1! In Proceedings of the OWLED 2007 Workshop on
OWL: Experiences and Directions, Innsbruck, Austria, Jun 6-7, of CEUR Workshop Proceedings Volume 258. Edited by:
Golbreich C, Kalyanpur A, Parsia B. Aachen, Germany: CEUR-WS.org; 2007. 21. Authors' contributions
JK RH
d AN
i
d Authors' contributions
JK, RH and AN conceived of the study, RH and AN formalized, implemented and evaluated the method, JK supervised the
project. RH and AN drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements Acknowledgements
We are grateful to Heinrich Herre, Roberto Poli and two anonymous reviewers for helpful comments on previous versions
of this manuscript. We acknowledge funding from the Max Planck Society and the University of Leipzig. Conclusions We present an ontology design pattern for the representation of functional abnormali-
ties. The design pattern is applicable to the Human Phenotype Ontology and the Mam-
malian Phenotype Ontology. We show how to model anatomical functions by using
processes from the Gene Ontology that may realize these functions. For this purpose, we
introduce a new relation between categories of anatomical structures and process cate-
gories. This relation states that an anatomical structure has some function that is real-
ized by a process of a certain kind. Using this relation, functions can be specified without
the explicit introduction of an ontology of anatomical functions. We evaluated our method by exploiting the naming of categories from the phenotype
ontologies to extract structure-process pairs that stand in the relation we introduced. We
extracted several structure-process pairs from the Mammalian Phenotype Ontology
together with the Adult Mouse Anatomy Ontology, and from the Human Phenotype
Ontology together with the Foundational Model of Anatomy. In our analyis, we found several problems with the phenotype ontologies. In particular,
we discovered ambiguous namings of the categories and suggest the use of a naming
convention for the categories in the phenotype ontologies. Additionally, we found a
number of problematic formal definitions of categories in the phenotype ontologies. Most of these are categories of the malfunctional type: the loss of the disposition to per-
form a certain function. The use of our ontological framework would permit an Page 14 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 improved ontological representation of functional phenotypes and better capabilities for
knowledge extraction from the phenotype ontologies. improved ontological representation of functional phenotypes and better capabilities for
knowledge extraction from the phenotype ontologies. Competing interests
The authors declare that they have no competing interests. References 1. Eilbeck K, Lewis SE, Mungall CJ, Yandell M, Stein L, Durbin R, Ashburner M: The Sequence Ontology: a tool for the
unification of genome annotations. Genome Biology 2005, 6(5):R44. 2. Bard J, Rhee SY, Ashburner M: An ontology for cell types. Genome Biology 2005, 6 2. Bard J, Rhee SY, Ashburner M: An ontology for cell types. Genome Biology 2005, 6(2):R21. Bard J, Rhee SY, Ashburner M: An ontology for cell types. Geno 3. Rosse C, Mejino JLV: A Reference Ontology for Biomedical Informatics: the Foundational Model of Anatomy. Journal of Biomedical Informatics 2003, 36(6):. 4. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA,
Hill DP, Tarver IL, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology:
tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25:25-29. 4. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA,
Hill DP, Tarver IL, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology
tool for the unification of biology The Gene Ontology Consortium Nat Genet 2000 25:25-29 4. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA,
Hill DP, Tarver IL, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene ontology:
tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000, 25:25-29. 5. Smith B, Ceusters W, Klagges B, Köhler J, Kumar A, Lomax J, Mungall C, Neuhaus F, Rector AL, Rosse C: Relations in
biomedical ontologies. Genome Biol 2005, 6(5):. 6. Robinson PN, Köhler S, Bauer S, Seelow D, Horn D, Mundlos S: The Human Phenotype Ontology: a tool for
annotating and analyzing human hereditary disease. American journal of human genetics 2008, 83(5):610-615. 7. Mungall C, Gkoutos G, Smith C, Haendel M, Lewis S, Ashburner M: Integrating phenotype ontologies across
multiple species Genome Biology 2010 11:R2 8. Hayamizu TF, Mangan M, Corradi JP, Kadin JA, Ringwald M: The Adult Mouse Anatomical Dictionary: a tool for
annotating and integrating data. Genome Biology 2005, 6(3):. 8. References Herre H, Heller B, Burek P, Hoehndorf R, Loebe F, Michalek H: General Formal Ontology (GFO) - A Foundation
Ontology Integrating Objects and Processes [Version 1.0]. Onto-med report, Research Group Ontologies in
Medicine, Institute of Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig; 2006. 22. Grenon P: BFO in a Nutshell: A Bi-categorial Axiomatization of BFO and Comparison with DOLCE. In Tech. rep
University of Leipzig, Leipzig; 2003. 23. Masolo C, Borgo S, Gangemi A, Guarino N, Oltramari A: WonderWeb Deliverable D18: Ontology Libra
Tech. rep Laboratory for Applied Ontology, ISTC-CNR, Trento, Italy; 2003. 23. Masolo C, Borgo S, Gangemi A, Guarino N, Oltramari A: WonderWeb Deliverable D18: Ontology Library (final). In
Tech rep Laboratory for Applied Ontology ISTC-CNR Trento Italy; 2003 Tech. rep Laboratory for Applied Ontology, ISTC-CNR, Trento, Italy; 20 p
y
pp
gy
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24. Hoehndorf R, Loebe F, Kelso J, Herre H: Representing default knowledge in biomedical ontologies: Application to 24. Hoehndorf R, Loebe F, Kelso J, Herre H: Representing default knowledge in biomedical on
the integration of anatomy and phenotype ontologies. BMC Bioinformatics 2007, 8:377. the integration of anatomy and phenotype ontologies. BMC Bioinformatics 2007, 8:377. 25. Hoehndorf R, Loebe F, Poli R, Kelso J, Herre H: GFO-Bio: A biological core ontology. Applied Ontology 2008,
3(4):219-227. Page 15 of 15 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 Hoehndorf et al. Journal of Biomedical Semantics 2010, 1:4
http://www.jbiomedsem.com/content/1/1/4 26. Suchanek FM, Ifrim G, Weikum G, Buitelaar P, Cimiano P, Loos B: LEILA: Learning to Extract Information by
Linguistic Analysis. 2006. 27. Ceusters W, Elkin P, Smith B: Referent Tracking: The Problem of Negative Findings. Stud Health Technol Inform
2006, 124:. doi: 10.1186/2041-1480-1-4
Cite this article as: Hoehndorf et al., Applying the functional abnormality ontology pattern to anatomical functions
Journal of Biomedical Semantics 2010, 1:4 doi: 10.1186/2041-1480-1-4
Cite this article as: Hoehndorf et al., Applying the functional abnormality ontology pattern to anatomical functions
Journal of Biomedical Semantics 2010, 1:4
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Besturent Concept, the Government Is Not Only Bound By Regulated Authorities, but the Government Has Free Authority to Act
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International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 www.ijassjournal.com Besturent Concept, the Government Is Not Only Bound By
Regulated Authorities, but the Government Has Free
Authority to Act Jose Aniceto Hornai Pereria
Magister of Law Programme , University of da Paz Dili, Timor-Leste (UNPAZ) Abstract: It is necessary to understand how the relationship of authority between the central government and
local governments in the State of Timor-Leste based on the RDTL Constitution, especially articles 5 and 72,
clearly discusses the terms regional authority or delegation of authority or decentralization based on democratic
values and reflects justice and harmony between governments. Central and local government in Timor-Leste. The authority given to regional leaders is currently not fully, administratively the regional leadership
is based on the existing law led by the Regent (the highest regional leader), this is regulated by government
regulation No.53 / 2020 regarding the first amendment to government regulation No. 11/2019 dated June 14
regarding the Organization of the Ministry of Home Affairs, in particular articles 28.28 and article 33 C-E
clearly discusses the issue of regional government development by means of the authority obtained to carry out
its duties and functions in accordance with existing authorities. Administratively, the regional government has the authority to administer the territory according to the existing
law, but does not yet fully have the authority to manage and develop the area, due to certain matters of regional
leadership having to wait from the central government, such as regarding regional development funds. Therefore, local governments are not free to regulate and develop areas. When viewed from the concept of besturen or delegation, it is a bit contradicting to the current
situation in Timor-Leste, especially regarding the issue of transferring authority to regional governments. The
delegation of authority by administrative law is a policy that can be used to facilitate the work performed by the
regional government wheels, but in practice in Timor-Leste, there has not been a complete delegation of
authority to regional government leaders to carry out their duties and functions freely, it still depends on the
decision. from the central government. Jose Aniceto Hornai Pereria I. Introduction One of the important aspects in a democratic law country is the principle of legality, which implies
that every government action must be based on the prevailing laws and regulations or based on the authority
granted by applicable regulations, namely the ability to carry out certain legal actions. Therefore, theoretically and practically, especially in a modern state, it is almost impossible to
formulate the functions and authorities of government in detail by determining the laws and regulations, this is
because the functions and authorities of government are closely related to services to the society which are Jose Aniceto Hornai Pereria Page 142 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 www.ijassjournal.com always developing, therefore there is a space of freedom or application of policies, as a relaxation (rigidity and
limitations) of written regulations. always developing, therefore there is a space of freedom or application of policies, as a relaxation (rigidity and
limitations) of written regulations. So that the concept of besturen emerged as an effort to delegate part of the authority from the central
government to local governments or departments under leadership to carry out their duties and functions without
having to be based on decisions of the central government or existing laws, but those concerned could take
certain policies or decisions to carry out their functions and duties properly and can be accounted for legally. The concept of besturen can also be interpreted as the principle of decentralization because part of
the authority of the central government is carried out by lower departmental leaders or leaders in certain areas
but without neglecting legal responsibilities to the central leadership. The concept of besturen, in theory, comes from the concept of personnel administration which in
Latin administrate which in Dutch means the same as besturen which means government function. So according
to R.D.H. Kusumaatmadja in Ragawino (2006: 18) says that administration in everyday life consists of two
meanings: In a narrow sense: administration is an activity of writing, taking notes in every activity or
administration. Jose Aniceto Hornai Pereria 4 "Timor-Leste's Constitution of 2002 - Constitute Project." 19 Feb. 2021,
https://www.constituteproject.org/constitution/East_Timor_2002.pdf?lang=en.Diaksespada 31 Mar. 2021.
Article 72: Local Government
1. Local government is constituted by corporate bodies endowed with representative organs, with the objective
of organizing the participation by citizens in solving the problems of their own community and promoting local
development without prejudice to the participation by the State. j
,
https://www.constituteproject.org/constitution/East_Timor_2002.pdf?lang=en.Diaksespada 31 Mar. 202
Article 72: Local Government 4 "Timor-Leste's Constitution of 2002 - Constitute Project." 19 Feb. 2021, I. Introduction In a broad sense: administration is an activity to achieve predetermined goals.1 The concept of besturen is a best practice in a government system because government activities are
not centered on the central government, but there is a delegation of some of the authority from the central
government to local governments or local officials to carry out their daily functions and tasks and the
government is not only bound by the regulated authority, but the government also has free authority to act. Basically, the concept of besturen or delegation of authority is a definition derived from the law of
governmental organizations, which can be explained as the overall rules relating to the acquisition and use of
governmental authority by public law subjects in public legal relations, authority in this connection is
connotated as the ability to carry out positive law. The authority itself contains rights and obligations which are essentially the ability to carry out
certain legal actions, namely actions that are intended to give rise to legal consequences, and include the arising
and disappearance of legal consequences. If examined legally, in Timor-Leste the issue of delegation of authority is also regulated in the
RDTL Constitution2, especially in article 5 paragraphs 1 & 23 and article 72 paragraph 1 & 2 4 regarding 1"10 BAB II LANDASAN TEORI 2.1.KonsepAdministrasi ...." http://repository.uin-
suska.ac.id/14032/7/7.%20BAB%20II__2018569ADN.pdf.Diaksespada 31 Mar. 202 2 "Timor-Leste's Constitution of 2002 - Constitute Project." 19 Feb. 2021,
https://www.constituteproject.org/constitution/East_Timor_2002.pdf?lang=en.Diaksespada 31 Mar. 2 3 "Timor-Leste's Constitution of 2002 - Constitute Project." 19 Feb. 2021,
https://www.constituteproject.org/constitution/East_Timor_2002.pdf?lang=en.Diaksespada 31 Mar. 2021Article 5: Decentralization 1. The State respects, on matters of territorial organization, the principle of the decentralization of
public administration. 2. The law determines and establishes and the characteristics of the different territorial levels and the
administrative competences of the respective organs. 3. OecussiAmbeno and Ataúro enjoy special administrative and economic treatment Page 143 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 www.ijassjournal.com territorial authority also regulated by government regulation No. 53/2020 dated 28 October the first amendment
to government regulation No. 11/2019, June 14 regarding the organization of the Estatal Administration
ministry and government regulation No. 54/2020 dated 28 October the second amendment to government
regulation No. II. Results and analysis Based on the explanation in the introduction, the writer determines the problems in the results and
analysis of the author, namely the provisions regarding the concept of besturen or the delegation of authority
based on the theory and rules of State administrative law and the Principle of Legality? The concept of besturen or delegation is one of the efforts of the central government by giving
authority or delegation of part of the authority from the central government to regional governments or regional
officials to carry out tasks and functions for the regions or departments they lead, without neglecting their
responsibilities. The concept of besturen can be interpreted as decentralization, which means that the delegation of
part of the authority from the central government to regional governments, but without neglecting responsibility
to the central government based on the principle of accountability. The concept of besturen gives authority to
the leadership of each department or regional leader to make certain decisions and conditions for the smooth
performance of the department led, even though it slightly ignores legal rules, but these decisions are in the
public interest and the success of the work team from the department and region they lead. The concept of besturen or delegation is the same as decentralization, which means restructuring or
reorganization of authority (the restructuring of reorganization of authority), which means a system that puts
forward joint responsibility (system of co-responsibility) and the principle of the subsidiary (the principle of
subsidiary). The delegation or besturen concept is a separate effort from regional regional leaders to take a stand
in decision making even though the policies or decisions taken are not stated in government regulations or
government regulations, but the decisions taken are useful and are not for the interests of individuals or groups,
but the existing authority it contains rights and obligations and is the ability to carry out certain legal actions,
namely actions intended to give rise to legal consequences, and include the arising and disappearance of legal
consequences. I. Introduction 3/2016, 16 March regarding the legalization of regional administration (district) and techniques
of interdepartmental groups through decentralization. The concept of besturen or delegation of authority here clearly talks about the efforts of regional
governments or government executives from each department in carrying out their daily duties and functions,
not always depending on existing laws and regulations (not dependent on legality principles). 2. The organization, competence, functioning and composition of the organs of local government shall be
defined by law. Jose Aniceto Hornai Pereria II. Results and analysis The concept of besturen or delegation is more likely to make work more effective if it is handled by
local governments or regional agents according to the 1999 UNDP report, apart from that this concept can also
improve quality and performance more effectively, because department leaders or regional regional leaders are
not always dependent on decisions from the central government, but has the capacity to make decisions in order
to achieve more efficient work results for the sake of the public interest. The concept of besturenor delegation is an authority or a power that only describes the right to do or
not to act or the right to act or not to act, therefore more clearly. According to BagirManan, authority in legal
language is not the same as power (macht). Power only describes the right to act or not to act. In law, authority
also means rights and obligations (rechten en plichten). In the study of administrative law, knowing the source
and method of obtaining the authority of government organs is important, because it relates to legal
accountability (rechtelijke verwoording) in the use of authority, in line with one of the principles in a
constitutional state "no authority without accountability" (geenbevoegdheidzonderveran -woordelijkheid or
there is no authority without responsibility). Talking about the concept of besturen or delegation means talking about authority or authority. The
term "authority" or "authority" can be found in both the concept of public law and private law. Therefore, Jose Aniceto Hornai Pereria Page 144 Page 144 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 International Journal of Arts and Social Science
ISSN: 2581-7922,
Volume 4 Issue 3, May-June 2021 www.ijassjournal.com according to MochtarKusumaatmadja, someone who has formal authority automatically has the power to carry
out certain actions in accordance with the legal provisions governing the granting of that authority. according to MochtarKusumaatmadja, someone who has formal authority automatically has the power to carry
out certain actions in accordance with the legal provisions governing the granting of that authority. Basically, juridically, the concept of authority is always related to power based on law, both how to
obtain it and how to use it. The power obtained and exercised based on such law in the literature is commonly
referred to as "legal power" or "rechtsmacht". III. Conclusion The development of regional areas is currently not running in accordance with the national
development plan, one of the inhibiting factors is that regional regional leaders have not obtained the authority
or there has been no delegation of full authority from the central government to manage regional areas, apart
from that regional development is not running due to dependence on the budget development of the central
government, in terms of the RDTL Constituency article 5 and article 72 and government regulation No.53 /
2020 regarding the first amendment to government regulation No. 11/2019 dated June 14 regarding the
Organization of the Department of Home Affairs, in particular articles 28.29 and articles 33 C-E clearly discuss
the problem of regional government development by means of the authority obtained to carry out its duties and
functions in accordance with the principles of decentralization. II. Results and analysis The term "power" in this case means: "an ability on the part of a
person to produce a change in a given legal relation by doing or not doing a given act" (Black, 1970: 1169). In the study of administrative law, knowing the sources and ways of obtaining the authority of
government organs is important, because it is related to legal accountability (rechtelijkeveranwoording) in the
use of authority, in line with one of the principles in a rule of law "there is no authority without accountability. This means that in every granting of authority to certain government officials, the accountability of the officials
concerned is implied. Not everyone gets the delegation of authority, someone will get the authority to do something must
l legitimacy, namely the authority granted by law. There are three (3) sources of authority, namely: Not everyone gets the delegation of authority, someone will get the authority to do something must
have legal legitimacy, namely the authority granted by law. There are three (3) sources of authority, namely: ⮚ Attribution authority is the granting of governmental authority by legislators to government
organs based on law. ⮚ Authority of delegation, namely the delegation of governmental authority from one organ of
government to another organ of government. Based on the Government Administration Law,
Delegation is the delegation of authority from higher government agencies and / or officials to
lower government agencies and / or officials with responsibility and accountability fully
transferred to the delegation recipient. ⮚ Authority to mandate occurs when the organ of government allows its authority to be exercised
by other organs on its behalf. Based on the Government Administration Law, mandate is the
delegation of authority from higher government agencies and / or officials to lower
government agencies and / or officials with responsibility and accountability remaining with
the mandate. The juridical consequences of an official's authority will be different. if the
authority comes from the delegation of authority (delegation) or assignment (mandate). IV. Closing Based on the abstraction, introduction and results and analysis, it is hoped that in the future the State
of Timor-Leste, legally and politically, will take a positive attitude through the state and government deposits, in
order to give authority or delegation of authority or to grant authority in the form of the concept of
decentralization to regional government leaders or departments under the leadership of the central government,
to manage their duties and functions independently, while still not neglecting legal responsibilities to the central
government, so that development is not only focused on certain areas but development reaches remote areas in
Timor-Leste. Reference or bibliography [1.] RDTL Constitution, 22 March 2002 [1.] RDTL Constitution, 22 March 2002 [2.] Government Regulation No. 53/2020 regarding the first amendments to government regulation No
11/2019 dated June 14 regarding the Organization of the Department of Home Affairs [3.] State Administrative Law Theory and Constitutional Law [3.] State Administrative Law Theory and Constitutional Law Jose Aniceto Hornai Pereria Page 145 Page 145
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Lipase Applications in Biodiesel Production
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7,200 Chapter 8 http://dx.doi.org/10.5772/52662 http://dx.doi.org/10.5772/52662 http://dx.doi.org/10.5772/52662 © 2013 Yücel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. Lipase Applications in Biodiesel Production Sevil Yücel, Pınar Terzioğlu and Didem Özçimen Sevil Yücel, Pınar Terzioğlu and Didem Özçimen Additional information is available at the end of the chapter Additional information is available at the end of the chapter Additional information is available at the end of the chapter 1. Introduction Because of the global warming and depletion of fossil fuels, in recent years, intensive inves‐
tigations are carried on for providing the greater use of sustainable biofuels instead of fossil
fuels. Biomass, which various biofuels are produced from, has an important role among oth‐
er alternative energy sources including wind energy, solar energy, geothermal energy, etc. Biodiesel is one of the important biofuels and a clean energy source as an alternative to pe‐
troleum-based diesel fuels. Biodiesel has some advantages and disadvantages. Transporta‐
bility, high combustion efficiency, low sulphur and aromatic content, high cetane number
and biodegradability are advantages of the biodiesel [1]. Disadvantages of biodiesel are high
viscosity, lower energy content, high cloud and pour point, high nitrogen oxide emission,
lower engine speed and power, injector cooking, high price and engine erosion [2]. lower engine speed and power, injector cooking, high price and engine erosion [2]. The flash point of biodiesel is higher than diesel fuel. This feature is important for fuel stor‐
age and transportation in the way of safety. Cetane number of biodiesel (~50) is higher than
diesel fuel [3]. Biodiesel does not include aromatic and sulphur content and contains oxygen
at the rate of 10-11% by mass [4]. Cetane number is an important factor to determine the
quality of diesel fuel, especially ignition quality of diesel fuel. In other words, it determines
the ignition tendency of fuel when being injected into engine. Ignition quality of biodiesel is
determined by the structure of methyl ester [5].Viscosity is also an important factor for bio‐
diesel. Viscosity affects mostly fuel injection equipment and the increase of fuel viscosity
changes the viscosity at low temperatures. High viscosity has an negative effect on fuel
spray atomization [6]. Amounts of elements and compounds in biodiesel and diesel fuel are
present in Table 1 [7]. Biodiesel has more polar structure than diesel fuel because of the oxy‐
gen, which is an electronegative element present in its structure, and therefore biodiesel has
higher viscosity comparing with diesel fuel. In addition, elemental oxygen content is respon‐
sible for lower heating value of biodiesel when compared with diesel fuel. [7-9]. Biodiesel
© 2013 Yücel et al.; licensee InTech. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction lower engine speed and power, injector cooking, high pric
The flash point of biodiesel is higher than diesel fuel. This
age and transportation in the way of safety. Cetane numb
diesel fuel [3]. Biodiesel does not include aromatic and sul
at the rate of 10-11% by mass [4]. Cetane number is an
quality of diesel fuel, especially ignition quality of diesel
the ignition tendency of fuel when being injected into eng
determined by the structure of methyl ester [5].Viscosity
diesel. Viscosity affects mostly fuel injection equipment
changes the viscosity at low temperatures. High viscosi
spray atomization [6]. Amounts of elements and compoun
present in Table 1 [7]. Biodiesel has more polar structure t
gen, which is an electronegative element present in its stru
higher viscosity comparing with diesel fuel. In addition, el
sible for lower heating value of biodiesel when compare
© 2013 Yücel et al.; licensee InTech. This is an open acce
Commons Attribution License (http://creativecommons
distribution, and reproduction in any medium, provided The flash point of biodiesel is higher than diesel fuel. This feature is important for fuel stor‐
age and transportation in the way of safety. Cetane number of biodiesel (~50) is higher than
diesel fuel [3]. Biodiesel does not include aromatic and sulphur content and contains oxygen
at the rate of 10-11% by mass [4]. Cetane number is an important factor to determine the
quality of diesel fuel, especially ignition quality of diesel fuel. In other words, it determines
the ignition tendency of fuel when being injected into engine. Ignition quality of biodiesel is
determined by the structure of methyl ester [5].Viscosity is also an important factor for bio‐
diesel. Viscosity affects mostly fuel injection equipment and the increase of fuel viscosity
changes the viscosity at low temperatures. High viscosity has an negative effect on fuel
spray atomization [6]. Amounts of elements and compounds in biodiesel and diesel fuel are
present in Table 1 [7]. Biodiesel has more polar structure than diesel fuel because of the oxy‐
gen, which is an electronegative element present in its structure, and therefore biodiesel has
higher viscosity comparing with diesel fuel. In addition, elemental oxygen content is respon‐
sible for lower heating value of biodiesel when compared with diesel fuel. [7-9]. Biodiesel Biodiesel - Feedstocks, Production and Applications 210 can be used in its pure form or when mixed with diesel fuel in certain proportions. 2. Lipases in biodiesel production Biocatalyst based biotechnological applications are receiving increasing attention. Lipases
(triacylglycerol acylhydrolases, EC 3.1.1.3) are the important biocatalysts because of their ex‐
cellent biochemical and physiological properties. Lipases are the hydrolytic enzymes that
can be used in various industrial applications for alcoholysis, acidolysis, amynolysis and hy‐
drolysis reactions. Biodiesel production is one of the stunning applications of lipase. Lipase
catalyzed biodiesel production was reported first by Mittelbach [16]. Lipase-catalyzed trans‐
esterification takes place in two steps, which involves hydrolysis of the ester bond and ester‐
ification with the second substrate [15]. A ping-ping bi bi mechanism generally used for
kinetic studies of enzyme catalyzed transesterification. Lipases can be isolated from many species of plants (papaya latex, oat seed lipase, and cas‐
tor seed lipase), animals (pig’s and human pancreatic lipases), bacteria, filamentous fungi
and yeast [17-19]. For industrial enzyme production generally microorganisms are preferred
because of their shortest generation time [20]. The other advantages of microorganisms can
be listed as high yield of conversion of substrate into product, great versatility to environ‐
mental conditions and, simplicity in genetic manipulation and in cultivation conditions [20]. Al h
h li
f
diff
bl
l
h
i
b
i l
d Lipases can be isolated from many species of plants (papaya latex, oat seed lipase, and cas‐
tor seed lipase), animals (pig’s and human pancreatic lipases), bacteria, filamentous fungi
and yeast [17-19]. For industrial enzyme production generally microorganisms are preferred
because of their shortest generation time [20]. The other advantages of microorganisms can
be listed as high yield of conversion of substrate into product, great versatility to environ‐
mental conditions and, simplicity in genetic manipulation and in cultivation conditions [20]. Although lipases from different sources are able to catalyze the same reaction, bacterial and
fungal lipases are mostly used in biodiesel production such as Aspergillus niger,Candida ant‐
arctica, Candida rugosa, Chromobacterium Viscosum, Mucor miehei, Pseudomonas cepacia, Pseudo‐ Lipases can be isolated from many species of plants (papaya latex, oat seed lipase, and cas‐
tor seed lipase), animals (pig’s and human pancreatic lipases), bacteria, filamentous fungi
and yeast [17-19]. For industrial enzyme production generally microorganisms are preferred
because of their shortest generation time [20]. The other advantages of microorganisms can
be listed as high yield of conversion of substrate into product, great versatility to environ‐
mental conditions and, simplicity in genetic manipulation and in cultivation conditions [20]. 1. Introduction Most
common biodiesel blends are B2 (2 % biodiesel, 98 % diesel), B5 (5 % biodiesel, 95% diesel),
B20 (20 % biodiesel, 80 % diesel) [10]. Biodiesel Content (%)
Diesel Content (%)
Carbon
79.6
86.4
Hydrogen
10.5
13.6
Oxygen
8.6
-
Nitrogen
1.3
-
C/H
7.6
6.5
n-Aliphatics
15.2
67.4
Olephenics
84.7
3.4
Aromatics
-
20.1
Naphtens
-
9.1
Table 1. The comparison of elemental and chemical content of diesel and biodiesel [7] Table 1. The comparison of elemental and chemical content of diesel and biodiesel [7] Table 1. The comparison of elemental and chemical content of diesel and biodiesel [7] The transesterification reaction can be influenced by several factors including molar ratio of
alcohol, catalyst, presence of water, free fatty acid in oil samples, temperature, time and agi‐
tation speed. In this context, an understanding of the factors affecting the process is very im‐
portant to make economically and environmentally biodiesel production [11]. To accelerate reaction rate, transesterification process is carried out in the presence of cata‐
lysts. So, biodiesel production is made by using chemical or enzymatic catalysts. Compared
to chemical, enzymatic reaction is more attractive because of ability of make a high quality
product, simplify the separation of products, mild reaction conditions, the reuse of the cata‐
lyst and especially environmental impact, although high conversion and reaction rate are
obtained with chemical catalysts [11-14]. Lipase is important enzyme catalyst that catalyzes
esterification and transesterification reaction to produce methyl esters (biodiesel). Figure 1
presents the enzymatic transesterification reaction [15]. Figure 1. Enzymatic transesterification reaction [15]. Figure 1. Enzymatic transesterification reaction [15]. In this study, enzymatic approach for biodiesel production was reviewed, and especially the
usage of lipases in biodiesel production and factors affecting the effectiveness of lipase in
reaction were explained in detail. Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662
211 2. Lipases in biodiesel production Although lipases from different sources are able to catalyze the same reaction, bacterial and
fungal lipases are mostly used in biodiesel production such as Aspergillus niger,Candida ant‐
arctica, Candida rugosa, Chromobacterium Viscosum, Mucor miehei, Pseudomonas cepacia, Pseudo‐
monas fluorescens, Photobacterium lipolyticum, Rhizopus oryzae, Streptomyces sp., and
Thermomyces lanuginose [21]. Candida rugosa, obtained from yeast, is the most used microor‐
ganism for lipase production [22]. Recently, Streptomyces sp. was investigated as a potent li‐
pase producing microbe for biodiesel production and found applicable in the field of
biodiesel [23]. Specificity of lipases has a great importance in the selection of the usage area of lipases. Li‐
pases can be divided into three groups due to their specificity as 1,3-specific lipases, fatty
acid-specific lipases and nonspecific lipases. Especially, 1,3-specific lipases which release fat‐
ty acids from positions 1 and 3 of a glyceride and hydrolyze ester bonds in these positions
such as Aspergillus niger, Rhizopus oryzae and Mucor miehei catalyze transesterification reac‐
tions efficiently [20,24]. The study of Du et al. [25], showed that higher yield (90%) was ach‐
ieved for biodiesel production by using a sn-1,3-specific lipase, Thermomyces lanuginosa
immobilized on silica gel (Lipozyme TL IM). Thus, the use of sn-1,3- specific lipases can give
rise to biodiesel yield of above 90% under appropriate conditions [24]. Substrate specificity
of lipases is also a crucial factor towards the biodiesel production which acts on the choice of
the proper enzyme based on the composition of raw materials by consisting in the capability
of distinguishing structural features of acyl chains [20,24]. Lipases from Pseudomonas fluores‐
cens, Pseudomonas cepacia, Candida rugosa, Candida antarctica and Candida cylindracea are suita‐
ble for transesterification reaction by displaying both wide substrate specificity and
regiospecificity [24]. Biodiesel - Feedstocks, Production and Applications 212 2.1. Immobilization of lipases The immobilization of enzymes, which is attracting worldwide attention, was firstly report‐
ed in 1971 at Enzyme Engineering Conference [26]. During the past decade, chemical modi‐
fication, physical modification, and gene expression techniques have been developed to
obtain more economic, active, selective, or stable lipases. Immobilization is a modification
method that can be defined as attaching the enzyme onto an insoluble solid support materi‐
al [18]. By immobilization more operational and temperature stable lipases can be obtained
and also lipases can be reused in the reactions. In addition, reusability of lipases will be a
possible solution to the high cost of the enzymes and make them suitable for applications in
industrial scale. The comparison of free enzymes and immobilized enzymes is given in Ta‐
ble 2. Methods for enzyme immobilization can be classified as adsorption, covalent bonding,
entrapment, and cross-linking. The selection of method and support material is a prominent
factor for obtaining an efficient lipase. The results of comparative studies revealed that the
same lipase molecule can show very different catalytic activities after immobilization onto
different supports [27]. Characteristics
Free Enzyme
Immobilized Enzyme
Price
High
Low
Efficiency
Low
High
Activity
Unstable
Stable
Reusability and recovery
Not possible
Possible
Tolerance to temperature, pH, etc. Low
High
To separate from the substrate
Difficult
Easy
To separate from the product
Difficult
Easy
Table 2. The comparison of free enzyme and immobilized enzyme [19] Table 2. The comparison of free enzyme and immobilized enzyme [19] 2.1.1. Adsorption technique Adsorption is the adhesion of lipase on the surface of the adsorbent by weak forces, such as
van der Walls, ionic and hydrophobic interactions, or dispersion forces [28]. Immobilization
via adsorption method is the simply mixing of an aqueous solution of enzyme with the car‐
rier material for a period and washing away the excess enzyme from the immobilized en‐
zyme on the carrier after a time [29]. The level of adsorption is strictly related to the pH,
temperature and ionic strength. Adsorption is the most widely employed method besides Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 213 other methods because of its special commercial advantages and simplicity. Adsorption is
the only reversible enzyme immobilization method. The advantages of adsorption is mild
and easy preparing conditions, low cost, no need for chemical additives, the carrier can be
recovered for repeated use, and high activity [30]. Various types of carriers used in immobilization of lipases. Acrylic resin, celite, polypro‐
pylene and textile membrane are broadly used carriers. Some of the reported results of
adsorption technique based immobilized enzymes used in biodiesel production are sum‐
marized in Table 3. As can be seen from table generally the biodiesel yields using the en‐
zymes obtained by adsorption method are higher than 85%. Novozym 435 is a
commercial lipase, which is obtained by immobilization of Candida antartica lipase on
acrylic resin and is a good catalyst that provides biodiesel yield higher than 90% with
vegetable oil or waste cooking oil as feedstock [31]. The other commercialized lipase is
known as Candida sp. 99–125 lipase immobilized on textile membrane, which can catalyze
lard, waste oil and vegetable oils with higher yields that is more than 87% [31]. Besides
many advantages of immobilization by adsorption method, the main disadvantage is that
desorption of the lipase from the carrier occurs because of the weak interactions between
the enzyme and support. 2.1.2. Covalent binding technique Another approach is covalent binding technique, which is the formation of covalent bonds
between the aldehyde groups of support surface and active amino acid residues on the sur‐
face of the enzyme [29]. A variety of supports have been used such inorganic materials, nat‐
ural polymers (agarose, chitin and chitosan), synthetic polymers (hydrophobic
polypeptides,nylon fibers) and Eupergit® (made by copolymerization of N,N’-methylene-
bis-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether and methacrylamide) for
immobilization of lipases by covalent binding [56].The main advantage of covalent binding
method is obtaining thermal and operational stable enzymes because of strong interactions
between the lipase and the carrier [31]. The comparison of biodiesel production performance
using immobilized lipase via covalent binding method is summarized in Table 4. Chitosan is
a promising carrier as a natural polymer due to its membrane forming and adhesion ability,
high mechanical strength and facility of forming insoluble in water thermally and chemical‐
ly inert films [57]. Xie and Wang [58], reported a technique for immobilization of Candida
rugosa lipase on magnetic chitosan microspheres for transesterification of soybean oil.The
immobilized enzyme was determined as an effective biocatalyst for the transesterification
reaction due to giving a good conversion of soybean oil and retaining its activity during the
four cycles [58]. Using two immobilized lipases with complementary position specificity instead of one li‐
pase is a new approach to produce a cost effective biodiesel [19]. Lipase from Rhizopus orizae
and Candida rugosa was covalently bound to the silica, which was used to produce biodiesel
from crude canola oil. Under optimum conditions, the conversion rate of degummed crude
canola oil to fatty acid methyl esters was 88.9%, which is higher than the conversion ob‐
tained by free enzyme mixture (84.25%) [59]. Biodiesel - Feedstocks, Production and Applications 214 Lipase Source
Carrier
Acid/Oil
Source
Alcohol
Maximum
Performance
(%)
Reference
Burkholderia sp. C20
Alkyl-functionalized
Fe3O4–SiO2
Olive
Methanol
92
(conversion)
[32]
Candida antartica
Acrylic resin
Soybean
Methanol
92(yield)
[33]
Candida antartica
Acrylic resin
Soybean and
rapeseed
Methanol
98.4
(conversion)
[34]
Candida antartica
Acrylic resin
Soybean and
rapeseed
Methanol
"/>95
(conversion)
[35]
Candida
antarctica B
Granular activated carbon
Palm
Isobutanol
100
(conversion)
[36]
Candida sp. 99–125
Textile membrane
Lard
Methanol
87.4
(yield)
[37]
Candida sp. 99–125
Textile (cotton) membrane Salad
Methanol
96
(conversion)
[38]
Candida sp. 2.1.2. Covalent binding technique 99–125
Textile membrane
Crude rice bran
Methanol
87.4
( yield)
[39]
Candida rugosa and
Pseudomonas fluorescens
Acurel
Palm
Ethanol
89 (yield)
[40]
Chromoacterlum viscosum
Celite-545
Jatropha
Ethanol
92 (yield)
[41]
Geobacillus
thermocatenulatus
Poly-hydroxybutyrate beadsBabassu
Ethanol
100 (yield)
[42]
Pseudomonas aeroginosa
Celite
Soybean
Methanol
80(yield)
[43]
Pseudomonas cepacia
Celite
Jatropha
Ethanol
98 (yield)
[44]
Pseudomonas cepacia
Electrospun
polyacrylonitrile fibers
Rapeseed
n-butanol
94
(conversion)
[45]
Pseudomonas cepacia
Polystyrene
Sapium
sebiferum
Methanol
96.22 (yield)
[46]
Pseudomonas cepacia
Ceramic beads
Waste cooking
Methanol
40 (yield)
[47]
Pseudomonas fluorescens
Porous kaolinite particle
Triglyceride
triolein
1-propanol
"/>90
(conversion)
[48]
Pseudomonas fluorescens
and Pseudomonas cepacia
Polypropylene powder
Soybean
Methanol
58
37
( yield)
[49]
Penicillium expansum
Resin D4020
Waste
Methanol
92.8 (yield)
[50]
Rhizomucor miehei
Hydrophilic resins
Olive husk
Ethanol
-
[51]
Rhizomucor miehei
Silica
Waste cooking
Methanol
91.08 (yield)
[52]
Rhizopus oryzae
Macroporous resin HPD-400Pistacia chinensis
bge seed
Methanol
94 (yield)
[53]
Saccharomyces cerevisiae
Mg–Al hydrotalcite
Rape
Methanol
96 (conversion)
[5454]
Thermomyces lanuginosus
(Lipozyme TL IM)
Hydrotalcite
Waste cooking
Methanol
95 (yield)
[55]
Table 3 Comparison of biodiesel production performance using immobilized lipase via adsorption method Lipase Source
Carrier
Acid/Oil Source Alcohol
Maximum
Performance
(%)
Reference
Burkholderia cepacia
Niobium Oxide (Nb2O5) Babassu
Ethanol
74.13
(yield)
[60]
Burkholderia cepacia
Polysiloxane–Polyvinyl
Alcohol (SiO2–Pva)
Babassu
Beef Tallow
Ethanol
100
89.7
(yield)
[60]
Candida rugosa
Chitosan Microspheres Soybean
Methanol
87
(conversion)
[58]
Candida rugosa
Chitosan Powder
Rapeseed
Soapstock
Methanol
95
(conversion)
[61]
Enterobacter aerogenesSilica
Jatropha
Methanol
94
(yield)
[62]
Porcine pancreatic
Chitosan Beads
Salicornia
Methanol
55
(conversion)
[63]
Pseudomonas
fluorescens
Toyopearl Af-
Amino-650m Resin
Babassu
Ethanol
94.9
(yield)
[64]
Rhizopus oryzae
Resin Amberlite Ira-93
Pistacia Chinensis
Bge Seed
Methanol
92
(yield)
[63]
Rhizopus oryzae
Polystyrene
Polymer(Amberlite
Ira-93)
Soybean
Methanol
90.05
(yield)
[65]
Rhizopus Orizae
+Candida rugosa
Silica
-
Methanol
"/>98
(conversion)
[66]
Rhizopus orizae
+Candida rugosa
Silica
Crude Canola
Methanol
88.9
(conversion)
[59]
Thermomyces
lanuginosus
Olive Pomace
Pomace
Methanol
93
(yield)
[67]
Thermomyces
lanuginosus
Polyglutaraldehyde
Activated Styrene-
Divinylbenzene
Copolymer
Canola
Methanol
97
(yield)
[68]
Thermomyces
lanuginosus
Toyopearl Af-
Amino-650m Resin
Palm
Ethanol
100
(yield)
[64]
Thermomyces
lanuginosus
Polyurethane Foam
Canola
Methanol
90
(yield)
[69]
Thermomyces
lanuginosus
Aldehyde-Lewatit
Soybean
Ethanol
100
(conversion)
[70]
Thermomyces
lanuginosus
Magnetic Fe3O4 Nano-
Particles
Soybean
Methanol
90
(conversion)
[71]
Table 4 Comparison of biodiesel prod ction performance
sing immobili ed lipase ia co alent binding method Biodiesel - Feedstocks, Production and Applications 216 2.1.3. Entrapment technique Entrapment method is based on capturing of the lipase within a polymer network that retains
the enzyme but allows the substrate and products to pass through [72]. This method can be
simply defined as mixing an enzyme with a polymer solution and then crosslinking the poly‐
mer to form a lattice structure that captures the enzyme [29]. Entrapment is often used for in‐
dustrial applications because the method is fast, cheap and can be carried out under mild
conditions [73]. Entrapment can be divided into three categories such as gel or fiber entrap‐
ping and microencapsulation [74]. A number of supports have been investigated such as algi‐
nate, celite, carrageenan, resins, acrylic polymers etc. Some carriers used for entrapment and
the biodiesel production yields obtained by these enzymes are displayed in Table 5. A disad‐
vantage of entrapment method is the mass transfer problem due to the act of support as a bar‐
rier, so the lipase became effective only for low molecular weight substrates [19,75]. Lipase Source
Carrier
Acid/Oil Source Alcohol
Maximum
Performance
(%)
Reference
Burkholderia cepacia
K-Carrageenan
Palm
Methanol
100
(conversion)
[76]
Burkholderia cepacia
Phyllosilicate
Sol–Gel
Tallow and
Grease
Ethanol
94
(yield)
[77]
Burkholderia cepacia
Mtms-Based Silica
Monolith Coated
With Butyl-Substituted
Silicates
Jatropha
Methanol
95
(yield)
[78]
Candida antarctica
Celite®
Triolein
Methanol
60
(conversion)
[79]
Candida rugosa
Calcium Alginate MatrixPalm
Ethanol
83
(yield)
[80]
Candida rugosa
Activated Carbon
Palm
Ethanol
85
(conversion)
[81]
Pseudomonas cepacia
Hydrophobic Sol–Gel
Soybean
Methanol
67
(conversion)
[82]
Pseudomonas fluorescens
Mtcc 103
Alginate
Jatropha
Methanol
72
(yield)
[83]
Via Encapsulation Method
Burkholderia cepacia
Silica Aerogels
Sunflower Seed
-
56
(conversion)
[84]
Burkholderia cepacia
K-Carrageenan
Palm
Methanol
100
(conversion)
[85]
Candida antartica
Silica Aerogels
Sunflower Seed
Methanol
90
(conversion)
[86]
Table 5. Comparison of biodiesel production performance using immobilized lipase via entrapment method Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662
217 2.1.4. Cross linking technique Cross-linking is another method for immobilization that can be defined as the interaction of
a three dimensional network within enzyme, coupling reagent, and carrier [19]. The advant‐
age of cross-linking is obtaining stable lipases due to the strong interaction between the li‐
pase and the carrier. On the other hand, the cross-linking conditions are intense and the
immobilized lipase shows lower activity [31]. The high free fatty acid content of waste cooking oil form water by esterification with alco‐
hol which cause agglomeration of lipase and lowering biocatalysis efficiency [87]. 2.1.3. Entrapment technique Hence,
free Geotrichum sp. lipase was not a suitable enzyme catalyst for transesterification of waste
cooking oil. Yan et al. [87], report a modification procedure for preparation of cross-linked
Geotrichum sp. The obtained lipase exhibited improved pH and thermostable stability com‐
pared to free lipase. The relative biodiesel yield was 85% for transesterification of waste
cooking oil with methanol. Kumari et al. [88] studied the preparation of Pseudomonas cepacia lipase cross-linked enzyme
aggregates. It was shown that cross linked lipases has a greater stability than free enzymes
to the denaturing conditions. The enzyme also used to catalyze madhuca indica oil, which’s
transesterification is difficult by chemical routes due to its high free fatty acid content. As a
result, 92% conversion was obtained after 2.5 h. Immobilization of Candida rugosa lipase on fine powder of Scirpus grossus L.f. by glutaralde‐
hyde by cross linked technique for biodiesel production from palm oil, as already investigat‐
ed by Kensingh et al. [89]. It was concluded that immobilized lipase yielded higher
conversion of biodiesel than that of free lipase. Lorena et al. [90] investigated the immobilization of the Alcaligenes spp. lipase on polyethyle‐
nimine agarose, glutaraldehyde agarose, octyl agarose, glyoxyl agarose, Sepabeads® by the
aggregation and crosslinking method. The transesterification of canola oil was achieved
with a yield 80% using a six-step addition of methanol and lipase immobilized on Sepa‐
beads® by the aggregation method. All these methods are shown schematically in Figure 2. All these methods are shown schematically in Figure 2. Figure 2. Schematic diagram of enzyme immobilization methods: a)Adsorption method b)Covalent binding method
c)Entrapment method d) Crosslinking method Figure 2. Schematic diagram of enzyme immobilization methods: a)Adsorption method b)Covalent binding method Figure 2. Schematic diagram of enzyme immobilization methods: a)Adsorption method b)Covalent binding method
c)Entrapment method d) Crosslinking method Biodiesel - Feedstocks, Production and Applications 218 2.1.5. Whole cell immobilization The applicability of lipases for the bulk production of fuels was limited significantly by the
high cost of lipases [91]. Utilizing microbial cells such as fungi, bacteria, and yeasts cells con‐
taining intracellular lipase instead of extracellular lipases (free and immobilized lipase) is an
easier and a cost effective way of enzymatic biodiesel production. Compared to conventional
enzymatic processes, the use of whole cells provides excellent operational stability and avoids
the complex procedures of isolation, purification and immobilization [91,92]. The general
preparation steps for immobilized extracellular enzymes and whole cell enzymes showed in
Figure 3. Biomass support particles have been used for immobilization of whole cells. (a)
(b)
Figure 3. The preparation steps of a) immobilized extracellular lipase and b) whole cell biocatalyst (b) (a) Figure 3. The preparation steps of a) immobilized extracellular lipase and b) whole cell biocatalyst Aspergillus and Rhizopus have been most widely used as whole cell biocatalyst. Ban et al. [93], used first a whole cell biocatalyst, immobilized Rhizopus oryzae IFO4697 (a 1,3-posi‐
tional specificity lipase) cells within biomass support particles, for the production of biodie‐
sel and 91.1% methyl ester content was attained which was a similar result as that using the
extracellular lipase. Many researchers have experimented on the use of whole cells to cata‐
lyze transesterification reaction summarized in Table 6. A technique using glutaraldehyde cross-linking treatment on whole cell catalyst for metha‐
nolysis of soybean oil was developed by Sun et al. [94]. The glutaraldehyde cross linking
treatment resulted in higher methanol tolerance and high catalytic activity (with the ratio of
methanol to oil reaching 3). Also, a novel methanol addition strategy was proposed as step‐
wise addition of different amounts of methanol (1.0, 1.2, 1.5, and 2.0M equivalent of oil) ev‐
ery 24 h. It was found that the highest methyl ester yield could reach 94.1% after 24 h
reaction by 1.2 mol, 1.5 M and 1.2 mol methanol additions at 0, 8, and 14 h. In general, the
whole cell catalyzed process is slower than extracellular lipase catalyzed process. Sun et al. [94], also reported that the reaction time could be shortened by this way. It is clear that sig‐
nificant reduction in the cost of biodiesel production can be achieved by combining the
whole cell biocatalyst process with stepwise addition of methanol. 2.1.5. Whole cell immobilization Lipase Source
Carrier
Acid/Oil Source
Alcohol
Maximum
Performance
(%)
Reference
Aspergillus niger
BSPsa
Waste Cooking
Methanol
86.4
(yield)
[96]
Aspergillus niger
Polyurethane
BSPsa
Palm
Methanol
>90
(yield)
[97]
Aspergillus niger
BSPsa
Palm
Methanol
87
(yield)
[98]
Aspergillus oryzae NS4 BSPsa
Soybean
Methanol
98
(conversion)
[99]
A. oryzae carrying r-
CALBb
BSPsa
Palm Soybean
Methanol
85
90
(conversion)
[100]
Aspergillus oryzae
expressing r-FHLc
BSPsa
Rapeseed
Methanol
Ethanol
1-Propanol 1-
Butanol
96 (yield)
94 (yield)
96 (yield)
97 (yield)
[101]
Escherichia coli BL21
-
Rapeseed
Methanol
97.7
(conversion)
[102]
Rhizopus chinensis
CCTCC M201021
-
Soybean
Methanol
"/>86
(yield)
[103]
Rhizomucor miehei
displaying Pichia
pastoris
-
Soybean
Methanol
83.14
(yield)
[104]
Rhizopus oryzae IFO
4697
BSPsa
Refined Rapeseed
Crude Rapeseed,
Acidified Rapeseed
Methanol
~60(yield)
~60(yield)
~70(yield)
[105]
Rhizopus oryzae IFO
4697
BSPsa
Soybean
Methanol
~90(yield)
[106]
Rhizopus oryzae IFO
4697
BSPsa
Soybean
Methanol
~85(yield)
[107]
Rhizopus oryzae
IFO4697
-
Soybean
Methanol
71
(conversion)
[108]
Rhizopus oryzae IFO
4697 and Aspergillus
oryzae niaD300
(combined use)
BSPsa
Soybean
Methanol
~100
(conversion)
[109] Biodiesel - Feedstocks, Production and Applications 220 Lipase Source
Carrier
Acid/Oil Source
Alcohol
Maximum
Performance
(%)
Reference
Rhizopus oryzae ATCC
24563
-
Soybean
(Free Fatty Acid Content
5.5%)
Methanol
97
(conversion)
[110]
Rhizopus oryzae IFO
4697
BSPsa
Soybean
Methanol
72
(yield)
[111]
Rhizopus oryzae
Polyurethane foam
BSPsa
Soybean
Methanol
90
(conversion)
[112]
Rhizopus oryzae
BSPsa
Jatropha Curcas
Methanol
80 (conversion)
[113]
Rhizopus oryzae IFO
4697
-
Soybean
Methanol
86
(yield)
[114]
Rhizopus oryzae
BSPsa
Rapeseed
Methanol,
Ethanol,
1-Propanol, 1-
Butanol
83,
79,
93,
69
(yield)
[101]
Serratia marcescens
YXJ-1002
-
Grease
Methanol
97
(yield)
[115]
aBSPs:Biomass support particles
br-CALB: Candida antarctica lipase B
cr-FHL: Fusarium heterosporum lipase
Table 6. Comparison of biodiesel production performance using whole cell biocatalysts Table 6. Comparison of biodiesel production performance using whole cell biocatalysts Whole cell biocatalysts will be a way to industrialization of biodiesel production but
the limited mass transfer efficiency of product and substrate is a hurdle to further in‐
vestigations [95]. 3.1. Vegetable oils Vegetable oils are candidates as alternative fuels for diesel engines with their high heat con‐
tent [118]. But, direct use of vegetable oils is not possible because of the high kinematics vis‐
cosity of them which are varies in the range of 30–40 cSt at 38 °C and are about 10 times
higher than of diesel fuel (Grade No. 2D) leads to many problems [118,119]. Therefore, mod‐
ification of vegetable oil is necessary and the valuable product of this modification is named
‘‘biodiesel’’. The edible vegetable oils such as soybean [120,121], sunflower [122-124], palm
[81,125], corn [126], cottonseed [127], canola [68,69,128] and olive [129,130] oils have been
widely used in enzymatic transesterification. In developed countries, edible oils constitute
more than 95% of biodiesel production feedstock because the produced biodiesel from these
oils have properties very similar to petroleum-based diesel [131]. Also, the country and its
climate, the oil percentage and the yield per hectare are effective parameters in selecting the
potential renewable feedstock of fuel [118,132]. For example, while rapeseed oil prevailing
the EU production, soybean oil prevailing the US and Latin American production, and palm
oil mainly being used in Asia [133]. Inedible oils do not find a place in human consumption due to including toxic components. Therefore, inedible oils do not compete with food crops. Thus, inedible vegetable oils are an
alternative feedstock for biodiesel production. Babassu (Orbinya martiana), Jatropha curcas
(Linnaeus), neem (Azadiracta indica), polanga (Calophyllum inophyllum),karanja (Ponga‐
mia pinnata), rubber seed tree (Hevea brasiliensis), mahua (Madhuca indica and Madhuca
longifolia), tobacco (Nicotina tabacum), silk cotton tree, etc. are promising inedible vegeta‐
ble oil sources. Jatropha curcas is an attractive feedstock between various oil bearing seeds
as it has been developed scientifically and found to give better biodiesel yield and produc‐
tivity [134]. Crude Jatropha oil contains about 14% of free fatty acid that is too high for alka‐
line catalyzed biodiesel production [118]. However, high free acid content is not a problem
in the production process of biodiesel via using enzyme catalysts. Besides Jatropha curcas,
26 species of fatty acid methyl ester of oils of including Azadirachta indica, Calophyllum in‐
ophyllum, and Pongamia pinnata were found most suitable for use as biodiesel, which ad‐
just to the major specification of biodiesel standards of European Standard Organization,
Germany, and USA [135]. Modi et al. 3. Feedstocks The main aim of researches is to obtain a biodiesel, which will have a competitive price com‐
pared to other conventional sources of energy [116]. At this point, selecting the feedstock,
represents more than 75-80% of the overall biodiesel production cost, is a vital step to ensure
a cost effective biodiesel production. Different kinds of feedstock with varied range of edible
and inedible vegetable oil, animal fats, waste oil, microbial oil and microalgae oil can be
used for enzyme catalyzed transesterification [117]. Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662
22 3.1. Vegetable oils reported conversion of crude oils of Pongamia pinnata
(karanj), Jatropha curcas (jatropha) via immobilized Novozym 435 to biodiesel fuel with
yield 90, and 92.7%, respectively [136]. 3.3. Waste oils/fats In general, around the world only half of the discharged edible oils recycled as animal feed
or as raw material for lubricant and paint and the remainder is discharged into the envi‐
ronment [143]. Hence, the use of waste oils/fats for biodiesel production is very important
to reduce and recycle the waste oil [143], to eliminate the environment and human health
risk caused by waste oils [144] and to lower the biodiesel production cost. Waste cooking
oil, animal fats, yellow grease, brown grease obtained from highly oxidized yellow grease
or recovered waste grease from plumbing trap and waste sludge or soap-stock from the
vegetable oil refining process were the major sources of waste oil have been used for bio‐
diesel production [145]. The selection of a catalyst to be used for the production of biodie‐
sel fuel is mostly influenced by the amount of free fatty acid content in various feedstocks
[146]. The lipase-catalyzed reaction is a promising method for converting waste oils which
contains high percentage of free fatty acids and high water content, into biodiesel with high
yield [145]. It has been reported that Novozym 435 is capable of converting the used olive
oils [129]. 3.2. Animal oils/fats Animal fats are another group of feedstock for biodiesel production. Animal fats used to
produce biodiesel via enzymatic route include lard [137], lamb meet [138] and beef tallow
[139]. Animal fats are economically feasible feedstocks compared to vegetable oils. Animal
fat methyl ester also has many favorably properties such as non-corrosive, high cetane num‐
ber, and renewable [140,141]. However, animal fats saturated compounds lead to a tendency
to oxidation and crystallization unacceptably at high temperatures [142]. Biodiesel - Feedstocks, Production and Applications 222 3.4. Algae oils There is a considerable interest in the use of algae (micro and macro) oils for synthesis of
biodiesel. Because these oils are cheap raw materials besides animal fats and have rapid
growth rate and productivity when compared to conventional forestry, agricultural crops,
high lipid content, tolerance for poor quality water, smaller land usage up to 49 or 132
times less when compared to rapeseed or soybean crops [142,147]. The smaller land usage
brings the advantage of reducing the competition for arable soil with other crops, in partic‐
ular for human consumption [147]. However, there are still some drawbacks for utilization
of algae for biodiesel production. A considerable investment in technological development
and technical expertise is needed to optimize the microalgae harvesting and oil extraction
processes, to use cheap sources of CO2 for culture enrichment [147]. Algae oils contain
about 20-40% oil [148]. Several researchers have been experimented on microalgal oils as
raw material for biodiesel production. Tran et al. [130], investigated the conversion of mi‐
croalgal oil from Chlorella vulgaris ESP-31 to biodiesel by using immobilized Burkholderia
lipase and a high fatty acid methyl esters conversion efficiency of 97.25 wt% oil (or 58.35 wt
% biomass) was obtained for 48 h reaction. It is proposed that microalgal oil has good po‐
tential for application in the commercial production of biodiesel. The enzymatic conversion
of microalgal oils to biodiesel in ionic liquids was firstly studied by Lai et al. [149]. Four
microalgae two strains of Botryococcus braunii (BB763 and BB764), Chlorella vulgaris, and
Chlorella pyrenoidosa have been catalyzed by two immobilized lipases, Penicillium expan‐
sum lipase and Candida antarctica lipase B (Novozym 435), in two solvent systems: an ion‐
ic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, [BMIm][PF6]) and an organic
solvent (tert-butanol). Penicillium expansum lipase was found more efficient for this appli‐
cation and the ionic liquid [BMIm] [PF6] showed a greater conversion yield (90.7% and
86.2%) obtained relative to the one obtained in the commonly used organic solvent tert-bu‐
tanol (48.6% and 44.4%). Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 4.1. The effect of temperature on enzymatic transesterification Enzymatic transesterification takes place at low temperatures varying from 25 to 60°C. In
general, initially the rate of reaction increases with rise in reaction temperature, because
of an increase in rate constants with temperature and less mass transfer limitations
[150,151]. Nevertheless, increased temperature after the optimum temperature promotes
to denaturation and higher thermal deactivation of the enzyme, since it decreased the
catalytic activity [152]. Various researches have been carried out to find out the effect of temperature on biodiesel
production with immobilized enzymes. It is clear that immobilization provide more temper‐
ature resistance compared to free enzymes due to supplying a more rigid external backbone
for lipase molecule [150,151]. However, optimum temperature is specific for each produc‐
tion. The studies about the effect of temperature for enzymatic transesterification are shown
in Table 7. Lipase
Oil Source
Alcohol
Performed
Temperatures In
The Range (°C)
Optimum
Temperature (°C)
Reference
Immobilized Aspergillus
niger
Palm
Methanol
25-50
40
[153]
Immobilized Aspergillus
niger
Waste Cooking
Methanol
25-50
30
[154]
Immobilized
Burkholderia cepacia
Babassu
Ethanol
39-56
39
[155]
Candida antarctica
Cotton Seed
T-Butanol
30-50
50
[156]
Candida antarctica
Acid
Methanol
30-50
30
[157]
Immobilized Candida Sp. 99–125
Salad
Methanol
27-50
40
[158]
Candida Sp. 4.1. The effect of temperature on enzymatic transesterification 99–125
Waste Cooking
Methanol
35-50
40-50
[159]
Immobilized
Enterobacter aerogenes
Jatropha
T-Butanol
30-55
55
[160]
Immobilized
Enterobacter aerogenes
Crude Rapeseed
Ethanol
25-50
35
[161]
Lipozyme RM IM
Soybean
Butanol
20-50
30
[162]
Lipozyme RM IM
Soybean
Methanol and
Ethanol
40–60
50
[163]
Lipozyme RM IM
Soybean Oil
Deodorizer
Distillate
Ethanol
45-78
50
[164]
Lipozyme TL IM
Rapeseed
N-Butanol
30-60
40
[165]
Lipozyme TL IM
Soybean
Ethanol
20-50
35
[162] Biodiesel - Feedstocks, Production and Applications 224 Lipase
Oil Source
Alcohol
Performed
Temperatures In
The Range (°C)
Optimum
Temperature (°C)
Reference
Lipozyme TL IM
Palm
Ethanol
30-78
50
[166]
Novozyme 435
Rapeseed
Methanol
25-55
40
[167]
Novozyme 435
Tung and Palm
Methanol and
Ethanol
45-55
55
[168]
Novozym 435
Cottonseed
-(Dimethyl
Carbonate As
Organic
Solvent)
30-55
50
[169]
Novozym 435
Canalo
Methanol
25-65
38
[170]
Novozym 435
Olive
Methanol
30-70
40
[129]
Novozym 435
Soybean
T-Amyl
30-60
40
[171]
Novozym 435
Sunflower
Methanol
25-65
45
[172]
Novozym 435
Stillingia
Methanol
30-60
40
[173]
Novozym 435
Cotton Seed
Methanol
30-70
50
[174]
Novozym 435, Lipozyme
TL IM and Lipozyme
RM IM
Soybean
Ethanol
25-60
25
[175]
Immobilized Penicillium
expansum
Waste
T-Amyl
25-55
35
[176]
Immobilized
Pseudomonas cepacia
Soybean
Methanol and
Ethanol
25–60
35
[177]
Pseudomonas cepacia
Soybean
Methanol
20–60
30
[178]
Immobilized
Pseudomonas
fluorescens
Triolein
1-Propanol
40-70
60
[48]
Pseudomonas
fluorescens
Soybean
Methanol
30-60
40
[49]
Rhizopus chinensis
CCTCC M201021
Soybean
Methanol
30-40
30
[179]
Thermomyces
lanuginosus
Canola
Methanol
30-70
40
[69]
Table 7. Data on optimum temperature for enzymatic biodiesel production Table 7. Data on optimum temperature for enzymatic biodiesel production 4.2. The effect of water content on enzymatic transesterification Water content is one of the key factors for enzymatic transesterification reaction that have a
strong effect on lipase’s active three-dimensional conformational state [21,180]. Biocatalysts,
needs a small amount of water to retain their activities [181]. Lipase has an unique feature
on the water-oil interface, and the lipase activity depends on this interface. The presence of
an oil–water interface required because it provides a suitable environment for enzyme acti‐
vation which occurs due to the unmasking and restructuring the active site through confor‐ Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 225 mational changes of the lipase molecule [182,183]. When the addition of water increased, the
amount of water available for oil to form oil–water droplets also increases, hence increasing
the available interfacial area [182]. Thus, enzymatic activity can not be possible in a water
free media. However, excess water cause reverse reaction of hydrolysis. The amount of re‐
quired water, to provide an optimum enzyme activity, differs according to the type of en‐
zyme and reaction medium composition. Enzymes, substrates, organic solvent and also
immobilized support have a crucial role on optimal water activity for lipase [184]. Optimum
water content not only provides keeping the hydrolysis of ester linkages at the minimum
level, but also ensures the highest degree of transesterification [24]. Thus, a better control of
water content is very important for enzymatic process. Water activity (aw) is defined as free (boundness) water in the system, which is a ratio of va‐
por pressure over the given system versus that over pure water [24]. Thermodynamic water
activity is the best predictor of reaction rate that can be determined in any phase by different
kinds of sensors such as holographic sensor, Weiss LiCl humidity sensor [180,185]. Also,
several methods have been developed for control of water activity, for example, equilibra‐
tion with saturated salt solutions [186], addition of salt hydrate pairs [187,188] and introduc‐
tion of air or nitrogen into the reactor [189]. Recently, Peterson et. al. developed a practical
way for control of water activity in large-scale enzymatic reactions by using a programma‐
ble logic controller. On the other hand, percentage water content is another expression
which is used widely in transesterification, generally assayed by Karl-Fischer coulometer. In general, lipases show higher activity with higher water activities in solvent free systems
instead of Candida antarctica lipase (Novozym 435) [184]. For Candida sp. 4.2. The effect of water content on enzymatic transesterification 99–125 lipase, the
optimum water content is 10–20% based on the oil weight to maintain the highest transester‐
ification activity [31]. Salis et al., investigated production of oleic acid alkyl esters by using Pseudomonas cepacia
and determined that aw in the range 0.4–0.6, 1-butanol:triolein 3:1 – were the best conditions
to reach maximum enzymatic activity. It was also found that at the higher values of water
activity, no hydrolysis reaction was occurred [190]. Noureddini and Philkana [82] tested immobilized Pseudomonas cepacia for the transesteri‐
fication of soybean oil with methanol and ethanol and observed that increased addition of
water provide a considerable increase in the ester yield. The optimal conditions were deter‐
mined for processing 10 g of soybean oil by 475 mg lipase in 1 h as 1:7.5 oil/methanol molar
ratio, 0.5 g water in the presence of methanol that resulted in 67 % yield and 1:15.2 oil/etha‐
nol molar ratio, 0.3 g water in the presence of ethanol that resulted in 65% yield. Al-Zuhair et al. studied the esterification of n-butyric acid with methanol in the presence of
Mucor miehei lipase, and found similar results with literature [191] that higher water con‐
tent, makes lipase more efficient [182]. Shah and Gupta used immobilized Pseudomonas cepacia lipase for ethanolysis of Jatropha
oil and noted that the best yield 98% gained by in the presence of 4–5% (w/w) water in 8 h. The yield was only 70% in absence of water [44]. Biodiesel - Feedstocks, Production and Applications 226 Kawakami et al. determined the effect of water content for transesterification of Jatropha oil
and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substi‐
tuted hydrophobic silica monolith. The authors reported that biodiesel yield reached 90%
with water content of 0.6% (w/w) after 12 h using a stoichiometric mixture of methanol and
oil (3:1) [78]. Kawakami et al. determined the effect of water content for transesterification of Jatropha oil
and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substi‐
tuted hydrophobic silica monolith. The authors reported that biodiesel yield reached 90%
with water content of 0.6% (w/w) after 12 h using a stoichiometric mixture of methanol and
oil (3:1) [78]. Chen et al. 4.2. The effect of water content on enzymatic transesterification investigated the effect of water content for production of biodiesel with oleic acid
with methanol catalyzed by soluble lipase NS81020, produced by modification of Aspergil‐
lus oryzae microorganism, in the biphasic aqueous-oil systems and found that the esterifica‐
tion yield is low if the water was scant. The higher reaction rate and fatty acid methyl ester
yield was obtained with 10 wt % water by oleic acid weight [192]. It is clear that during the past decade numerous investigations have been made to determine
the optimal water content for transesterification. As a result, the necessary amount of water
content is an important factor to create an interfacial surface between oil and water and to
ensure optimal enzymatic activity. Also, water has a strong influence on structural integrity,
active site polarity, and protein stability of lipase [21,193]. However, it differs from enzyme
to reaction conditions. 4.3. The effect of acyl acceptors on enzymatic transesterification Methanol, short chain alcohol, usually used as an acyl acceptor due to its low price and
availability. Insoluble and a relatively high amount of methanol with respect to oil, have a
negative influence on the stability of lipases and could be solved by a stepwise addition of
the alcohols [15, 194]. To eliminate inhibitory effects of methanol some co-solvents are add‐
ed to the reaction mixture. Tert-butanol is one of the important co-solvents which is added
to enzymatic reaction. Usage of tert-butanol, a polar solvent, is also a possible solution for
eliminating the inhibitory effects of methanol and glycerol (both of them soluble in tert-bu‐
tanol) and suggested instead of using butanol [195]. Liu et al. [196], transesterified waste
baked duck oil by three different commercial immobilized lipases (Novozym 435, Lipozyme
TLIM and Lipozyme RMIM) with different monohydric alcohols (methanol, ethanol, propa‐
nol, isopropanol, isobutanol, isoamyl alcohol) and fusel oil-like alcohol mixture (containing
15% isobutanol, 80% isoamyl alcohol, 5% methanol) in solvent-free and tert-butanol sys‐
tems. It was reported that each lipase presented a different kinetic pattern depending on the
monohydric alcohols. The results showed that Lipozyme TL IM and Novozym 435 gave
high conversion rate with isobutanol and isoamyl alcohol either in solvent-free or in tert-bu‐
tanol system. Thus, the combined use of lipases, Novozym 435 and Lipozyme TLIM, as cata‐
lyst and fusel oil-like mixture as raw material for biodiesel synthesis was found effective in
view of cost saving of biodiesel production [195]. Recently, novel acyl acceptors were investigated such as ethyl acetate, methyl acetate, butyl
acetate, vinyl acetate [197], dimethyl carbonate [198]. Du and coworkers demonstrated the
positive effect of methyl acetate, on enzymatic activity of Novozym 435 and found that li‐
pase could be reused directly without any additional treatment [199]. The advantage of us‐
ing methyl acetate is that the cost of the catalyst can be reduced dramatically due to the
longer operational life and reusability of lipase. The byproduct of the system is triacetylgly‐ Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 227 cerol, which does not have any negative effect on the fuel property, and also no glycerol
produced [200]. Hence, these advantages will provide industrial implementation of enzy‐
matic biodiesel production. Dimethyl carbonate is another promising alternative acyl ac‐
ceptor, which is eco-friendly, odorless, cheap, non-corrosive, and non-toxic [200]. 4.4. Effects of the solvent on enzymatic transesterification reaction In enzymatic transesterification reaction, excess of alcohol increases reaction efficiency, but
if alcohol doesn’t dissolve in reaction medium it can disrupt the enzyme activity. Methanol
and vegetable oil in the values close to 1:1 molar ratio forms a solution in 40°C. Solvent is
added into the reaction medium to increase the solubility of alcohol and thus it allows first
step enzymatic transesterification by blocking degradation lipase catalytic activity [24]. To
overcome deactivation of lipase activity and improve the lipase activity, various organic sol‐
vents have been used for enzymatic biodiesel synthesis. These solvents have been listed in
Table 8. Cyclohexane, n-hexane, tert-butanol, petroleum ether, isooctane and 1,4-dioxane are
mainly studied hydrophilic and hydrophobic organic solvents in enzymatic biodiesel pro‐
duction. In organic solvent medium, overall alcohol is added at the beginning of the reac‐
tion. In solvent free reaction medium, alcohol is added in several portions to prevent
enzyme activity with high alcohol concentration [24]. Hexane is generally preferred because of its low cost and easily availability in the market. Some studies were performed in hexane solvent systems with soybean and tallow oil using
monohydric alcohols [70,201, 202]. Nelson et al. performed transesterification of tallow with
monohydric alcohols by Lipozyme IM 60 (M. miehei) and Novozyme SP435 (C. antarctica)
in hexane and a solvent-free system. They compared the transesterification yields of two dif‐
ferent systems. The yields with higher than 95% were obtained with methanol, ethanol and
butanol with Lipozyme IM 60 lipase under hexane system (Table 8) while reaction yields
under solvent-free system were 19% for methanol, 65.5% for ethanol, and 97.4% for isobuta‐
nol [201]. Similar results were found by Rodrigues et al. [70]. They compared the yields of
transesterification of soybean with ethanol by Lipozyme TL IM. In the presence of n-hexane
with 7.5:1 molar ratio of ethanol:soybean oil, the transesterification conversion was found to
be as 100% while in solvent-free system the yield was 75%. At stoichiometric molar ratio, the
yield was 70% conversion after 10 h of reaction in both systems. Transesterification conver‐
sion was obtained as 80% by three stepwise addition of ethanol, while a two step ethanolysis
produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems. In enzyme catalyzed reaction, both alcohol amount and low glycerol solubility in biodiesel
have negative effects on enzyme activity. 4.3. The effect of acyl acceptors on enzymatic transesterification The
transesterification reaction is irreversible, because carbonic acid monoacyl ester, the inter‐
mediate compound, immediately decomposes to carbon dioxide and alcohol [200]. The fatty
acid methyl ester yield is higher for lipase-catalyzed transesterification of vegetable oils with
dimethyl carbonate besides conventional acyl acceptors (methanol and methyl acetate) [200]. Only, the higher price of acyl acceptor besides alcohols is a disadvantage [194]. 4.4. Effects of the solvent on enzymatic transesterification reaction Deposit of glycerol coating the immobilized cata‐
lyst is formed during the process, which reduces the enzymes activity [203]. The solubility of Biodiesel - Feedstocks, Production and Applications 228 methanol and glycerol in hydrophobic solvents is low. For this reason, this problem may oc‐
cur in hydrophobic solvent system. The enzymatic alcoholysis of triglyceride also was studied with petroleum ether, isooctane,
cyclo hexane,1,4-dioxane (Table 4-2) [16,48,204]. Iso et al. [48], reported that when methanol
and ethanol were used as alcohol in enzymatic transesterification, the reactions need an ap‐
propriate organic solvent [48]. On the other hand, the reaction could be performed without
solvent when 1-propanol and 1-butanol was used. They also used, benzene, chloroform and
tetrahydrofuran as solvent and immobilized P. fluorescens lipase as catalyst at 50°C to com‐
pare the results that of the 1,4 dioxane. The highest enzymatic activity was observed with
1,4-dioxane. The enzymatic activity increased with the high amount of 1,4-dioxane. But high
conversion of oil (app.90%) to biodiesel was obtained with high proportion of 1,4 diox‐
ane(90%). Although usage of high amount of solvents is not preferable in industry solvents
can be recovered together with methanol after transesterifiation reaction. Hydrophilic organic solvents can interact with water molecule in enzyme and this may af‐
fect the catalytic activity of enzyme. However, as shown in Table 8 high performance was
ensured with hydrophilic solvents such as 1,4-dioxane and tert-butanol [48,156, 205-208]. Some studies were performed in the presence of t-butanol solvent because of positive effects
on enzymatic catalyzed reaction. T-butanol has moderate polarity so methanol and glycerol
are easily soluble in tertiary butanol. Solubility of methanol prevents enzyme inhibition and
solubility of glycerol prevents accumulation on the enzyme carrier material. Another ad‐
vantage of this solvent is sinteric hindrance. Due to this property, tert-butanol is not accept‐
ed by the lipase. High yield and conversions were obtained in the presence of t-butanol with
various vegetable oils and immobilized lipases shown in Table 4-2. For example, Liu et al.,
[196] studied biodiesel synthesis by immobilized lipases in solvent-free and tert-butanol me‐
dia. Each lipase showed a different conversion depending on the monohydric alcohols and
immobilized lipase in solvent-free medium and tert-butanol system. For methanolysis, re‐
gardless of the lipase type, the conversion rate is higher in tert-butanol than that in solvent-
free medium. Novozym 435 showed higher conversion rate with straight monoalcohols in
tert-butanol medium. 4.4. Effects of the solvent on enzymatic transesterification reaction Lipozym RM IM and Lipozyme TL IM showed lower conversion with
straight and branched monoalcohols (except methanol) in solvent free system. Similar re‐
sults were obtained by Halim and Kamaruddin [208], in transesterification of waste cooking
palm oil using various commercial lipases (Lipozyme RM IM, Lipozyme TL IM and Novo‐
zyme 435) in tert-butanol as reaction medium. Novozyme 435 was found to be more effec‐
tive in catalyzing the transesterification with methanol in in-tert-butanol medium. It was
also been demonstrated that even 3:1 methanol to oil molar ratio didn’t inhibit the Novo‐
zyme 435 in tert-butanol system. Du et al. [209], showed that Lipozyme TL IM could be used
without loss of lipase activity for 200 batches in tert-butanol system. Li et al. [210], used ace‐
tonitrileand tert-butanol mixture as co-solvent in transesterfication of stillingia oil with
methanol. The highest biodiesel yield (90.57%) was obtained in co-solvent with 40% tert-bu‐
tanol and 60% acetonitrile (v/v) with co-solvent. They also reported that co-solvent (as a
mixture)enhance the tolerance of lipase to the methanol than the pure tert-butanol. Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662
229 Solvent
Oil
Alcohol
Lipase
Temp/
Time
Reaction mixture Performance
(%)
Ref. Tert-butanol
Cotton seed
Methanol
Novozyme
435
(Candida
antarctica)
50 °C /
24h
13.5% meth., 54%
oil
32.5% tert-
butanol,
Lipase:1.7%
(wt of oil)
97 (yield)
[156]
Tert-butanol
Cotton seed
Methanol
Pancreatic
lipase
37 °C /
4 h
Methanol :oil mol
ratio:1:15
Lipase:0.5%
enzyme
(wt of oil)
water conc.5%
(wt of oil)
75–80
(conversion)
[205]
Tert-butanol
Rapeseed
Methanol
Novozyme435
& Lipozyme TL
IM
35 °C /
12 h
Methanol: oil mol. ratio 4:1
tert-butanol/oil
vol. 1:1
Lipase:
3% Lipozyme TL IM
1% Novozym 435
(wt of oil)
95 (conversion)
[206]
Tert-butanol
Soybean and
deodorizer
distillate
Methanol
Lipozyme TL
IM
Novozym 435
40°C/
12 h
Methanol:oil molar
ratio 3.6:1
Lipase :3%
Lipozyme TL IM
2% Novozym 435
tert-butanol: 80%
(wt of oil)
84 ( yield)
[207]
Tert-butanol
Waste cooking
palm
Methanol
Novozyme 435 40°C /
12 h
Methanol:oil mol. ratio 4:1,
Lipase:4% (wt of
oil)
88(yield)
[208]
Tert-butanol
Waste baked
duck
Methanol
Novozym 435
Lipozyme TL
IM
45 °C /
20 h
Methanol:oil mol. 4.5. The effect of molar ratio of alcohol to oil on enzymatic transesterification Biodiesel yield always increased due to the molar excess of alcohol over fatty acids in trigly‐
cerides in traditional transesterification system [15]. The transesterification reaction is rever‐
sible and so, an increase in the amount of one of the reactants will result in higher ester yield
and minimally 3 molar equivalents of methanol are required for the complete conversion of
methyl ester [174]. Conversely, for enzyme catalyzed transesterification, insoluble excess
methanol which exists as fine droplets demonstrates negative effects on enzyme activity and
also decrease the production yield [211]. The reaction medium is an important factor during
the determination of the optimum molar of alcohol to oil. The inactivation of lipases occurs
by contact with insoluble alcohol because the highly hydrophilic alcohol eliminates the layer
of essential water from the enzymes [212]. Thus, stepwise addition of alcohol is a potential
approach for ratio optimizing the molar ratio in solvent free systems [15]. Whilst, higher re‐
action rates could be obtained with a slight excess of alcohol in organic solvent systems [15]. The two-step reaction system was reported to avoid the inactivation of the lipase by addi‐
tion of excess amounts of methanol in the first-step reaction, and by addition of vegetable oil
and glycerol in the second-step reaction [213]. Watanabe et al. [213], used a two-step reac‐
tion system for methyl esterification of free fatty acids and methanolysis of triacylglycerols
using immobilized Candida antarctica lipase. The first step reaction was methyl esterifica‐
tion of free fatty acids that was performed by treating a mixture of 66 wt % acid oil and 34
wt % methanol with 1 wt % immobilized lipase. The second step reaction was conducted to
convert triacylglycerols to fatty acid methyl esters. In this step, a mixture of 52.3 wt % dehy‐
drated first-step product, 42.2 wt% rapeseed oil, and 5.5 wt% methanol using 6 wt% immo‐
bilized lipase in the presence of additional 10 wt % glycerol was treated. The contents of
fatty acid methyl esters was 91.1wt.% after the second step reaction was repeated by the use
of immobilized lipase for 50 cycles using recovered glycerol. 4.4. Effects of the solvent on enzymatic transesterification reaction ratio 4:1,
Lipase: 5 wt%(wt
of oil)
85.4,
78.5,
(conversion)
[196]
Hexane
Tallow
Methanol
Ethanol
Propanol
Lipozyme IM
60
45 C/
5 h
0.34 M tallow in
hexane
(8 mL),
Lipase: 10
(wt of oil)
200rpm
94.8,
98.0,
98.5
(conversion )
[201] Biodiesel - Feedstocks, Production and Applications 230 Solvent
Oil
Alcohol
Lipase
Temp/
Time
Reaction mixture Performance
(%)
Ref. Hexane
Soybean
Methanol
Lipozyme IM
77
36.5°C/
3h
Methanol :oil mol
ratio:3.4:1
Lipase:0.9BAUN*of
lipase;
water 5.8% (wt%
of oil)
92.2 (yield)
[202]
Hexane
Soybean
Ethanol
Lipozyme TL
IM
30 °C/
10 h
Ethanol:oil
mol.ratio:7.5:1
Lipase: 15 %(wt of
oil). 4% water
100
(conversion)
[70]
Cyclo
hexane
Sunflower
Methanol
Lipase AK
Lipozyme TL
IM
Lipozyme RM
IM
40°C/
24 h
Volume of organic
solvent/ oil: 2
ml/0.2 mmol
Lipase: 10% (wt of
oil)
65,
75,
35
(conversion)
[204]
Acetonitrile
60%and 40%
t-butanol (v/v)
Stillingia
Methanol
Novozym 435
and Lipozyme
TL IM
40°C/
24h
Methanol:oil mol
ratio: 6.4:1
Lipase:
4% (w/w) of
multiple-lipase
(1.96% Novozym
435+2.04%
Lipozyme TL IM)
90.57
(yield)
[210]
Petroleum
ether
Sunflower
Ethanol
Lipozyme IM
Lipase AK
45°C /
5h
Ethanol:oil mol. ratio:11:1
Lipase:20%
(wt of oil)
82,
99,
(yield)
[16]
I-octane
Sunflower
Methanol
Lipase AK
Lipozyme TL,
IM
Lipozyme
RM,IM
40 °C
Methanol: oil mol
ratio::3:1
Vol. of organic
solvent/oil: 2
ml/0.2 mmol
80,
65,
60,
(yield)
[204]
1,4-dioxane
Triolein
Methanol
Lipase AK
50°C /
80h
Methanol:oil mol. ratio: 3:1
90% solvent
~70 (conversion) [48]
*BAUN:Batch Acidolysis Units Novo
Table 8. Effect of the solvent on the performance of enzymatic transesterification reaction Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 231 Although positive effects of the usage of the solvents on the transesterification reaction,
some drawbacks has also been known) such as; extra reactor volume, solvent toxicity and
emissions, solvent recovery and loss cost [133]. 4.5. The effect of molar ratio of alcohol to oil on enzymatic transesterification Moreno-Pirajan and Giraldo [81], added different amounts of alcohol varied from 2.7 to 13.7
molar equivalents for methanol and from 5.7 to 26.7 molar equivalents for ethanol, based on
the moles of triglycerides toward the transesterification of palm oil catalyzed by Candida ru‐
gosa lipase and 10.4 molar ratio for all alcohols to palm oil was determined as optimal alco‐
hol requirement resulted in 85 mol% of methyl esters yield with n-butanol. Lipase catalyzed esterification of palmitic acid with ethanol in the presence of Lipozyme IM
20 in a solvent free medium was investigated by Vieira et al. [212]. Different acid/alcohol
molar ratios were tried as 0.16, 0.5, 1.0, 1.5, and 1.84. The best result was obtained with 0.5
acid/alcohol molar ratio. Zaidi et al. [214], explained the correlation existing between the kinetic parameters and the
chain-length of the substrates in esterification of oleic acid using nylon-immobilized lipase
in n-hexane. It is observed that the inhibition coefficient of the alcohol increased from 0.034 Biodiesel - Feedstocks, Production and Applications 232 to 0.42 mol l−1, when the number of carbon atoms increased from 1(methanol) to 18 (oleyl
alcohol), respectively. Dizge and Keskinler [69], used immobilized Thermomyces lanuginosus lipase to produce bi‐
odiesel with canola oil with methanol and investigated the role of substrate molar ratio. The
biodiesel production was conducted at 1:1, 1:2,1:3,1:4,1:5;1:6 and 1:10 oil/alcohol molar ratios
at 40°C. The highest methyl ester yield (85.8%) was obtained at the oil/methanol molar ratio
of 1:6. Two important result from this study can be concluded as (i) an increase in the num‐
ber of moles of methanol resulted in an increase in the ester production, (ii) when the forma‐
tion of esters reached a maximum level the further increases in the methanol concentrations
cause a decrease in the formation of esters due to enzyme inactivation. Thus, the actual amount of alcohol needed varies significantly depending on the origin of
the lipase and fat. 5. Reactors for enzymatic transesterification Through the industrialization of enzymatic biodiesel production, it is necessary to show the
applicability of enzymes in reactor systems. Various reactors, including batch reactors,
packed bed reactors and supercritical reactors have been investigated by researchers. Most
of the investigations on enzymatic synthesis of biodiesel have been performed in batch reac‐
tors and packed bed reactors. Batch reactors are simple designs used in the laboratory. In batch reactors, methanol shows
a good dispersion in the oil phase. But the physical agitation caused by shear stress from the
stirring would disrupt the enzyme carrier which shortens the enzymes life [31]. On the other
hand, batch operation is labor intensive, and not suitable for automation [215]. Packed bed
reactors are alternative of batch reactors which are substantially faster and more economical
continuous reactors [216]. A packed-bed reactor system is most widely used in biotechnolo‐
gy, as it is easy to operate and scale up these systems. In addition, these systems have high
bed volume. The most important advantage of these systems is that it is lowering shear
stress on immobilized enzymes which leads to long-term enzyme stability [217]. Further‐
more, stepwise addition of alcohol can be performed to reduce the inactivation of the en‐
zyme caused by excess alcohol. One of the encountered problems with an immobilized
lipase is the inhibition of the enzyme due to the cloggage of the catalyst by accumulation of
the glycerol by-product inside the reactor [218]. Also, the separation of glycerol which re‐
mains in the bottom of the reactor can be achieved in a simple way by using more than one
column. Recently, a packed-bed reactor system, in which a reactant solution is pumped
through a column containing biomass support particles immobilized recombinant Aspergil‐
lus oryzae and the effluent from the column is recycled into the same column with a step‐
wise addition of methanol was developed by Yoshida et al. [219]. In this system, lipase
retains its activity for five batch cycles and 96.1% methyl ester content was obtained with a
residence time of 140 min per pass and stepwise addition of 4.25 molar equivalents of meth‐
anol to oil for 6 passes. The methanolysis of soybean oil in packed bed reactor system using Lipase Applications in Biodiesel Production
http://dx.doi.org/10.5772/52662 233 Rhizopus oryzae whole cell was studied by Hama et al. [112]. 5. Reactors for enzymatic transesterification The final methyl ester content
was over 90% at a flow rate of 25 l/h in the first cycle and also, after 10 cycles approximately
80% conversion was achieved. Wang et al. [216], developed Pseudomonas cepacia lipase –
Fe3O4 nanoparticle biocomposite based packed bed reactors. A single-packed-bed reactor
and the four-packed-bed reactor were used to produce biodiesel by using refined soybean
oil. A high conversion rate (over 88%, 192 h) and great stability was achieved with the four-
packed-bed reactor compared to single-packed-bed reactor. It is considered that the four-
packed-bed reactor supplied a longer residence time of the reaction mixture in the reactor
and lowered the inhibition of the lipase by products [216]. By this way, the reaction efficien‐
cy was improved. Additionally, the cost of biodiesel production can be reduced by the effec‐
tive recycling of the enzyme catalysts [184]. Supercritical reactors also have been investigated by researchers for enzymatic biodiesel
production. D. Oliveira and J. V. Oliveira [220], produced biodiesel from palm kernel oil in
the presence of Novozym 435 and Lipozyme IM in supercritical carbon dioxide in the tem‐
perature range of 40−70 °C and from 60 to 200 bar using a water concentration of 0−10 wt %
and oil/ethanol molar ratios from 1:3 to 1:10. Lipozyme IM showed better results and the
highest reaction conversion was obtained as 77.5 %. It was observed that lipase structure
changed at pressures beyond 200 bar. Madras et al. [221], synthesized biodiesel from sun‐
flower oil in supercritical carbon dioxide catalyzed by Novozym. However, the obtained
conversions, when the reaction was conducted in supercritical methanol and ethanol at the
optimum conditions, were 23 and 27%, respectively [221]. Enzymatic transesterification of
lamb meat fat in supercritical carbon dioxide was investigated by Taher et al. [222].The max‐
imum conversion (49.2%) was obtained at 50◦C, with 50% Novozym 435 loading, 4:1 molar
ratio, within 25 h reaction. Supercritical reactors could not commercialized according to the
low conversion rate and cost of the system. Consequently, packed bed reactor systems seem to be a practical transesterification reactor
system with high transesterification efficiency. These systems will bring industrial scale up
enzymatic biodiesel production in an economic way. 6. Conclusion Today, the growing energy necessity and environmental pollution problem requires the use
of renewable alternative energy sources to become less dependent on fossil resources. As
known, biodiesel is an important alternative energy resource and seems to be the fuel of fu‐
ture because it is an environmentally friendly, nontoxic, renewable, and biodegradable fuel. Conventionally, biodiesel production is achieved by mainly alkaline or acid catalysts. The
interest in the use of biocatalyst for biodiesel production has been an increasing trend due to
its many advantages. Biodiesel have been shown to be effectively produced by enzymatic catalyst and also, nu‐
merous researches have been performed to obtain highly active lipases and to optimize Biodiesel - Feedstocks, Production and Applications 234 process conditions for biodiesel production. Besides many advantages, to produce biodiesel
by enzyme catalysts on an industrial scale, it is necessary to reduce the high cost of enzymes
and obtain lipases with better features. The immobilization of lipases and genetic engineer‐
ing methods seems to be an attractive way to obtain more active, stable, and reusable lipases
in organic solvents and alcohols. Also, selection of alternative acyl-acceptors is an option for
eliminating the negative effects of methanol on lipase activity. It can be concluded that in enzyme catalyzed biodiesel production significant progresses
have been made but further improvements such as novel reactor design should be ad‐
dressed and emphasized in the future research in order to ensure industrial enzymatic bio‐
diesel production. By making novel improvements, much attention will be focused on
enzyme usage in biodiesel production, and especially lipase reactions will be applied much
more in this area. Author details Sevil Yücel1*, Pınar Terzioğlu1,2 and Didem Özçimen1 Sevil Yücel1*, Pınar Terzioğlu1,2 and Didem Özçimen1 *Address all correspondence to: yuce.sevil@gmail.com syucel@yildiz.edu.tr *Address all correspondence to: yuce.sevil@gmail.com syucel@yildiz.edu.tr 1 Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Bioengin‐
eering Department, Istanbul, Turkey 1 Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Bioengin‐
eering Department, Istanbul, Turkey 1 Yıldız Technical University, Faculty of Chemical and Metallurgical Engineering, Bioengin‐
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Epidemiologic characteristics of renal cell carcinoma in Brazil
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International Braz J Urol
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ABSTRACT Purpose: In Brazil, National data regarding the epidemiology of renal cell carcinoma (RCC) are scarce. The aim of this
study was to describe the demographic, clinical, and pathologic characteristics of RCC diagnosed and treated by members
of the SBU - Brazilian Society of Urology. Materials and Methods: For this cross-sectional study, data were collected through an on line questionnaire available to
the members of the Brazilian Society of Urology (SBU). Between May 2007 and May 2008, voluntary participant urolo
gists collected data on demographic, clinical and pathological characteristics from patients diagnosed with RCC in their
practice. Results: Fifty SBU affiliated institutions contributed with patient information to the study. Of the 508 patients, 58.9%
were male, 78.9% were white, and the mean age was 59.8 years. Smoking history, high blood pressure and a body mass
index above 30 kg/m2 were present in 14.8%, 46.1% and 17.9% of the patients, respectively. Abdominal ultrasound and
computed tomography were the main diagnostic methods. The majority of the cases were localized tumors and metastasis
were presented in 9.5% of the patients; 98.4% underwent nephrectomy. Clear cell carcinoma was the most common his
tological type. In comparison with private institutions, stage IV disease was less frequent among patients treated at public
health services (P = 0.033). Conclusions: RCC in Brazil is more common in white men in their sixth decade of life. Ultrasound is the main diagnostic
tool for the diagnosis of clear cell carcinoma and we found that localized disease was predominant. A national registry of
RCC is feasible and may provide valuable information. E
International Braz J Urol
Clinical Urology E
International Braz J Urol
Clinical Urology Vol. 36 (2): 151-158, March - April, 2010
doi: 10.1590/S1677-55382010000200004 Vol. 36 (2): 151-158, March - April, 2010 Key words: carcinoma; renal cell carcinoma; cross sectional studies; epidemiology; kidney neoplasms
Int Braz J Urol. 2010; 36: 151-8 Epidemiologic Characteristics of Renal Cell Carcinoma in
Brazil Brazilian Society of Urology Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil pain, weight loss, fever, and night sweats), the exam
leading to diagnosis (clinical findings, laboratory tests,
or imaging studies), and type of medical assistance
(public heath care, private care, or third-party pay
ment by insurance companies; the latter two were
combined for analysis under the category “private
institutions”). In Brazil, data regarding the epidemiology of
RCC on a national scale are currently scarce, since
the disease is not among the 10 most frequent tumor
types annually reported by the National Cancer In
stitute (6). Data from regional surveys suggest that
RCC represents approximately 1.2% of all cases of
cancer in the country (7). Recognizing the need for
better information about the burden of RCC in Brazil,
the Brazilian Urological Society (SBU) led the effort
to produce this first-ever survey on a national scale
describing the epidemiology of RCC in Brazil. Body-mass index (BMI), the weight in kilo
grams divided by the square of the height in meters,
was calculated for each patient. TNM stage was de
termined according to the 2002 classification of renal
tumors (8). Tumor histology was classified according
to the Heidelberg classification (9). Five subtypes of
RCC were reported in the study: clear-cell, papillary,
chromophobe, collecting-duct, and unclassified car
cinomas. The aim of this study was to describe the de
mographic, clinical, and pathologic characteristics of
RCC diagnosed and treated by urologists associated
to SBU in Brazil. Data Collection In addition to descriptive statistics of the
demographic, clinical, and pathologic variables, ex
ploratory analyses were conducted for comparisons
between groups of patients. The chi-square or Fisher’s
exact tests were used to compare the frequency of
categorical variables between groups, and Student’s-
t-test or analysis of variance were used to compare
continuous variables. All P values are two sided, and
P < 0.05 was considered significant. All data analysis
was conducted using the MedCalc software, version
9.6.0.0 (MedCalc, Mariakerke, Belgium). To perform a cross-sectional study, a web-
based survey containing 75 questions was made
available to approximately 3,700 physicians affiliated
to SBU, who were invited to participate in the study
through mailed announcements and frequent advertis
ing of the study on the SBU website. The question
naire was available on the website for the duration of
the study, and could also be sent by regular mail or fax
upon request (www.sbu.org.br). Participant physicians
were instructed to collect data from patients seeking
medical attention between May 2007 and May 2008. All completed questionnaires were registered in a cen
tral database that was under the supervision of SBU. The study was approved by the institutional review
boards affiliated with the centers where participants
were enrolled. An informed consent was offered for all
patients and signed by them. The study was sponsored
by Pfizer Brazil. INTRODUCTION decades, since this rise in incidence is mainly a result
of the increasing detection of small tumors (4). How
ever, at least in the United States, both incidence of
late-stage RCC and mortality rates due to the disease
have also been rising, implying that improvements in
the ability to diagnose RCC are being outweighed by
the increasing prevalence of some of the risk factors
for this tumor (5). The incidence of renal cell carcinoma (RCC)
is increasing globally (1,2). Currently, the disease rep
resents approximately 2.0% of all new cases of cancer
and over 100.000 deaths worldwide (3). Improve
ments in imaging diagnosis may have contributed
to the rising incidence of RCC over the past three 151 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Table 1 – Distribution of patients according to Federation
States. State of Enrollment
Number
Percent
Bahia
1
0.2
Paraíba
1
0.2
Paraná
2
0.4
Goiás
3
0.6
Mato Grosso do Sul
4
0.8
Distrito Federal
6
1.2
Pará
6
1.2
Rio Grande do Sul
8
1.6
Sergipe
9
1.8
Piauí
12
2.4
Minas Gerais
18
3.5
Rio de Janeiro
19
3.7
Pernambuco
24
4.7
Espírito Santo
26
5.1
São Paulo
369
2.6
Total
508
100.0 Table 1 – Distribution of patients according to Federation
States. (41.3%) were the most frequent, whereas the classic
triad of hematuria, flank pain and palpable flank mass
was present in only 4.5% of the cases (Figure-1). The
most common procedure leading to the diagnosis of
RCC was an abdominal ultrasound (73.4%), followed
by computed tomography (CT) scan of the abdomen
(19.1%) and physical exam (3%). Diagnosis through
intravenous urography was anecdotal (0.6%). Three-
quarters of patients had localized disease (i.e., TNM
stage I and II) and the most common histological type
was clear cell carcinoma; virtually all patients under
went nephrectomy (radical or partial) for management
of their RCC. Other patient and disease characteristics
are shown in Table-2. Patient and Disease Profile Fifty SBU affiliated institutions from 14
Brazilian States contributed with patient data for this
study. Each institution was represented by at least one
physician and a total of 508 patients were enrolled. Approximately three-quarters of patients were seen
at institutions from the State of São Paulo. Table-1
shows the distribution of patients according to State. Patient demographics and tumor characteristics are
given in Table-2. Slightly more patients were male,
nearly 80% were white and 83.5% were assisted by Data collected from each patient included
age, gender, race, state of origin, weight, height,
tumor-node-metastases (TNM) stage, histological
subtype, the presence of known risk factor for RCC
(history of smoking, hypertension, obesity, diabetes,
hypercholesterolemia, end-stage renal disease, and
Von Hippel-Lindau disease), signs and symptoms
present at diagnosis (hematuria, palpable mass, flank 152 Exploratory Analyses The presence of most risk factors for RCC
varied little according to age. However, there were
significantly higher proportion of patients with a
history of hypertension (P < 0.0001) or diabetes (P
= 0.0011) among subjects aged 60 years or older, in
comparison with younger patients. The proportion of
cases diagnosed by ultrasound or by CT scan did not
differ among patients seen at public health services or
in private institutions (P = 0.631). Metastatic disease public health services. The mean age was close to
60 years, and the most prevalent risk factor for RCC
was hypertension. With regard to symptoms and signs
upon presentation, hematuria (42.9%) and flank pain public health services. The mean age was close to
60 years, and the most prevalent risk factor for RCC
was hypertension. With regard to symptoms and signs
upon presentation, hematuria (42.9%) and flank pain Figure 1 – Frequency of signs and symptoms at presentation of renal cell carcinoma. Figure 1 – Frequency of signs and symptoms at presentation of renal cell carcinoma. 153 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Table 2 – Patient and tumor characteristics. Characteristic
Number
Percent
Age, years
Mean (± SD), range
59.8 (± 12.3), 24.6 to 96.8
Sex
Female
209
41.1
Male
299
58.9
Race
White
401
78.9
Black
33
6.5
Asian
3
0.6
Mixed race
71
14.0
Body mass index (BMI), kg/m2
Mean (± SD)
26.8 (± 5.2)
History of smoking
75
14.8
Hypertension
234
46.1
BMI > 30 kg/m2
91
17.9
End-stage renal disease
11
2.2
Von Hippel-Lindau disease
1
0.2
TNM stage
I
189
37.2
II
124
24.4
III
61
12
IV
48
9.5
Unknown
86
16.9
Histological subtype
Clear-cell carcinoma
374
73.6
Papillary carcinoma
33
6.5
Chromophobe carcinoma
46
9.1
Collecting-duct carcinoma
2
0.4
Unclassified
52
10.2
Unknown
1
0.2
Radical or partial nephrectomy
500
98.4
Type of medical assistance
Public health
424
83.5
Private
14
2.8
Insurance company
69
13.6
Unknown
1
0.2
SD = standard deviation. Table 2 – Patient and tumor characteristics. Number
Percent SD = standard deviation. Characteristic Age, years
Mean (± SD), range
Sex
Female
Male
Race
White
Black
Asian
Mixed race
Body mass index (BMI), kg/m2
Mean (± SD)
History of smoking
Hypertension
BMI > 30 kg/m2
End-stage renal disease
Von Hippel-Lindau disease
TNM stage
I
II
III
IV
Unknown
Histological subtype
Clear-cell carcinoma
Papillary carcinoma
Chromophobe carcinoma
Collecting-duct carcinoma
Unclassified
Unknown
Radical or partial nephrectomy
Type of medical assistance
Public health
Private
Insurance company
Unknown
SD = standard deviation. 59.8 (± 12.3), 24.6 to 96.8 TNM stage
I
189
37.2
II
124
24.4
III
61
12
IV
48
9.5
Unknown
86
16.9
Histological subtype
Clear-cell carcinoma
374
73.6
Papillary carcinoma
33
6.5
Chromophobe carcinoma
46
9.1
Collecting-duct carcinoma
2
0.4
Unclassified
52
10.2
Unknown
1
0.2
Radical or partial nephrectomy
500
98.4
Type of medical assistance
Public health
424
83.5
Private
14
2.8
Insurance company
69
13.6
Unknown
1
0.2
SD = standard deviation Unknown 154 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil Table 3 – Exploratory analyses of TNM stage distribution according to clinical characteristics, presence of risk factors,
histological subtype, and type of medical assistance. Table 3 – Exploratory analyses of TNM stage distribution according to clinical characteristics, presence of risk factors,
histological subtype, and type of medical assistance. Table 3 – Exploratory analyses of TNM stage distribution according to clinical characteristics, presence of risk factors,
histological subtype, and type of medical assistance. TNM Stage (%)
p Value
Factors
I
II
III
IV
Age, mean (years)
58.9
59.6
61.3
60.6
0.578
Sex (%)
Female
45.1
33.1
11.4
10.3
0.302
Male
44.5
26.7
16.6
12.1
History of smoking (%)
24.4
23.0
35.3
27.0
0.089
Hypertension (%)
43.8
49.2
42.4
37.8
0.563
BMI > 30 kg/m2 (%)
18.1
21.1
11.7
17.0
0.478
End-stage renal disease (%)
2.1
1.7
1.7
0
0.799
Histological subtype (%)
Clear-cell
42.4
29.1
16.1
12.3
0.1626
Others
51.9
30.2
9.4
8.5
Medical assistance (%)
Public health
83.6
92.7
83.6
22.9
0.033
Private institution
16.4
7.3
16.4
77.1
BMI = body mass index. was less frequent among patients seen at public health
services (P = 0.033), in comparison with those seen
in private institutions. On the other hand, TNM stage
was not associated with any of the other patient de
mographic characteristics, tumor histological subtype
or risk factors (Table-3). of the patients presented with localized disease (37%
with stage I RCC). Characteristic The predominance of early-stage
disease is corroborated by the fact that abdominal
ultrasound (73.4%) or abdominal CT scans (19.1%)
were main diagnostic methods in contrast to physi
cal examination (3.0%) and intravenous urography
(0.6%). Tumor stage is considered one of the most
important prognostic factors in RCC (10,11). In our
study, the proportion of patients with metastatic dis
ease (9.5%) was similar to that reported in other clini
cal series (12,13). On the other hand, this proportion
is lower than expected from population-based studies,
in which patients with metastatic disease comprise
between 25% and 30% of cases upon presentation
(14). We did not find an association between TNM
stage and patient or tumor-related characteristics. It is generally acknowledged that men present with
more advanced disease at diagnosis. Aron et al., ana
lyzing 35,336 cases of RCC from the Surveillance,
Epidemiology, and End Results (SEER) registries
database from 1973 to 2004, reported that male gender
was associated with higher stage at presentation and COMMENTS This study provides a cross-sectional view of
RCC in Brazil, a country where no national incidence
rates for the disease are currently available (6,7). In
an attempt to overcome the paucity of data in our
country, SBU carried out a nationwide study on the
epidemiologic and clinical features of RCC in Brazil. As a result, a total of 508 patients were enrolled by
physicians from 50 different institutions. Most de
mographic characteristics of the study cohort were
quite similar to those in the existing literature (5),
since male patients predominated, the majority was
white, and the mean age was close to 60 years. Also,
similar to what occurs in developed countries, most 155 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil poorer overall survival, compared with women (15). However, we did not find the same association in our
sample. Similarly, there was no association between
stage and the presence of risk factors for RCC in the
present study. Smoking, hypertension and obesity are
the most frequently recognized risk factors for RCC
(16-18). Yet, the presence of risk factors did not seem
to correlate with stage at presentation in the present
series. tively, patients in the private sector could have under
gone more extensive imaging assessment, leading to
stage migration in this health care sector. The latter
hypothesis, however, does not seem to be supported
by our own finding of no significant difference in the
proportion of cases diagnosed by ultrasound or by
CT scan in the two sectors. Finally, the discrepancies
in the proportions of patients with metastatic disease
may have resulted from the play of chance or from
the lack of data for complete classification of nearly
17% of patients. Several studies have evaluated the prognos
tic value of histological subtype in RCC. Although
some studies have suggested that clear-cell histology
is associated with a poorer survival (19), Patard et
al. reported that in a multivariate analysis includ
ing TNM stage, Fuhrman nuclear grade and Eastern
Cooperative Oncology Group performance status
(PS), histological subtypes of RCC did not have an
independent prognostic significance (12). Although
we did not collect data on Fuhrman nuclear grade
and PS, we did not find an association between the
presence of clear-cell histology and more advanced
disease. An obvious limitation of our study design is
the lack of central pathologic review; therefore, we
cannot exclude the possibility of histological subtype
misclassification in some cases. COMMENTS The major drawback of the study was that
participation of the urologists was on a volunteer
basis and it did not include all the cases seen during
the period. All SBU members were invited by mail to
participate in this epidemiological survey. From the 76
institutions affiliated with the SBU, 50 (65.8%) took
part in this study. Although we had the participation
of institutions from 14 Brazilian States, 72.6% of
patients that were enrolled in the study were from one
single State, São Paulo. Probably due the fact that São
Paulo has the majority (31.6%) of total SBU affiliated
Institutions (24 of 76 institutions).i Although it could be filled out in a relative
short period of time, the questionnaire used in this
survey was rather long and required the review of
medical charts, laboratory findings, imaging studies
and pathology reports. Perhaps the use of shorter
questionnaires aiming at more specific questions will
be better accepted. Other strategies to increase partici
pation of Brazilian urologists of the whole country in
epidemiological surveys should be discussed. We found an association between TNM stage
and type of medical assistance. Surprisingly, there was
a higher proportion of early stage disease, especially
stage II, among patients seen in public health care
facilities, whereas patients seen at private institutions
were more likely to present with metastatic disease
(P = 0.033). Such findings are in contrast to those
obtained in another epidemiologic study conducted
by SBU. In that cross-sectional survey of prostate
cancer in the State of São Paulo, 30% of the patients
seen in public institutions were diagnosed with lo
cally advanced or metastatic disease, compared with
21% of patients treated in the private sector (20). The
reasons for these findings are not clear, and we may
only speculate as to possible explanations. One reason
for the higher frequency of metastatic disease in the
private sector could be referral bias, leading to more
patients with advanced disease in the public institu
tions being referred to medical oncologists or pallia
tive care. Another explanation could be the migration
of patients diagnosed with metastatic disease to the
private sector seeking for faster assistance. Alterna Medical associations may provide many
opportunities for productive research, especially for
physicians not currently affiliated to academic insti
tutions. COMMENTS Despite the limitations of the method, this
study represents the largest collection of RCC cases
in Brazil to date and shows that a registry is feasible
and may provide valuable information regarding RCC
in Brazil. We hope the present work will stimulate
further participation of Brazilian urologists in future
projects. CONFLICT OF INTEREST None declared. 156 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil dividual survival for patients with renal cell carcinoma. J Clin Oncol. 2002; 20: 1368-74. REFERENCES 1. Hollingsworth JM, Miller DC, Daignault S, Hollen
beck BK: Rising incidence of small renal masses: a
need to reassess treatment effect. J Natl Cancer Inst. 2006; 98: 1331-4. 12. Patard JJ, Leray E, Rioux-Leclercq N, Cindolo L,
Ficarra V, Zisman A, et al.: Prognostic value of histo
logic subtypes in renal cell carcinoma: a multicenter
experience. J Clin Oncol. 2005; 23: 2763-71. 13. Scoll BJ, Wong YN, Egleston BL, Kunkle DA, Saad
IR, Uzzo RG: Age, tumor size and relative survival of
patients with localized renal cell carcinoma: a surveil
lance, epidemiology and end results analysis. J Urol. 2009; 181: 506-11. 2. Mathew A, Devesa SS, Fraumeni JF Jr, Chow WH:
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database. Eur Urol. 2008; 54: 133-40. y
5. McLaughlin JK, Lipworth L, Tarone RE: Epidemio
logic aspects of renal cell carcinoma. Semin Oncol. 2006; 33: 527-33. 16. Bergström A, Hsieh CC, Lindblad P, Lu CM, Cook NR,
Wolk A: Obesity and renal cell cancer--a quantitative
review. Br J Cancer. 2001; 85: 984-90. 6. Brasil. Ministério da Saúde. Instituto Nacional do
Câncer. Estimativa 2008: Incidência de Câncer no
Brasil. Available at http://www.inca.gov.br/estima
tiva/2008 (Acessed 21/10/2008). 17. Hunt JD, van der Hel OL, McMillan GP, Boffetta P,
Brennan P: Renal cell carcinoma in relation to cigarette
smoking: meta-analysis of 24 studies. Int J Cancer. 2005; 114: 101-8. 7. Mirra AP, Latorre MRDO, Veneziano DB. (ed.); In
cidência de Câncer no Município de São Paulo, Brasil
1997-1998: mortalidade de Câncer no Município de
São Paulo, Brasil: tendência no Período de 1969-1998. Brasília, DF: Ministério da Saúde, 2001.i 18. EDITORIAL COMMENT clear cell carcinoma; virtually all patients underwent
nephrectomy (radical or partial) for management of
their RCC. The predominance of early-stage disease
is corroborated by the fact that abdominal ultrasound
(73.4%) or abdominal CT scans (19.1%) were main
diagnostic methods in contrast to physical exami
nation (3.0%) and intravenous urography (0.6%). Unfortunately, 85% of the patients in the study were
from the southeast region of the country. Another
obvious limitation of the study design is also the lack
of a central pathologic review. A possible reason for
the higher frequency of metastatic disease in private
sector could be a referral bias, leading patients with
more advanced disease to look for other specialists
in private health care sector. Our approach to kidney cancer has changed
considerably in recent years. More and more tumors
are now diagnosed at earlier stages. For example,
smaller masses are often benign justifying the use
of needle biopsy to confirm the diagnosis. This pro
cedure was unthinkable in previous eras when most
tumors were considered malignant until proved oth
erwise after the analysis of abdominal ultrasound or
CT scans. Similarly, this increased the indications for
partial nephrectomy, as well as less aggressive meth
ods, such as cryoablation. The authors presented an
epidemiologic study on renal cell carcinoma in Brazil. It is very important that we can consult these data to
plan a strategy to treat our patients. Three-quarters
of patients had localized disease (i.e., TNM stage I
and II) and the most common histological type was Dr. Antonio A. Ornellas
National Cancer Institute
Rio de Janeiro, RJ
E-mail: ornellasa@hotmail.com CONFLICT OF INTEREST van Dijk BA, Schouten LJ, Kiemeney LA, Goldbohm
RA, van den Brandt PA: Relation of height, body mass,
energy intake, and physical activity to risk of renal cell
carcinoma: results from the Netherlands Cohort Study. Am J Epidemiol. 2004; 160: 1159-67. 8. Sobin LH, Wittekind C: TNM Classification of Ma
lignant Tumours. 6th ed. Hoboken, New Jersey: John
Wiley & Sons. 2002. 19. Amin MB, Amin MB, Tamboli P, Javidan J, Stricker
H, de-Peralta Venturina M, et al.: Prognostic impact
of histologic subtyping of adult renal epithelial neo
plasms: an experience of 405 cases. Am J Surg Pathol. 2002; 26: 281-91. 9. Kovacs G, Akhtar M, Beckwith BJ, Bugert P, Cooper
CS, Delahunt B, et al.: The Heidelberg classification
of renal cell tumours. J Pathol. 1997; 183: 131-3. 20. Nardi AC, Moreira Filho DC, Nardozza, A ; Rios, LAS,
Zequi, SC. Perfil do Câncer de Próstata no Estado de
São Paulo- Detectado pela Sociedade Brasileira de
Urologia-Secção São Paulo. 1st. ed. São Paulo: Lado
a Lado Comunicação ed, 2005. v. 1. pp. 117. 10. Kattan MW, Reuter V, Motzer RJ, Katz J, Russo P:
A postoperative prognostic nomogram for renal cell
carcinoma. J Urol. 2001; 166: 63-7. 11. Zisman A, Pantuck AJ, Dorey F, Chao DH, Gitlitz BJ,
Moldawer N, et al.: Mathematical model to predict in Accepted after revision:
October 18, 2009 Accepted after revision:
October 18, 2009 Correspondence address:
Dr. Stênio de Cássio Zequi
Sociedade Brasileira de Urologia
Rua Bambina, 153
Rio de Janeiro, RJ, 22251-050, Brazil
Fax: + 55 21 2246-4194
E-mail: steniozequi@uol.com.br 157 Epidemiologic Characteristics of Renal Cell Carcinoma in Brazil REPLY BY THE AUTHORS Although the vast majority of the cases were
from the southwest region, it reflects the participation
rates of our urological community. The opportunities
for study participation were rigorously the same for all
members of the Brazilian Society of Urology (SBU). We must remember that more than half of SBU mem
bers are in the Brazil southeast states. Based on this
inedited data, the SBU must develop new approaches to attract more Brazilian urologists for National trials
and surveys. Although the lack of central pathologic re
view be a limitation of the study design, as already
discussed in the paper, it may represents a new op
portunity to SBU to promote an approach with other
National medical societies, specially the Brazilian
Society of Pathology. The Authors 158
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Association between Myopia and Glaucoma; A Cross-sectional Study
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Pakistan journal of health sciences
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cc-by
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*Corresponding Author: University Medical and Dental Collage, Faisalabad,
Pakistan University Medical and Dental Collage, Faisalabad,
Pakistan naseerfatima08@gmail.com naseerfatima08@gmail.com th
Received Date: 30 March, 2023
rd
Acceptance Date: 23 April, 2023
th
Published Date: 30 April, 2023 th
Received Date: 30 March, 2023
rd
Acceptance Date: 23 April, 2023
th
Published Date: 30 April, 2023 th
Received Date: 30 March, 2023
rd
Acceptance Date: 23 April, 2023
th
Published Date: 30 April, 2023 th
Received Date: 30 March, 2023
rd
Acceptance Date: 23 April, 2023
th
Published Date: 30 April, 2023 1
2*
3
4
4
Maryam Jabbar , Naseer Fatima , Muhammad Siddique , Faisal Rashid , Faryal Qureshi and 2*
3
4
4
5
atima , Muhammad Siddique , Faisal Rashid , Faryal Qureshi and Abdul Mateen Bodla ¹The University of Faisalabad, Faisalabad, Pakistan
²University Medical and Dental Collage, Faisalabad, Pakistan
³Sheikh Zayed Medical College/Hospital Rahim Yar Khan, Rahim Yar Khan, Pakistan
⁴Ophthalmology Department, Services Hospital, Lahore, Pakistan
⁵Optometrist, Ophthalmology Department, Services Hospital Lahore
⁶ Pak Red Crescent Medical & Dental College, Kasur, Pakistan Key Words: Myopia is a major cause of worldwide avoidable blindness and its prevalence increasing rapidly. Objective: To investigate the prevalence of glaucomatous optic nerve damage with various
myopia levels as well as the relationship between myopia and glaucoma. Methods: A multi-
centered cross-sectional study was conducted from January 2022 to July 2022. 250 individuals
between the ages of 40 and 65 were recruited using non-probability purposive sampling
technique. Mild, moderate and severe degree of myopia who had never had any surgery were
included. All subjects underwent refraction and the optic disc ratio was assessed by slit lamp
biomicroscopy, perimetry was performed to evaluate the visual ïeld defects and IOP was
determined using air puff tonometer. Frequency of glaucoma indicated by presence of visual
ïeld defects, glaucomatous optic disc. SPSS software was used for data analysis. Results: 145
(58%) of the 250 total subjects were female and 105(42%) were male. 67 (26.8%) people had
refractive errors of mild myopia up to 3D. While 85 (34%) had a severe degree of myopia
(refractive error greater than 6D) and 98 (39.2%) had a moderate degree. No intragroup's
intraocular pressure showed a signiïcantly distinct conïguration. Age-related VF anomalies (a
larger blind region, a vertical cup to disc ratio, and an unjustiïed defect) were associated with
both glaucoma and high myopia. Findings from the study indicated that glaucoma risk increased
for those with high myopia (p=0.001). Conclusion: High myopia is strongly associated with
glaucomatous changes and a high prevalence of optic disc damage. Myopia, Glaucoma, Optic Disc, Refraction, Visual
acuity DOI: https://doi.org/10.54393/pjhs.v4i04.667 DOI: https://doi.org/10.54393/pjhs.v4i04.667 How to Cite: Jabbar, M. ., Fatima, N. ., Siddique, M. ., Rashid, F. .,
Qureshi, F. ., & Mateen Bodla, A. . (2023). Association
between Myopia and Glaucoma; A Cross-sectional
Study: Association between Myopia and Glaucoma. Pakistan Journal of Health Sciences, 4(04). https://doi.org/10.54393/pjhs.v4i04.667 Association between Myopia and Glaucoma; A Cross-sectional 1
2*
3
4
4
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Maryam Jabbar , Naseer Fatima , Muhammad Siddique , Faisal Rashid , Faryal Qureshi and Abdul Mateen Bodla I N T R O D U C T I O N discovery of new risk factors might enable earlier and more
thorough screening of populations at risk. Additionally, it
might shed light on the pathophysiology of the disease [9]. Myopia is the most typical type of vision impairment that
affects people globally [10-12], and in recent decades, its
incidence has gone up dramatically, especially in Asian
countries [13-15]. There are several explanations for the
rising prevalence of myopia, including more study and
near-work time, decreased outdoor time, greater
education levels, and genetic factors [16-19]. Iris colour and Glaucoma is the most common cause of permanent
blindness in the world [1]. Even if individuals with visual
ïeld anomalies are not aware of their diagnosis, it can have
a negative inðuence on their quality of life [2,3]. The most
prevalent type of the disease is POAG in various
populations [4].The main therapy objective of therapy is to
lower the IOP which cause no further glaucomatous
changes [5, 6]. Although the pathophysiology of glaucoma
is currently unknown, genetic and environmental factors
are likely to have an impact on it [7, 8]. Therefore, the PJHS VOL. 4 Issue. 4 April 2023 Copyright © 2023. PJHS, Published by Crosslinks International Publishers 133 DOI: https://doi.org/10.54393/pjhs.v4i04.667
Association between Myopia and Glaucoma Jabbar M et al., mm Hg were excluded from this study. All subjects
underwent visual acuity assessment, refraction evaluation
and slit lamp biomicroscopy examination. In addition
tonometry and perimetry was also performed. The visual
acuity of each individual was tested using a logMar chart
which is a standardized chart used to test visual acuity. Then perform a refraction analysis (objective and
subjective). To achieve the best corrected visual acuity,
objective refraction was performed using an auto
refractometer in conjunction with subjective refraction. auto-refractometer, which is a computerized instrument
that measures the refractive error of the eye without any
input from the patient. Subjective refraction was
performed by asking the patient to choose between
different lenses to obtain the best possible visual acuity. A
slit lamp biomicroscopy to examine the anterior and
posterior segments of the eye in details. Its was also used
to evaluate the optic disc ratio and identify glaucomatous
optic discs. Visual ïeld abnormalities were assessed using
perimetry. Intraocular pressure (IOP) was measured using
an air puff tonometer. The data were compiled using a self-
structured proforma, and SPSS software was used for
descriptive statistics and chi square analysis. I N T R O D U C T I O N A p-value of
0.05 or less was regarded as signiïcant. myopia development are also correlated [20]. Numerous
ocular pathologies, such as cataracts [21] and retinal
detachment [22] have been linked to high myopia (6 D). Myopia may or may not be a risk factor or a predictor for the
initial onset and progression of glaucomatous optic nerve
injury, according to the results of earlier investigations [23-
25]. The optic nerve head (ONH), a structure in the posterior
ocular fundus, mediates the entry and exit of the retinal
blood vessels as well as the ejection of the retinal ganglion
cell axons. It is located 4-5 mm nasally and somewhat
superiorly from the fovea in emmetropic eyes (mean disc-
fovea angle) [26, 27]. Bruch's membrane (BM), the choroid,
and the peripapillary scleral ðange, respectively,
constitute the inner, middle, and external layers of the ONH
canal anatomically [28]. The phrase "optic disc" describes
the entire area, including the lamina cribrosa at its base,
and can be used to describe the size and shape of the
structure. The average inter-individual variability in optic
disc size among Caucasians is 1:7 [29]. The optic disc in
extremely myopic eyes enlarges with longer axial length or
greater myopic refractive error, starting at a cut-off value
of roughly eight diopters or an axial length of about 26.5 mm
[30]. The size of the disc is likely not a determinant in the
development of glaucoma in eyes that are not severely
myopic, as there are typically no noticeable disc size
differences between primary and secondary open-angle
glaucoma groups [31]. A greater incidence of
glaucomatous optic neuropathy is associated with the size
and existence of a secondary macrodisc in eyes with
severe myopia [32]. This cross-sectional study's objectives
were to assess the relationship between myopia and
glaucoma as well as the prevalence of glaucomatous optic
nerve injury among myopic individuals. The ïndings of this
study may help determine whether or not myopia indicates
a signiïcant risk for glaucoma, which is the primary cause
of permanent blindness worldwide. Even if patients are
ignorant of their visual impairment, myopia has a severe
impact on their quality of life. R E S U L T S A multi-centered cross sectional study was conducted
from January 2022 to July 2022. 250 people between the
ages of 40 and 65 were recruited using a non-probability
purposive sampling technique. Age distribution was among
5 groups (Figure 1) as follow 41-45 years, 46-50 years, 51-55
years, 56-60 years and 61-65years. 90
80
70
60
50
40
30
20
10
0
41-45 Years
46-50 Years
51-55 Years
56-60 Years
61-65 Years
80
61
49
29
31
Figure 1: Age Distribution
In this study, 145(58%) of the 250 total subjects were female
and 105(42%) were male (Figure 2). Figure 2: Gender Distribution
Female
58%
Male
42%
Female
Male
hed by Crosslinks International Publishers
134 90
80
70
60
50
40
30
20
10
0
41-45 Years
46-50 Years
51-55 Years
56-60 Years
61-65 Years
80
61
49
29
31
Figure 1: Age Distribution
In this study, 145(58%) of the 250 total subjects were female
and 105(42%) were male (Figure 2). D I S C U S S I O N The latest ïndings are consistent with signiïcant
population-based research on myopia and glaucoma from
decades previously. According to the BMES [33], glaucoma
has been connected to the development of matched optic
disc cupping with rim thinning (cup-to-disc ratio 0.7, or
cup-to-disc asymmetry 0.3), as well as detectable visual
ïeld loss on automated perimetry. The most recent
ïndings concur with signiïcant population-based
research on glaucoma and myopia that were done decades
ago. The development of matched optic disc cupping with
rim thinning (cup-to-disc ratio 0.7, or cup-to-disc
asymmetry 0.3) and detectable visual ïeld loss on
automated perimetry have both been associated with
glaucoma, according to the BMES [33]. According to the
Tajimi Study in Japan and the Aravind Comprehensive Eye
Survey in India, there is a link between POAG as evaluated by All authors have read and agreed to the published version of
the manuscript. C o n i c t s o f I n t e r e s t
The authors declare no conðict of interest. S o u r c e o f F u n d i n g S o u r c e o f F u n d i n g The authors received no ïnancial support for the research,
authorship and/or publication of this article. C O N C L U S I O N S High myopia is strongly associated with glaucomatous
changes and a higher prevalence of optic disc damage
compared to low or moderate myopia. Older age was linked to both glaucoma and high myopia-
related VF abnormalities (a larger blind area, a vertical disc
ratio and an unidentiïed defect). According to study
ïndings, people with high myopia were more likely to get
glaucoma (p 0.001). A u t h o r s C o n t r i b u t i o n
Conceptualization: MJ
Methodology: NF, FQ
Formal analysis: MS
Writing-review and editing: FR, AMB A u t h o r s C o n t r i b u t i o n
Conceptualization: MJ
Methodology: NF, FQ
Formal analysis: MS
Writing-review and editing: FR, AMB M E T H O D S There was a signiïcant
relationship at 5% signiïcant level between high myopia
and glacuomatous changes of respondents (Table 1). According to the present study, frequency of glaucoma, as
indicated by presence of visual ïeld abnormalities,
glaucomatous optic disc and may or may not raise IOP. Chi-
square statistics were used to examine degree of myopia
and glacuomatous changes. There was a signiïcant
relationship at 5% signiïcant level between high myopia
and glacuomatous changes of respondents (Table 1). Table 1: Degree of Myopia and Glaucoma Association (Optic Disc
Enlargement, visual ïeld defects and raised IOP) Degrees of
myopia
Optic Disc
Enlargement
Visual Field
Defects
Raised
IOP
p-value
g
Mild Myopia
Moderate Myopia
High Myopia
5
53
91
2
29
51
6
48
85
0.13
0.06
0.001 M E T H O D S A multi-centered cross sectional study was conducted
from January 2022 to July 2022. 250 subjects between the
ages of 40 and 65 were recruited using a non-probability
purposive sampling technique. With the support of the
Raosoft sample size calculator, the sample for this study
has been estimated. Mild (up to 3D) moderate (3D to 6D) and
severe degree (more than 6D) of myopia who had never had
a cataract or refractive surgery were included. This study
omitted pathological myopia, secondary myopia, ocular
illnesses (amblyopia, strabismus, congenital ocular
disease), and history of ocular interventions (LASIK,
cataract surgery). Patients whose IOP was greater than 40 Figure 1: Age Distribution
In this study, 145(58%) of the 250 total subjects were female
and 105(42%) were male (Figure 2). Figure 2: Gender Distribution
Female
58%
Male
42%
Female
Male Figure 2: Gender Distribution
Female
58%
Male
42%
Female
Male Figure 2: Gender Distribution PJHS VOL. 4 Issue. 4 April 2023 Copyright © 2023. PJHS, Published by Crosslinks International Publishers 134 DOI: https://doi.org/10.54393/pjhs.v4i04.667
Association between Myopia and Glaucoma Jabbar M et al., In current study, 67 (26.8%) people had refractive errors of
mild myopia up to 3 D. While 85 (34%) had a severe degree of
myopia (refractive error greater than 6D) and 98 (39.2%) had
a moderate degree (Figure 3). a thorough ophthalmologic exam and myopia greater than
one degree [34, 35]. According to the BES in China [33],
extremely high myopia (higher than 6 D), the onset of
glaucomatous optic nerve, anomalies of the visual ïeld,
and increased IOP have all been connected. The Rotterdam
Eye Study in the Netherlands found a connection between
adverse myopia greater than 4 D and glaucomatous visual
ïeld loss [36, 37]. The ïndings of the present study suggest
that deïned to high myopia may be an indicator of risk for
glaucoma, while low to moderate myopia may not have a
signiïcant impact on glaucoma. If myopia has been
categorised as low to moderate myopia and marked or high
myopia after just a myopic refractive error of 6 diopters. a thorough ophthalmologic exam and myopia greater than
one degree [34, 35]. According to the BES in China [33],
extremely high myopia (higher than 6 D), the onset of
glaucomatous optic nerve, anomalies of the visual ïeld,
and increased IOP have all been connected. M E T H O D S The Rotterdam
Eye Study in the Netherlands found a connection between
adverse myopia greater than 4 D and glaucomatous visual
ïeld loss [36, 37]. The ïndings of the present study suggest
that deïned to high myopia may be an indicator of risk for
glaucoma, while low to moderate myopia may not have a
signiïcant impact on glaucoma. If myopia has been
categorised as low to moderate myopia and marked or high
myopia after just a myopic refractive error of 6 diopters. The results of the current study are consistent with a 30
000-person eye survey undertaken in Malmö before the
Early Manifest Glaucoma Experiment. According to the
Malmö Eye Survey [38], as myopia increased, so did the
prevalence of glaucoma. At initial intraocular pressure
levels, the relationship between myopia and glaucoma was
strong, but it gradually weakened as intraocular pressure
climbed. People with high myopia may need more frequent
eye health management because they may be more
susceptible to glaucoma and optic nerve damage. Individuals with high myopia should undergo thorough eye
exams frequently to check for any warning signs of optic
nerve damage or glaucomatous changes. 120
100
80
60
40
20
10
0
67
85
98
MILD
MYOPIA
MODERATE
MYOPIA
HIGH
MYOPIA
DEGREE OF MYOPIA
Figure 3: Degree of Myopia 120
100
80
60
40
20
10
0
67
85
98
MILD
MYOPIA
MODERATE
MYOPIA
HIGH
MYOPIA
DEGREE OF MYOPIA
Figure 3: Degree of Myopia y p
j
y p
p
The results of the current study are consistent with a 30
000-person eye survey undertaken in Malmö before the
Early Manifest Glaucoma Experiment. According to the
Malmö Eye Survey [38], as myopia increased, so did the
prevalence of glaucoma. At initial intraocular pressure
levels, the relationship between myopia and glaucoma was
strong, but it gradually weakened as intraocular pressure
climbed. People with high myopia may need more frequent
eye health management because they may be more
susceptible to glaucoma and optic nerve damage. Individuals with high myopia should undergo thorough eye
exams frequently to check for any warning signs of optic
nerve damage or glaucomatous changes. Figure 3: Degree of Myopia According to the present study, frequency of glaucoma, as
indicated by presence of visual ïeld abnormalities,
glaucomatous optic disc and may or may not raise IOP. Chi-
square statistics were used to examine degree of myopia
and glacuomatous changes. R E F E R E N C E S Gan K, Liu Y, Stagg B, Rathi S, Pasquale LR, Damji K. Telemedicine for glaucoma: guidelines and
recommendations. Telemedicine and e-Health. 2020
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[22] Bullimore MA, Ritchey ER, Shah S, Leveziel N, Bourne
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] Lee K, Yang H, Kim JY, Seong GJ, Kim CY, Bae HW. Risk factors associated with structural progression
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[23] Morgan IG and Jan CL. 10.1016/j.ophtha.2006.03.059 Ramakrishnan R, Nirmalan PK, Krishnadas R,
Thulasiraj RD, Tielsch JM, Katz J, et al. Glaucoma in a
rural population of southern India: the Aravind
comprehensive eye survey. Ophthalmology. 2003
Aug; 110(8): 1484-90. doi: 10.1016/S0161-6420(03)
00564-5
[35] Seol BR, Park KH, Jeoung JW. Optic disc tilt and
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May; 60(6): 2127-33. doi: 10.1167/iovs.18-25839
[25] Czudowska MA, Ramdas WD, Wolfs RC, Hofman A, De
Jong PT, Vingerling JR, et al. Incidence of
glaucomatous visual ïeld loss: a ten-year follow-up
from the Rotterdam Study. Ophthalmology. 2010 Sep;
117(9): 1705-12. doi: 10.1016/j.ophtha.2010.01.034
[36] Jonas RA, Wang YX, Yang H, Li JJ, Xu L, Panda-Jonas
S, et al. Optic disc-fovea angle: the Beòing Eye Study
2011. PLoS One. 2015 Nov; 10(11): e0141771. doi:
10.1371/journal.pone.0141771
[26] Jiang X, Varma R, Wu S, Torres M, Azen SP, Francis
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[37] Jonas RA, Wang YX, Yang H, Li JJ, Xu L, Panda-Jonas
S, Jonas JB. Optic disc-fovea distance, axial length
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[27] Leske MC, Heòl A, Hussein M, Bengtsson B, Hyman L,
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glaucoma trial. Archives of ophthalmology. 2003 Jan;
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[38] Wang YX, Yang H, Luo H, Hong SW, Gardiner SK,
Jeoung JW, et al. Peripapillary scleral bowing
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[28] Omodaka K, An G, Tsuda S, Shiga Y, Takada N, Kikawa
T, et al. Classiïcation of optic disc shape in glaucoma
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https://openalex.org/W2810164683
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https://peerj.com/articles/5071v0.2/submission
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English
| null |
Peer Review #1 of "Methods matter: the relationship between strength and hypertrophy depends on methods of measurement and analysis (v0.1)"
| null | 2,018
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cc-by
| 13,018
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Manuscript to be reviewed Methods matter: The relationship between strength a
hypertrophy depends on methods of measurement an
analysis
Andrew D Vigotsky Corresp., 1 , Brad J Schoenfeld 2 , Christian Than 3 , J Mark Brown 3
1 Department of Biomedical Engineering, Northwestern University, Evanston, Illinois, United States of America
2 Department of Health Sciences, City University of New York, Herbert H. Lehman College, Bronx, New York, United States
3 School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia
Corresponding Author: Andrew D Vigotsky
Email address: avigotsky@gmail.com Methods matter: The relationship between strength and
hypertrophy depends on methods of measurement and
analysis 1 Department of Biomedical Engineering, Northwestern University, Evanston, Illinois, United States of America
2 Department of Health Sciences, City University of New York, Herbert H. Lehman College, Bronx, New York, United States
3 School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia
Corresponding Author: Andrew D Vigotsky
Email address: avigotsky@gmail.com 1 Department of Biomedical Engineering, Northwestern University, Evanston, Illinois, United States of America
2 Department of Health Sciences, City University of New York, Herbert H. Lehman College, Bronx, New York, United States
3 School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia
Corresponding Author: Andrew D Vigotsky
Email address: avigotsky@gmail.com Corresponding Author: Andrew D Vigotsky
Email address: avigotsky@gmail.com Corresponding Author: Andrew D Vigotsky
Email address: avigotsky@gmail.com Purpose: The relationship between changes in muscle size and strength may be affected by both
measurement and statistical approaches, but their effects have not been fully considered or quantified. Therefore, the purpose of this investigation was to explore how different methods of measurement and
analysis can affect inferences surrounding the relationship between hypertrophy and strength gain. Methods: Data from a previous study – in which participants performed eight weeks of elbow flexor
training, followed by an 8-week period of detraining– were reanalyzed using different statistical models,
including standard between-subject correlations, analysis of covariance, and hierarchical linear modeling. Results: The associative relationship between strength and hypertrophy is highly dependent upon both
method/site of measurement and analysis; large differences in variance accounted for (VAF) by the
statistical models were observed (VAF = 0–24.1%). Different sites and measurements of muscle size
showed a range of correlations coefficients with one another (r = 0.326–0.945). Finally, exploratory
analyses revealed moderate-to-strong relationships between within-individual strength-hypertrophy
relationships and strength gained over the training period (ρ = 0.36–0.55). Conclusions: Methods of measurement and analysis greatly influence the conclusions that may be
drawn from a given dataset. Analyses that do not account for inter-individual differences may
underestimate the relationship between hypertrophy and strength gain, and different methods of
assessing muscle size will produce different results. It is suggested that robust experimental designs and
analysis techniques, which control for different mechanistic sources of strength gain and inter-individual
differences (e.g., muscle moment arms, muscle architecture, activation, and normalized muscle force),
be employed in future investigations. PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 25
ABSTRACT
26
Purpose: The relationship between changes in muscle size and strength may be affected by bo
27
measurement and statistical approaches, but their effects have not been fully considered
28
quantified. Therefore, the purpose of this investigation was to explore how different methods
29
measurement and analysis can affect inferences surrounding the relationship between hypertrop
30
and strength gain. 31
Methods: Data from a previous study – in which participants performed eight weeks of elbo
32
flexor training, followed by an 8-week period of detraining – were reanalyzed using differe
33
statistical models, including standard between-subject correlations, analysis of covariance, a
34
hierarchical linear modeling. 35
Results: The associative relationship between strength and hypertrophy is highly dependent up
36
both method/site of measurement and analysis; large differences in variance accounted for (VA
37
by the statistical models were observed (VAF = 0–24.1%). Different sites and measurements
38
muscle size showed a range of correlations coefficients with one another (r = 0.326–0.945
39
Finally, exploratory analyses revealed moderate-to-strong relationships between within-individu
40
strength-hypertrophy relationships and strength gained over the training period (ρ = 0.36–0.55)
41
Conclusions: Methods of measurement and analysis greatly influence the conclusions that may
42
drawn from a given dataset. Analyses that do not account for inter-individual differences m
43
underestimate the relationship between hypertrophy and strength gain, and different methods
44
assessing muscle size will produce different results. It is suggested that robust experimen
45
designs and analysis techniques, which control for different mechanistic sources of strength ga
46
and inter-individual differences (e.g., muscle moment arms, muscle architecture, activation, a
47
normalized muscle force), be employed in future investigations. 48 Manuscript to be reviewed 1
Methods Matter: The relationship between strength and hypertrophy depends on meth
2
of measurement and analysis
3
4
Andrew D. Vigotsky1, Brad J. Schoenfeld2, Christian Than3, J. Mark Brown3
5
6
1 Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA
7
2 Department of Health Science, Lehman College, Bronx, NY, USA
8
3 School of Biomedical Sciences, The University of Queensland, St. Lucia 4072, Australia
9
10
11
12
13
14
15
16
17
18
Corresponding author:
19
Andrew Vigotsky
20
ORCID: 0000-0003-3166-0688
21
avigotsky@gmail.com
22
23
Running title: Methods Matter 1
Methods Matter: The relationship between strength and hypertrophy depends on methods
2
of measurement and analysis PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) 25
ABSTRACT 26
Purpose: The relationship between changes in muscle size and strength may be affected by both
27
measurement and statistical approaches, but their effects have not been fully considered or
28
quantified. Therefore, the purpose of this investigation was to explore how different methods of
29
measurement and analysis can affect inferences surrounding the relationship between hypertrophy
30
and strength gain. 31
Methods: Data from a previous study – in which participants performed eight weeks of elbow
32
flexor training, followed by an 8-week period of detraining – were reanalyzed using different
33
statistical models, including standard between-subject correlations, analysis of covariance, and
34
hierarchical linear modeling. 31
Methods: Data from a previous study – in which participants performed eight weeks of elbow
32
flexor training, followed by an 8-week period of detraining – were reanalyzed using different
33
statistical models, including standard between-subject correlations, analysis of covariance, and
34
hierarchical linear modeling. 35
Results: The associative relationship between strength and hypertrophy is highly dependent upon
36
both method/site of measurement and analysis; large differences in variance accounted for (VAF)
37
by the statistical models were observed (VAF = 0–24.1%). Different sites and measurements of
38
muscle size showed a range of correlations coefficients with one another (r = 0.326–0.945). 39
Finally, exploratory analyses revealed moderate-to-strong relationships between within-individual
40
strength-hypertrophy relationships and strength gained over the training period (ρ = 0.36–0.55). 41
Conclusions: Methods of measurement and analysis greatly influence the conclusions that may be
42
drawn from a given dataset. Analyses that do not account for inter-individual differences may
43
underestimate the relationship between hypertrophy and strength gain, and different methods of
44
assessing muscle size will produce different results. It is suggested that robust experimental
45
designs and analysis techniques, which control for different mechanistic sources of strength gain
46
and inter-individual differences (e.g., muscle moment arms, muscle architecture, activation, and
47
normalized muscle force), be employed in future investigations. 35
Results: The associative relationship between strength and hypertrophy is highly dependent upon
36
both method/site of measurement and analysis; large differences in variance accounted for (VAF)
37
by the statistical models were observed (VAF = 0–24.1%). Different sites and measurements of
38
muscle size showed a range of correlations coefficients with one another (r = 0.326–0.945). 25
ABSTRACT 39
Finally, exploratory analyses revealed moderate-to-strong relationships between within-individual
40
strength-hypertrophy relationships and strength gained over the training period (ρ = 0.36–0.55). 41
Conclusions: Methods of measurement and analysis greatly influence the conclusions that may be
42
drawn from a given dataset. Analyses that do not account for inter-individual differences may
43
underestimate the relationship between hypertrophy and strength gain, and different methods of
44
assessing muscle size will produce different results. It is suggested that robust experimental
45
designs and analysis techniques, which control for different mechanistic sources of strength gain
46
and inter-individual differences (e.g., muscle moment arms, muscle architecture, activation, and
47
normalized muscle force), be employed in future investigations. 35
Results: The associative relationship between strength and hypertrophy is highly dependent upon
36
both method/site of measurement and analysis; large differences in variance accounted for (VAF)
37
by the statistical models were observed (VAF = 0–24.1%). Different sites and measurements of
38
muscle size showed a range of correlations coefficients with one another (r = 0.326–0.945). 39
Finally, exploratory analyses revealed moderate-to-strong relationships between within-individual
40
strength-hypertrophy relationships and strength gained over the training period (ρ = 0.36–0.55). 41
Conclusions: Methods of measurement and analysis greatly influence the conclusions that may be
42
drawn from a given dataset. Analyses that do not account for inter-individual differences may
43
underestimate the relationship between hypertrophy and strength gain, and different methods of
44
assessing muscle size will produce different results. It is suggested that robust experimental
45
designs and analysis techniques, which control for different mechanistic sources of strength gain
46
and inter-individual differences (e.g., muscle moment arms, muscle architecture, activation, and
47
normalized muscle force), be employed in future investigations. 35
Results: The associative relationship between strength and hypertrophy is highly dependent upon
36
both method/site of measurement and analysis; large differences in variance accounted for (VAF)
37
by the statistical models were observed (VAF = 0–24.1%). Different sites and measurements of
38
muscle size showed a range of correlations coefficients with one another (r = 0.326–0.945). 39
Finally, exploratory analyses revealed moderate-to-strong relationships between within-individual
40
strength-hypertrophy relationships and strength gained over the training period (ρ = 0.36–0.55). 41
Conclusions: Methods of measurement and analysis greatly influence the conclusions that may be
42
drawn from a given dataset. 50
Introduction 51
The combined actions of neural input, muscles, and the joint(s) about which those muscles
52
act serve to produce sufficient endpoint force for physical function, allowing the performance of
53
activities of daily living, as well as the spectrum of athletic endeavors. Due to the complexity of
54
the neuromuscular and musculoskeletal systems, many factors can influence strength, including,
55
but not limited to, muscle moment arm, muscle size, activation, muscle architecture, and
56
normalized muscle force (or specific tension) (Vigotsky et al. 2015). Muscle size is of particular
57
interest, as 1) it is highly plastic (Fluck & Hoppeler 2003) and 2) a clear positive relationship exists
58
between baseline muscle cross-sectional area (CSA) and strength, with greater CSAs correlating
59
with greater strength capacities (Maughan & Nimmo 1984; Maughan et al. 1984; Schantz et al. 60
1983). However, this relationship is not necessarily linear, as several additional factors
61
interactively influence strength capacity (Vigotsky et al. 2015); studying the role of and
62
relationship between muscle size and strength is therefore less straightforward under longitudinal
63
contexts. 64
While the cross-sectional correlation between muscle mass and strength remains well-
65
established, some researchers have recently challenged the belief that resistance training (RT) -
66
induced hypertrophy significantly impacts the ability to produce force, claiming improvements in
67
these outcomes are separate and unrelated adaptations (Buckner et al. 2016a). Indeed, data remain
68
somewhat equivocal on the relationship between changes in size and changes in strength resulting
69
from regimented RT: A considerable range of correlation coefficients have been observed, from
70
~0 to ~0.6 (Ahtiainen et al. 2016; Appleby et al. 2012; Baker et al. 1994; Balshaw et al. 2017;
71
Cribb et al. 2007; Erskine et al. 2014; Erskine et al. 2010; Loenneke et al. 2017; Maeo et al. 2018;
72
Pope et al. 2016; Rasch & Morehouse 1957; Watanabe et al. 2018). The discrepancies in findings
73
between studies may be related, in part, to the statistical measures employed to analyze
74
relationships between muscle hypertrophy and strength gain. For instance, analyses in a majority
75
of studies are based on between-subject data using only two time points, but within-subject
76
analyses are more appropriate for the question at hand. Inferentially, drawing individual-level
77
conclusions from group-level data is a statistical fallacy, known as the ecological fallacy
78
(Robinson 1950). PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) 25
ABSTRACT Analyses that do not account for inter-individual differences may
43
underestimate the relationship between hypertrophy and strength gain, and different methods of
44
assessing muscle size will produce different results. It is suggested that robust experimental
45
designs and analysis techniques, which control for different mechanistic sources of strength gain
46
and inter-individual differences (e.g., muscle moment arms, muscle architecture, activation, and
47
normalized muscle force), be employed in future investigations. 48 PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 81
analyses answer the question, “Is the growth of one’s muscle related to their increases in strength?”
82
Due to individual differences, the former (between-subject) may not necessarily map to the latter
83
(within-subject). For example, if subject A has a 30% larger muscle moment arm than subject B,
84
then one may expect subject A to have a 30% greater slope between increases in muscular strength
85
(force) and externally-measured strength (moment), all else being equal. To address the ecological
86
fallacy and answer the within-subject question, more sophisticated statistical approaches are
87
needed (Goldstein 2011; Jackson et al. 2006; Robinson 1950). 88
A hierarchical approach can assist in avoiding the pitfall of the ecological fallacy
89
(Goldstein 2011; Jackson et al. 2006). Traditionally, each participant’s change in strength and
90
change in size, from pre- to post-intervention, are calculated and regressed among one another
91
(Ahtiainen et al. 2016; Appleby et al. 2012; Baker et al. 1994; Balshaw et al. 2017; Cribb et al. 92
2007; Erskine et al. 2014; Erskine et al. 2010; Maeo et al. 2018; Pope et al. 2016; Rasch &
93
Morehouse 1957; Watanabe et al. 2018). However, a hierarchical modeling approach allows for
94
one to look at time points nested within participants, such that each participant’s points are kept
95
“separate” from other participants (Gelman & Hill 2007; Goldstein 2011; Raudenbush & Bryk
96
2002). Within the hierarchical model, each participant can receive varying intercepts and/or
97
varying slopes, which allows for inter-individual differences to be appropriately accounted for
98
(Gelman & Hill 2007; Goldstein 2011; Raudenbush & Bryk 2002). To carry out hierarchical
99
modeling with varying slopes and intercepts, multiple (≥ 3) time points are required (i.e., to
100
quantify model variance), so most training datasets cannot be used to answer this question, as a
101
majority only collect data at two time points (pre- and post-intervention). To date, only one study
102
has employed a within-subject analysis: Loenneke et al. (2017) used analysis of covariance
103
(ANCOVA) (Bland & Altman 1995a) and found appreciably greater coefficients of determination
104
in within- relative to between-subject models for the same muscle and strength test (e.g., R2 =
105
0.004 vs. 0.35). However, in contrast to hierarchical linear models, ANCOVA has an affine
106
assumption; participants receive different intercepts, but all are constrained to the same slope
107
(Bland & Altman 1995a). 50
Introduction Pragmatically, this problem can be better understood by differentiating between
79
the question that each analysis addresses. Between-subject analyses answer the question, “Do
80
those who grow more also get stronger than those who grow less?” Conversely, within-subject PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 112
periods of both training and detraining. It was hypothesized that different statistical models would
113
produce different outcomes, with between-subject correlations showing the weakest relationships
114
and hierarchical linear modeling showing the strongest. 115 112
periods of both training and detraining. It was hypothesized that different statistical models would
113
produce different outcomes, with between-subject correlations showing the weakest relationships
114
and hierarchical linear modeling showing the strongest. 115 117
Participants 118
The study reanalyzed data from a previously published study, the methods of which have
119
been described (Than et al. 2016). In brief, young, recreationally active individuals (mean ± SD,
120
age = 24 ± 3 years, BMI = 22 ± 2, n = 19) were recruited for participation in the study. Participants
121
reported exercising at least 3 times per week via various sporting activities but did not perform
122
resistance training for the elbow flexors. Informed consent was obtained for all participants. The
123
original study was approved by the University of Queensland Medical Research Ethics Committee
124
(no. 2014001416). Manuscript to be reviewed Therefore, further work is needed to understand how model choice
108
affects the strength of the relationship between hypertrophy and changes in strength. 109
The purpose of this study was to investigate the relationship between changes in muscle
110
size and strength in the elbow flexors using a variety of statistical and measurement approaches,
111
while also employing both between- and within-subject analyses over multiple time-points during PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) 144
Resistance training protocol 145
Resistance training for the non-dominant brachium was carried out five days per week for
146
the initial 8 weeks of the study, followed by a subsequent 8-week detraining period. Training
147
consisted of unilateral dumbbell elbow flexion performed with a supinated forearm. During each
148
session, participants performed 9 sets of 12 repetitions with a 90-second rest interval afforded
149
between sets. Loads were based on maximal voluntary isometric contraction (MVIC) values that
150
were obtained each week using a Sundoo SN Analogue Force Gauge (model number SN-500) at
151
90° elbow flexion. Subjects began each workout using 70% of that week’s MVIC recording. If the
152
full number of target repetitions (i.e., 12) was not achieved on a given set, the load was lowered to
153
the next level of load until completion – e.g., if a participant achieved 8 repetitions at 70%, the
154
load was decreased to 50% so that all 12 reps could be performed. Loads were progressively
155
lowered on successive sets to 50% and 30% of MVIC as needed so that subjects could complete
156
the target repetition range with proper form. The dominant brachium of each subject served as the
157
control for the study throughout the training and detraining periods. Subjects were instructed to
158
refrain from exercise involving the elbow flexors, other than activities of daily living, throughout
159
the 16-week study period. 160 Manuscript to be reviewed Manuscript to be reviewed 126
Muscle size 127
Measures of muscle thickness were obtained via B-mode ultrasound imaging (Mindray
128
DP-50) using a 7.5 MHz linear transducer probe. Images were taken at baseline and after each
129
week of training throughout the 16-week study period. Scanning was carried out by a trained
130
sonographer on both the dominant and non-dominant elbow flexors at 30, 50, and 70% of total
131
length of the biceps brachii whilst participants were seated with the antebrachium in a neutral
132
position. After weeks 4, 8, and 16, CSA scans were acquired for both upper limbs via panoramic
133
B-mode ultrasound (S3000 Siemens/Acuson system) using a 4–9 MHz linear transducer operating
134
at 9 MHz. Imaging for CSA was obtained via lateral acquisition at 50% width of the biceps brachii. 135
Values for both muscle thickness and CSA were determined using ImageJ (version 1.48; National
136
Institutes of Health, Bethesda, MD). Muscle thickness was not assessed for Week 4 due to a
137
conflict in scheduling with CSA ultrasounds. All ultrasound measures were completed by a paid
138
qualified professional, and not by the researchers of the paper. If the probe lost contact at any point
139
during the measurement, the measurement was retaken. Test-retest intraclass correlation
140
coefficients (ICC; model 2,1) of 0.99 and 0.97 for CSA and muscle thickness, respectively, have
141
been previously reported (Jenkins et al. 2015). Because an ICC(2,1) model was used, these results PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 173
analysis, strength was treated as a dependent variable, participants were treated as a categorical
174
factor (dummy-coded), and size was treated as a covariate. Variance accounted for (VAF) was
175
calculated using the formula
, where SS is type III sum of squares (Bland &
VAF =
SSsize
SSsize + SSresidual
176
Altman 1995a). This is equivalent to a partial η2 for the size covariate. Lastly, because the
177
ANCOVA method has a number of assumptions and does not allow for varying slopes, a more
178
robust hierarchical linear model was used for the final analysis (Quené & van den Bergh 2004). In
179
this analysis, the outcome measure (yij) was the net joint moment during MVIC, and muscle size
180
was used as a level-one predictor variable (xij), which were group-mean centered for analyses. 181
Subject was treated as a level-two variable. Finally, varied slopes and intercepts were permitted,
182
creating the final model:
183
184
Level 1
185
yij = β0j + β1jxij + ϵij
186
187
Level 2
188
β0j = γ00 + r0j
β1j = γ10 + r1j
189 173
analysis, strength was treated as a dependent variable, participants were treated as a categorical
174
factor (dummy-coded), and size was treated as a covariate. Variance accounted for (VAF) was
175
calculated using the formula
, where SS is type III sum of squares (Bland &
VAF =
SSsize
SSsize + SSresidual 176
Altman 1995a). This is equivalent to a partial η2 for the size covariate. Lastly, because the
177
ANCOVA method has a number of assumptions and does not allow for varying slopes, a more
178
robust hierarchical linear model was used for the final analysis (Quené & van den Bergh 2004). In
179
this analysis, the outcome measure (yij) was the net joint moment during MVIC, and muscle size
180
was used as a level-one predictor variable (xij), which were group-mean centered for analyses. 181
Subject was treated as a level-two variable. Finally, varied slopes and intercepts were permitted,
182
creating the final model: 190
The model was fit using restricted maximum likelihood in the lme4 package (Bates et al. 2015). 161
Statistical analysis 162
Several statistical analyses were carried out to investigate how methods of both
163
measurement and analysis may affect the conclusions drawn from a study investigating the
164
relationship between strength and hypertrophy. All analyses were carried out in R (version 3.4.3)
165
(R Core Development Team 2017). First, standard bivariate linear regression analyses of pre- and
166
post-measures were utilized to investigate the relationship between muscle size (thickness or CSA)
167
and strength, using a between-subject model. This was done for two different conditions: training
168
and detraining. For each condition, a data point (Δsize, Δstrength) was calculated for each participant,
169
where, in the general case, Δ = post – pre, where pre and post are the values before and after a
170
given condition (training or detraining), respectively, as has been done in a number of previous
171
investigations (Ahtiainen et al. 2016; Erskine et al. 2014; Loenneke et al. 2017). Second, an
172
ANCOVA was utilized to replicate the method of analysis used by Loenneke et al. (2017). In this PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 191
Sample variance of the residuals (s²) were used to calculate VAF (or R2) using the following
192
formula:
, where
is the sample variance of the residuals in the unconditional
VAF = 1 -
s2
s
2
uncond
s
2
uncond 190
The model was fit using restricted maximum likelihood in the lme4 package (Bates et al. 2015). 191
Sample variance of the residuals (s²) were used to calculate VAF (or R2) using the following
192
formula:
, where
is the sample variance of the residuals in the unconditional
VAF = 1 -
s2
s
2
uncond
s
2
uncond 193
model, which contained only varied intercepts and no fixed effects (i.e., the same model, but with
194
β1j = 0). This approach is mathematically equivalent to the VAF found for the ANCOVA using
195
type III sums of squares (see Appendix A). Intraclass correlation coefficients (ICC) were
196
calculated on the unconditional models to estimate the proportion of original variance explained
197
by subject. To estimate 95% confidence intervals (CI) of the VAFs, each model was bootstrapped
198
2000 times with replacement. The 0.025 and 0.975 quantiles of the VAF estimates were calculated
199
as the lower and upper bounds of each estimate’s 95% CI. 193
model, which contained only varied intercepts and no fixed effects (i.e., the same model, but with
194
β1j = 0). This approach is mathematically equivalent to the VAF found for the ANCOVA using
195
type III sums of squares (see Appendix A). Intraclass correlation coefficients (ICC) were
196
calculated on the unconditional models to estimate the proportion of original variance explained
197
by subject. To estimate 95% confidence intervals (CI) of the VAFs, each model was bootstrapped
198
2000 times with replacement. The 0.025 and 0.975 quantiles of the VAF estimates were calculated
199
as the lower and upper bounds of each estimate’s 95% CI. 200
To understand how the different measures of hypertrophy relate to one another, within-
201
and between-subject correlation matrices were constructed using the different thickness measures PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 202
and CSA. The between-subject analysis included all thickness and CSA measures, across all
203
subjects, for any time point at which both CSA and thickness were measured. The within-subject
204
correlation matrix was constructed in a similar manner: 1) a correlation coefficient was calculated
205
for each participant (ri); 2) using a Fisher z-transformation, ri was transformed to a z-score (zi); 3)
206
a weighted average was obtained using the number of points (ni) from each participant (
,
z =
∑zi(ni - 3)
∑(ni - 3)
207
for i participants); and 4) was transformed back to Pearson’s r (Borenstein et al. 2009; Cooper et
z
208
al. 2009; Corey et al. 1998; Hedges & Olkin 1985). Because CSA measures were only taken with
209
thickness at two time points, within-subject correlation coefficients could not be estimated between
210
CSA and muscle thickness. 202
and CSA. The between-subject analysis included all thickness and CSA measures, across all
203
subjects, for any time point at which both CSA and thickness were measured. The within-subject
204
correlation matrix was constructed in a similar manner: 1) a correlation coefficient was calculated
205
for each participant (ri); 2) using a Fisher z-transformation, ri was transformed to a z-score (zi); 3)
206
a weighted average was obtained using the number of points (ni) from each participant (
,
z =
∑zi(ni - 3)
∑(ni - 3)
207
for i participants); and 4) was transformed back to Pearson’s r (Borenstein et al. 2009; Cooper et
z
208
al. 2009; Corey et al. 1998; Hedges & Olkin 1985). Because CSA measures were only taken with
209
thickness at two time points, within-subject correlation coefficients could not be estimated between
210
CSA and muscle thickness. 210
CSA and muscle thickness. 211
Further exploratory analyses were performed to investigate if those with stronger strength-
212
hypertrophy relationships also got stronger. To do this, Pearson correlation coefficients were
213
calculated for each individual across the entire study (i.e., including both training and detraining
214
periods). The resulting correlation coefficients were then correlated with Δstrength from the training
215
period using Spearman’s rank-order correlations (ρ). Spearman’s ρ was used due to the
216
heteroscedastic nature of the residuals. Qualitative interpretations of correlation coefficients and
217
VAFs can be found in Table 1, which are in accordance with Hopkins (2002). Manuscript to be reviewed R code for all
218
procedures can be found in the Supplemental Files. 219
220
* Table 1 here *
221
222
223
Results
224
Differences in VAFs ranged from zero to an order of magnitude (Table 2). Similar differences
225
were also observed between different statistical models for a given measure (Table 2). Intraclass
226
correlation coefficients from the hierarchical linear models suggest that most of the original
227
variance could be accounted for by including a level for subject (ICC = 0.89–0.91). Heterogeneity
228
in correlation coefficients was observed when comparing different measures of muscle thickness,
229
which ranged from r = 0.503 to r = 0.945 for between-subject correlations and from r = 0.326 to
230
r = 0.875 for weighted within-subject correlations (Table 3). Finally, Pearson’s r of each
231
individual’s strength-hypertrophy relationship was a moderate to strong predictor of strength for 211
Further exploratory analyses were performed to investigate if those with stronger strength-
212
hypertrophy relationships also got stronger. To do this, Pearson correlation coefficients were
213
calculated for each individual across the entire study (i.e., including both training and detraining
214
periods). The resulting correlation coefficients were then correlated with Δstrength from the training
215
period using Spearman’s rank-order correlations (ρ). Spearman’s ρ was used due to the
216
heteroscedastic nature of the residuals. Qualitative interpretations of correlation coefficients and
217
VAFs can be found in Table 1, which are in accordance with Hopkins (2002). R code for all
218
procedures can be found in the Supplemental Files. 219 224
Differences in VAFs ranged from zero to an order of magnitude (Table 2). Similar differences
225
were also observed between different statistical models for a given measure (Table 2). Intraclass
226
correlation coefficients from the hierarchical linear models suggest that most of the original
227
variance could be accounted for by including a level for subject (ICC = 0.89–0.91). Heterogeneity
228
in correlation coefficients was observed when comparing different measures of muscle thickness,
229
which ranged from r = 0.503 to r = 0.945 for between-subject correlations and from r = 0.326 to
230
r = 0.875 for weighted within-subject correlations (Table 3). Finally, Pearson’s r of each
231
individual’s strength-hypertrophy relationship was a moderate to strong predictor of strength for PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 232
all measurements (US30% ρ = 0.644; US50% ρ = 0.356; US70% ρ = 0.413; USavg ρ = 0.480; CSA ρ =
233
0.449). 234
235
* Table 2 here *
236
237
* Table 3 here *
238
239
Discussion 232
all measurements (US30% ρ = 0.644; US50% ρ = 0.356; US70% ρ = 0.413; USavg ρ = 0.480; CSA ρ =
233
0 449) Manuscript to be reviewed It is unclear from where these differences arise;
273
that is, if they are due to measurement technique, differences in mechanisms of strength gain,
274
differences in upper vs. lower extremities, or some other factor. However, our data provide a
275
methodological proof of principle by delineating how different statistical models may drastically
276
affect the conclusions formed from a given dataset, even when performed on the same set of
277
regressors. Due to the robustness of hierarchical linear models, it is recommended that such
278
analyses are used over ANCOVAs for future investigations with similar methods. 279
How muscle size is assessed will likely affect the strength of the relationship between
280
changes in muscle size and strength. The measurement techniques utilized by previous and present
281
investigations (Ahtiainen et al. 2016; Appleby et al. 2012; Baker et al. 1994; Balshaw et al. 2017;
282
Buckner et al. 2016a; Cribb et al. 2007; Erskine et al. 2014; Loenneke et al. 2017; Pope et al. 2016)
283
have been limited in that they do not account for changes in architectural characteristics (Lieber &
284
Ward 2011). There are several ways to measure muscle size, including limb circumference
285
(DeLorme 1945), estimates of total and segmental muscle mass (dual-energy X-ray absorptiometry
286
and bioelectrical impedance analysis) (Karelis et al. 2013), muscle thickness (Than et al. 2016),
287
anatomical CSA (Erskine et al. 2014; Trezise et al. 2016), muscle volume (Balshaw et al. 2017;
288
Erskine et al. 2014; Erskine et al. 2010), and PCSA (Erskine et al. 2010). There are strong
289
physiological and mechanical rationales with basic science evidence to suggest that not all of these
290
measures are equal, even when accounting for measurement error (Lieber & Ward 2011; Powell
291
et al. 1984). For example, although muscle volume appears to be a strong predictor of strength in
292
some contexts (even greater than anatomical CSA) (Akagi et al. 2009; Fukunaga et al. 2001), it
293
does not perform as well in others (Baxter & Piazza 2014), perhaps at least partly due to inter- and 279
How muscle size is assessed will likely affect the strength of the relationship between
280
changes in muscle size and strength. The measurement techniques utilized by previous and present
281
investigations (Ahtiainen et al. 2016; Appleby et al. 2012; Baker et al. 1994; Balshaw et al. 2017;
282
Buckner et al. 239
Discussion 240
To the authors’ knowledge, this is the first study to investigate the relationship between
241
hypertrophy and changes in muscle strength using hierarchical linear modeling, which allows for
242
robust within-individual analysis, in addition to the use of multiple types of measures of muscle
243
size. Our results demonstrate that not only does measurement approach substantially affect
244
outcomes, but so does the type of statistical model employed. These findings have important
245
methodological implications for improving our understanding of the associative relationship
246
between hypertrophy and changes in strength. 247
Previous literature has approached the question of how changes in muscle size relate to
248
changes in strength from a between-subject perspective. However, it can be argued that a repeated-
249
measures design allows for a more direct evaluation of the strength-hypertrophy relationship. 250
Individual differences in muscle moment arms (MA), normalized muscle force (NMF), pennation
251
angles (θp), voluntary activation (α), et cetera will greatly confound the relative relationship
252
between changes in strength and muscle size (in this case, physiological CSA(PCSA)). All of the
253
aforementioned components are multipliers in the formula used to calculate a muscle’s
254
contribution to a joint moment (
) (Vigotsky et al. 2015). To
M = α ⋅PCSA ⋅NMF ⋅cos θp ⋅MA
255
date, only one previous investigation has utilized a quantitative within-subject approach to
256
investigate the relationship between hypertrophy and changes in strength (Loenneke et al. 257
2017); although, qualitative within-subject changes are depicted in a classic study by DeLorme
258
(1945). Specifically, Loenneke et al. (2017) employed an ANCOVA with subject as a factor and
259
muscle size as a covariate; from the resulting sum of squares, VAF could be calculated (Bland &
260
Altman 1995a). ANCOVA is limited, however, in that it, in its basic form, assumes parallelism
261
between all relationships, has several assumptions that may confound results (e.g., sphericity,
262
compound symmetry, and homoscedasticity), and is not robust to missing data points (Bland & PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 263
Altman 1995a; Bland & Altman 1995b; Quené & van den Bergh 2004). The parallel or affine
264
assumption is of particular interest because there are several heterogeneities that confound this
265
assumption (i.e., α, MA, NMF, and θp). Repeated-measures hierarchical models are a robust way
266
to investigate longitudinal relationships within a group or person (Gelman & Hill 2007;
267
Raudenbush & Bryk 2002). By comparing these statistical models, a clear difference is apparent
268
(Table 2). For all measurements, the hierarchical linear model resulted in greater VAFs than the
269
ANCOVA (Table 2). These differences may be due to the hierarchical linear model allowing for
270
varying slopes or, alternatively, some of the inherent assumptions and limitations of ANCOVAs
271
(Quené & van den Bergh 2004). Interestingly, the VAFs found in this present study are much
272
lower than those found by Loenneke et al. (2017). It is unclear from where these differences arise;
273
that is, if they are due to measurement technique, differences in mechanisms of strength gain,
274
differences in upper vs. lower extremities, or some other factor. However, our data provide a
275
methodological proof of principle by delineating how different statistical models may drastically
276
affect the conclusions formed from a given dataset, even when performed on the same set of
277
regressors. Due to the robustness of hierarchical linear models, it is recommended that such
278
analyses are used over ANCOVAs for future investigations with similar methods. 263
Altman 1995a; Bland & Altman 1995b; Quené & van den Bergh 2004). The parallel or affine
264
assumption is of particular interest because there are several heterogeneities that confound this
265
assumption (i.e., α, MA, NMF, and θp). Repeated-measures hierarchical models are a robust way
266
to investigate longitudinal relationships within a group or person (Gelman & Hill 2007;
267
Raudenbush & Bryk 2002). By comparing these statistical models, a clear difference is apparent
268
(Table 2). For all measurements, the hierarchical linear model resulted in greater VAFs than the
269
ANCOVA (Table 2). These differences may be due to the hierarchical linear model allowing for
270
varying slopes or, alternatively, some of the inherent assumptions and limitations of ANCOVAs
271
(Quené & van den Bergh 2004). Interestingly, the VAFs found in this present study are much
272
lower than those found by Loenneke et al. (2017). Manuscript to be reviewed 294
intra-muscular variation in architecture (Blazevich et al. 2006; Lieber & Ward 2011; Ward et al. 295
2009) and adaptation (Earp et al. 2015; Ema et al. 2013; Franchi et al. 2017; Narici et al. 1996;
296
Wakahara et al. 2013; Wakahara et al. 2012). Muscle volume is not only sensitive to changes in
297
sarcomeres in parallel (PCSA), but also sarcomeres in series (fiber length). Sarcomeres in parallel
298
will contribute to the magnitude of force production, while sarcomeres in series will affect the
299
shapes of the force-length and force-velocity curves. Functionally speaking, not all muscle volume
300
is equal (Lieber & Ward 2011). Importantly, in series hypertrophy appears to be limited to the
301
initial weeks of commencing resistance training, further reinforcing potential issues when
302
extrapolating correlative findings from novice to trained individuals (Blazevich et al. 2007). 303
Similarly, thickness and anatomical CSA, as measured in this study, are also limited, as they only
304
represent one part of the muscle and do not account for the intricacies of muscle architecture. This
305
is further evidenced by Franchi et al. (2017), who found that, cross-sectionally, muscle thickness,
306
anatomical CSA, and muscle volume are related, but the relative changes between muscle
307
thickness and muscle volume did not strongly correlate following a training period. This is
308
important when considering the formula for PCSA, in that the volume of the entire muscle must
309
be taken into account (Lieber & Ward 2011); not just thickness or anatomical CSA. Moreover, the
310
variability in correlation coefficients between these measures may be a cause for concern (Table
311
3), in that it suggests not all measures of muscle size are necessarily capturing the same effects,
312
which is elucidated further by the statistical models (Table 2). Since PCSA has been shown to be
313
a strong predictor of force production both in vivo (Fukunaga et al. 1996) and in vitro (Powell et
314
al. 1984), it is considered the gold standard for relating muscle form (architecture) to function
315
(force production) (Lieber & Ward 2011). PCSA is, in essence, the “effective” CSA, as it is the
316
average CSA perpendicular to the fibers’ line of action. Manuscript to be reviewed Thus, PCSA controls for pennation and is
317
representative of the number of sarcomeres in parallel, making it highly indicative of a muscle’s
318
potential to generate force through the tendon (Lieber & Ward 2011). It is imperative to consider
319
these differences in measurement techniques in the context of this study and similar investigations
320
(Ahtiainen et al. 2016; Erskine et al. 2014; Erskine et al. 2010; Loenneke et al. 2017). Although
321
this study (Table 2) and others (Loenneke et al. 2017) have observed what is analogous to a strong
322
correlation (r ≥ 0.5) (Hopkins 2002) with repeated-measures designs, substandard measurements
323
of muscle size were used in the present study. Therefore, it is likely that PCSA measurements
324
would produce different results (Aagaard et al. 2001). While PCSA is expensive to obtain and 294
intra-muscular variation in architecture (Blazevich et al. 2006; Lieber & Ward 2011; Ward et al. 295
2009) and adaptation (Earp et al. 2015; Ema et al. 2013; Franchi et al. 2017; Narici et al. 1996;
296
Wakahara et al. 2013; Wakahara et al. 2012). Muscle volume is not only sensitive to changes in
297
sarcomeres in parallel (PCSA), but also sarcomeres in series (fiber length). Sarcomeres in parallel
298
will contribute to the magnitude of force production, while sarcomeres in series will affect the
299
shapes of the force-length and force-velocity curves. Functionally speaking, not all muscle volume
300
is equal (Lieber & Ward 2011). Importantly, in series hypertrophy appears to be limited to the
301
initial weeks of commencing resistance training, further reinforcing potential issues when
302
extrapolating correlative findings from novice to trained individuals (Blazevich et al. 2007). 303
Similarly, thickness and anatomical CSA, as measured in this study, are also limited, as they only
304
represent one part of the muscle and do not account for the intricacies of muscle architecture. This
305
is further evidenced by Franchi et al. (2017), who found that, cross-sectionally, muscle thickness,
306
anatomical CSA, and muscle volume are related, but the relative changes between muscle
307
thickness and muscle volume did not strongly correlate following a training period. This is
308
important when considering the formula for PCSA, in that the volume of the entire muscle must
309
be taken into account (Lieber & Ward 2011); not just thickness or anatomical CSA. Manuscript to be reviewed 2016a; Cribb et al. 2007; Erskine et al. 2014; Loenneke et al. 2017; Pope et al. 2016)
283
have been limited in that they do not account for changes in architectural characteristics (Lieber &
284
Ward 2011). There are several ways to measure muscle size, including limb circumference
285
(DeLorme 1945), estimates of total and segmental muscle mass (dual-energy X-ray absorptiometry
286
and bioelectrical impedance analysis) (Karelis et al. 2013), muscle thickness (Than et al. 2016),
287
anatomical CSA (Erskine et al. 2014; Trezise et al. 2016), muscle volume (Balshaw et al. 2017;
288
Erskine et al. 2014; Erskine et al. 2010), and PCSA (Erskine et al. 2010). There are strong
289
physiological and mechanical rationales with basic science evidence to suggest that not all of these
290
measures are equal, even when accounting for measurement error (Lieber & Ward 2011; Powell
291
et al. 1984). For example, although muscle volume appears to be a strong predictor of strength in
292
some contexts (even greater than anatomical CSA) (Akagi et al. 2009; Fukunaga et al. 2001), it
293
does not perform as well in others (Baxter & Piazza 2014), perhaps at least partly due to inter- and PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Third,
350
moment arm measures should be subject-specific and occur over the duration of an experiment, as
351
moment arms may change with training (Sugisaki et al. 2015; Vigotsky et al. 2015). Finally, longer
352
duration studies may be more appropriate for several reasons: 1) individual response trajectories
353
will vary, as evidenced by the high ICCs in this present investigation and the heterogeneous rank
354
orders between time points in previous work (Churchward-Venne et al. 2015); 2) edema can
355
greatly confound gross imaging measures of muscle size, depending on when the measurements 330
The question of how changes in strength and changes in muscle size are related is one with broad
331
clinical implications, ranging from the treatment and prevention of sarcopenia and dynapenia to
332
exercise prescription for strength athletes. Clinically, if changes in muscle size are not important
333
for strength, then exercise programs need not focus on variables that are more important for
334
hypertrophy than strength, such as volume (Ralston et al. 2017; Schoenfeld et al. 2017). Changes
335
in strength do indeed arise from non-hypertrophic factors (Folland & Williams 2007), including a
336
myriad of neural adaptations (Enoka 1988), in addition to changes in muscle moment arms
337
(Sugisaki et al. 2015; Vigotsky et al. 2015) and normalized muscle force production (Erskine et
338
al. 2010), in which lateral force transmission has been suggested to play a role (Jones et al. 1989). 339
This implies that changes in strength are interactive rather than linear. As such, how this
340
relationship is investigated and modeled should reflect such complexities. First, with more
341
reductionist strength testing (i.e., single-joint isometric testing), it can be argued that the “skill”
342
component of strength is less relevant (as opposed to one-repetition maximum tests (Buckner et
343
al. 2016b)), since little coordination is necessary and even untrained individuals see little-to-no
344
changes in voluntary activation and co-contraction (Behm 1995; Erskine et al. 2014; Erskine et al. 345
2010; Noorkoiv et al. 2014). Moreover, neural measures, such as voluntary activation, can be more
346
accurately assessed during isometric efforts than during dynamic efforts (Farina 2006; Vigotsky
347
et al. 2017) and thus can more easily be incorporated into a final model. Second, measures of
348
muscle size should reflect those in the model (i.e., using PCSA). Manuscript to be reviewed Moreover, the
310
variability in correlation coefficients between these measures may be a cause for concern (Table
311
3), in that it suggests not all measures of muscle size are necessarily capturing the same effects,
312
which is elucidated further by the statistical models (Table 2). Since PCSA has been shown to be
313
a strong predictor of force production both in vivo (Fukunaga et al. 1996) and in vitro (Powell et
314
al. 1984), it is considered the gold standard for relating muscle form (architecture) to function
315
(force production) (Lieber & Ward 2011). PCSA is, in essence, the “effective” CSA, as it is the
316
average CSA perpendicular to the fibers’ line of action. Thus, PCSA controls for pennation and is
317
representative of the number of sarcomeres in parallel, making it highly indicative of a muscle’s
318
potential to generate force through the tendon (Lieber & Ward 2011). It is imperative to consider
319
these differences in measurement techniques in the context of this study and similar investigations
320
(Ahtiainen et al. 2016; Erskine et al. 2014; Erskine et al. 2010; Loenneke et al. 2017). Although
321
this study (Table 2) and others (Loenneke et al. 2017) have observed what is analogous to a strong
322
correlation (r ≥0 5) (Hopkins 2002) with repeated-measures designs substandard measurements PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 325
typically relies on MRI, newer technologies, such as 3D ultrasound, show promise as valid,
326
affordable alternatives to MRI for estimating muscle volume and PCSA (Barber et al. 2009; Barber
327
et al. 2011; Haberfehlner et al. 2016). Moving forward, it seems prudent that investigators utilize
328
PCSA rather than other measures of muscle size, as the theory that hypertrophy leads to strength
329
gains is predicated on this measure rather than other measures of muscle size. 330
The question of how changes in strength and changes in muscle size are related is one with broad
331
clinical implications, ranging from the treatment and prevention of sarcopenia and dynapenia to
332
exercise prescription for strength athletes. Clinically, if changes in muscle size are not important
333
for strength, then exercise programs need not focus on variables that are more important for
334
hypertrophy than strength, such as volume (Ralston et al. 2017; Schoenfeld et al. 2017). Changes
335
in strength do indeed arise from non-hypertrophic factors (Folland & Williams 2007), including a
336
myriad of neural adaptations (Enoka 1988), in addition to changes in muscle moment arms
337
(Sugisaki et al. 2015; Vigotsky et al. 2015) and normalized muscle force production (Erskine et
338
al. 2010), in which lateral force transmission has been suggested to play a role (Jones et al. 1989). 339
This implies that changes in strength are interactive rather than linear. As such, how this
340
relationship is investigated and modeled should reflect such complexities. First, with more
341
reductionist strength testing (i.e., single-joint isometric testing), it can be argued that the “skill”
342
component of strength is less relevant (as opposed to one-repetition maximum tests (Buckner et
343
al. 2016b)), since little coordination is necessary and even untrained individuals see little-to-no
344
changes in voluntary activation and co-contraction (Behm 1995; Erskine et al. 2014; Erskine et al. 345
2010; Noorkoiv et al. 2014). Moreover, neural measures, such as voluntary activation, can be more
346
accurately assessed during isometric efforts than during dynamic efforts (Farina 2006; Vigotsky
347
et al. 2017) and thus can more easily be incorporated into a final model. Second, measures of
348
muscle size should reflect those in the model (i.e., using PCSA). While this is expensive and time
349
consuming, it will provide more appropriate biomechanical insight (Lieber & Ward 2011). Manuscript to be reviewed 356
are performed (Damas et al. 2016); 3) the magnitude of the difference between measurement points
357
will be greater, which in turn will decrease the relative role of measurement error in parameter and
358
VAF estimates (Fuller 1987); and 4) to understand the extent to which contributions may or may
359
not change over time. While this present study did not incorporate these recommendations, since
360
it was based on previously collected data (Than et al. 2016), future studies should do so to properly
361
isolate the associative contribution of muscle size (PCSA) to strength increases. 362
Thus far, our discussion has primarily focused on the associative, rather than causal,
363
relationship between hypertrophy and strength gain. A conducive discussion of the causal nature
364
of this relationship requires an operational definition of causality. In formal logic, causality is often
365
broken down into two conditions: 1) necessary conditions, which state that B will not occur without
366
A (“if not A, then not B”); and 2) sufficient conditions, which state that A will result in B (“if A,
367
then B”) (Epp 2011; Hall 1987). However, a less formal concept of causality is also possible
368
without these conditions having been met, in the form of contributory causality. A contributory
369
cause is neither necessary nor sufficient (Hall 1987; Riegelman 1979). Those who experience an
370
effect need not experience its putative cause, and those who experience the putative cause need
371
not experience its effect (Riegelman 1979). For instance, although smoking causes lung cancer,
372
not all of those who smoke develop lung cancer (i.e., it is not sufficient), and not all of those who
373
develop lung cancer are smokers (i.e., it is not necessary); therefore, smoking may be viewed as a
374
contributory cause of lung cancer (Riegelman 1979). The arguments put forth by Buckner et al. 375
(2016a); Dankel et al. (2018); Mattocks et al. (2017) do indeed rule out hypertrophy as being a
376
necessary or sufficient cause for strength gain, but we suggest that the contributory nature of
377
hypertrophy to strength should not be dismissed on this basis. In other words, changes in strength
378
can occur without changes in muscle size and vice versa, but this does not preclude muscle size
379
from contributing to strength. Manuscript to be reviewed While this is expensive and time
349
consuming, it will provide more appropriate biomechanical insight (Lieber & Ward 2011). Third,
350
moment arm measures should be subject-specific and occur over the duration of an experiment, as
351
moment arms may change with training (Sugisaki et al. 2015; Vigotsky et al. 2015). Finally, longer
352
duration studies may be more appropriate for several reasons: 1) individual response trajectories
353
will vary, as evidenced by the high ICCs in this present investigation and the heterogeneous rank
354
orders between time points in previous work (Churchward-Venne et al. 2015); 2) edema can
355
greatly confound gross imaging measures of muscle size, depending on when the measurements PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 387
incorporate all of the measures included in the formula to determine strength (PCSA, MA,
388
activation and co-contraction, synergist characteristics, and NMF) are likely needed to better
389
understand the emergent properties of strength. Finally, because the problem is so complex, the
390
contributory role of hypertrophy in strength gain may not be able to be fully established from one
391
study or line of evidence. Instead, a body of literature consisting of many forms of evidence—
392
ranging from animal and agent-based models to observational and experimental human studies—
393
may be required to elucidate the contributory role of hypertrophy in strength gain. 394
This study and its discussion have focused primarily on single muscle group hypertrophy
395
and single-joint isometric strength gain. The larger question of multi-joint and dynamic strength
396
gain is perhaps more relevant, but unfortunately much more complex (Vigotsky et al. 2018). 397
Starting with relatively simpler systems and research questions may bear more fruit, while also
398
providing a conceptual basis that can be used when studying more complex systems and research
399
questions. 387
incorporate all of the measures included in the formula to determine strength (PCSA, MA,
388
activation and co-contraction, synergist characteristics, and NMF) are likely needed to better
389
understand the emergent properties of strength. Finally, because the problem is so complex, the
390
contributory role of hypertrophy in strength gain may not be able to be fully established from one
391
study or line of evidence. Instead, a body of literature consisting of many forms of evidence—
392
ranging from animal and agent-based models to observational and experimental human studies—
393
may be required to elucidate the contributory role of hypertrophy in strength gain. 394
This study and its discussion have focused primarily on single muscle group hypertrophy
395
and single-joint isometric strength gain. The larger question of multi-joint and dynamic strength
396
gain is perhaps more relevant, but unfortunately much more complex (Vigotsky et al. 2018). 397
Starting with relatively simpler systems and research questions may bear more fruit, while also
398
providing a conceptual basis that can be used when studying more complex systems and research
399
questions. 400
This is the first study to utilize repeated-measures hierarchical linear modeling to
401
investigate the relationship between muscle size and strength. Manuscript to be reviewed We herein demonstrate that
402
repeated-measures hierarchical linear models produce different results than other within-subject
403
models (ANCOVA), in addition to between-subject models, which is in line with previous work
404
by Loenneke et al. (2017). Moreover, it was found that different measures of muscle size can
405
produce vastly different results. As such, we have advocated for more rigorous and reductionist
406
experimental designs to better understand the mechanistic origins of single-joint strength following
407
exercise programs, by suggesting that researchers measure PCSA and single-joint isometric
408
strength, in addition to potential confounding variables1. These findings are important for the
409
interpretation of previous studies, in addition to the design of future studies, on this same topic. 410 Manuscript to be reviewed Experimentally, it is important to consider the emergent, nonlinear,
380
and interactive properties of strength; there are many moving parts that should be accounted for
381
when attempting to understand such a complex system, which may concurrently change in
382
different directions (e.g., increase in size but decrease agonist activation). Indeed, a systems rather
383
than reductionist approach may be most appropriate for understanding strength emergence. In
384
studying this system, it is necessary to measure all factors (confounders) that may contribute to
385
strength to truly understand the role of hypertrophy, especially because different protocols may
386
elicit differential adaptations (Jenkins et al. 2017). Thus, longitudinal, within-subject studies that PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) 1 Note that these recommendations only apply to studies that are investigating the strength-hypertrophy relationship
with a reductionist approach. We are in no way suggesting that PCSA and single-joint isometric measures be used
for all resistance training studies. 411
Conclusions 412
The strength of the associational relationship between muscle hypertrophy and strength gain is
413
highly dependent upon the statistical model employed. We have demonstrated that hierarchical
414
linear modeling, which allows for varying slopes and intercepts, provides greater estimates of the
415
strength of the relationship between muscle hypertrophy and strength gain. Moreover, different 1 Note that these recommendations only apply to studies that are investigating the strength-hypertrophy relationship
with a reductionist approach. We are in no way suggesting that PCSA and single-joint isometric measures be used
for all resistance training studies. PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Manuscript to be reviewed 416
assessments of muscle size do not perfectly correlate, and therefore, different methods of
417
assessment may lead to different conclusions. These findings should be taken into consideration
418
when planning and interpreting studies on the relationship between muscle hypertrophy and
419
strength gain. PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) 421
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638 PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Table 1(on next page) Correlation coefficient and variance accounted for interpretations. Adapted from Hopkins (2002). Note that all intervals are of the form xlow ≤ xo < xhigh. PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed 2
Adapted from Hopkins (2002). Note that all intervals are of the form xlow ≤ xo Manuscript to be reviewed Table 2(on next page) Manuscript to be reviewed 1
Table 1. Correlation coefficient and variance accounted for interpretations. Interpretation
Correlation coefficient (r or ρ)
Variance accounted for (%)
Trivial
[0, 0.1)
[0, 1)
Small
[0.1, 0.3)
[1, 9)
Moderate
[0.3, 0.5)
[9, 25)
Large/strong
[0.5, 0.7)
[25, 49)
Very large/strong
[0.7, 0.9)
[49, 81)
Nearly perfect
[0.9, 1)
[81, 100)
Perfect
1
100
2
Adapted from Hopkins (2002). Note that all intervals are of the form xlow ≤ xo < xhigh. orrelation coefficient and variance accounted for interpretations. PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Table 2(on next page) Percent (%) variance accounted for (95% CI) using different types of models. 30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average
represents the average of all three of the measured thicknesses at a given time point. Cross-
sectional area was measured at 50%. ANCOVA = analysis of covariance; HLM = hierarchical
linear model PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1
Table 2. Percent (%) variance accounted for (95% CI) using different types of models. Between-subjects
Within-subjects
Measure
Training
Detraining
ANCOVA
HLM
Thickness (30%)
3.6 (0–61.9)
1.0 (0–45.1)
0.2 (0–6.1)
7.4 (0.8–16.0)
Thickness (50%)
0.8 (0–21.6)
0.0 (0–23.7)
0.3 (0–9.7)
24.1 (6.7–42.0)
Thickness (70%)
1.4 (0–39.1)
1.6 (0–38.0)
2.2 (0–10.9)
7.5 (2.1–23.7)
Thickness (Average)
0.4 (0–21.1)
0.0 (0–26.4)
1.2 (0–12.9)
18.1 (6.6–30.4)
Cross-sectional area
0.4 (0–32.2)
1.2 (0–35.4)
11.7 (1.1–34.2)
12.1 (2.0–69.5)
2
30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average represents the average
3
of all three of the measured thicknesses at a given time point. Cross-sectional area was measured at 50%. ANCOVA
4
= analysis of covariance; HLM = hierarchical linear model 2
30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average represents the average
3
of all three of the measured thicknesses at a given time point. Cross-sectional area was measured at 50%. ANCOVA
4
= analysis of covariance; HLM = hierarchical linear model PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Table 3(on next page) Table 3(on next page) Correlation matrix of measures of muscle size. 30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average
represents the average of all three of the measured thicknesses at a given time point. Cross-
sectional area was measured at 50%. * = between-subject correlation; † = weighted within-
subject correlation PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018) Manuscript to be reviewed Manuscript to be reviewed 1
Table 3. Correlation matrix of measures of muscle size. Thickness
(30%)
Thickness
(50%)
Thickness
(70%)
Thickness
(Average)
Cross-sectional
area
Thickness
(30%)
0.503 *
0.618 *
0.778 *
0.557 *
Thickness
(50%)
0.344 †
0.869 *
0.916 *
0.742 *
Thickness
(70%)
0.326 †
0.687 †
0.945 *
0.730 *
Thickness
(Average)
0.659 †
0.875 †
0.871 †
0.773 *
Cross-
sectional area
2
30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average represents the average
3
of all three of the measured thicknesses at a given time point. Cross-sectional area was measured at 50%. * = between-
4
subject correlation; † = weighted within-subject correlation 1
Table 3. Correlation matrix of measures of muscle size. 2
30%, 50%, and 70% represent the position of the ultrasound probe on the brachium. Average represents the average
3
of all three of the measured thicknesses at a given time point. Cross-sectional area was measured at 50%. * = between-
4
subject correlation; † = weighted within-subject correlation PeerJ reviewing PDF | (2018:02:24413:1:1:NEW 1 May 2018)
|
https://openalex.org/W3032748148
|
https://aia.springeropen.com/track/pdf/10.1186/s42774-019-0026-3
|
English
| null |
Discrete unified gas kinetic scheme for multiscale anisotropic radiative heat transfer
|
Advances in aerodynamics
| 2,020
|
cc-by
| 8,688
|
RESEARCH r *Correspondence:
zlguo@hust.edu.cn
1State Key Laboratory of Coal
Combustion, School of Energy and
Power Engineering, Huazhong
University of Science and
Technology, 430074 Wuhan, China
Full list of author information is
available at the end of the article Discrete unified gas kinetic scheme for
multiscale anisotropic radiative heat transfer Xinliang Song1, Chuang Zhang1, Xiafeng Zhou2 and Zhaoli Guo1* Xinliang Song1, Chuang Zhang1, Xiafeng Zhou2 and Zhaoli Guo1* © The author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. Abstract In this work, a discrete unified gas kinetic scheme (DUGKS) is developed for radiative
transfer in anisotropic scattering media. The method is an extension of a previous one
for isotropic radiation problems [1]. The present scheme is a finite-volume
discretization of the anisotropic gray radiation equation, where the anisotropic
scattering phase function is approximated by the Legendre polynomial expansion. With the coupling of free transport and scattering processes in the reconstruction of
the flux at cell interfaces, the present DUGKS has the nice unified preserving properties
such that the cell size is not limited by the photon mean free path even in the optical
thick regime. Several one- and two-dimensional numerical tests are conducted to
validate the performance of the present DUGKS, and the numerical results demonstrate
that the scheme is a reliable method for anisotropic radiative heat transfer problems. Keywords: Gray radiative transfer equation, Anisotropic scattering, Scattering phase
function, Legendre polynomial Song et al. Advances in Aerodynamics (2020) 2:3
https://doi.org/10.1186/s42774-019-0026-3 Song et al. Advances in Aerodynamics (2020) 2:3
https://doi.org/10.1186/s42774-019-0026-3 Advances in Aerodynamics Open Access International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. © The author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
d
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mmons license, and indicate if changes were made. 1
Introduction Radiative heat transfer appears in many engineering applications, such as short-pulsed
laser in turbid media [2], radiative base heating from rocket exhaust plums [3], radiation in
liquid rocket engines [4], nonequilibrium radiative hypersonic flows [5], and some other
processes [6–9]. In practical applications, the scattering media are usually anisotropic
[10], and some studies have been reported in the literature. For instance, it is found that
radiative heat transfer can be greatly influenced in aerosol media due to the anisotropy
properties [11]. The physically realistic approach for the scattering behaviour of coal com-
bustion particles is the anisotropic, strongly forward scattering [12]. The liquid aluminum
oxide particles produced from combustion of solid propellant exhibit a strong forward
scattering characteristic [13]. Radiative heat transfer in anisotropic media can be described by the radiative transfer
equation (RTE) for radiation intensity of the photon, which is a high dimensional integral-
differential equation. As the scattering effect of the media is strong (optical thick), i.e.,
the photon mean free path (MFP) λ is much smaller than the characteristic macroscopic
length L, the radiation behaves diffusively. On the other hand, the radiation can transport
freely with the light speed when the scattering effect is weak (optical thin). Many prac-
tical media contain both strong and weak scattering regimes, such that it is necessary to
design numerical schemes which can capture the radiation transport accurately in both Song et al. Advances in Aerodynamics (2020) 2: Page 2 of 15 (2020) 2:3 Song et al. Advances in Aerodynamics cases uniformly. The widely used stochastic Monte Carlo method (MCM) [14–16], which
simulates the RTE by tracking the transport process of simulated photons with a mesh
size smaller than the MFP, is inefficient in optical thick media. The classical determinis-
tic discrete ordinates method (DOM) [17–19] and finite volume method (FVM) [10, 20–
22] also suffer similar challenges. Therefore, it is necessary to develop multiscale schemes
which are suitable for problems with arbitrary optical thickness without the limitation of
the mesh size by the MFP. The asymptotic preserving (AP) scheme for the linear kinetic equation is one of the
idealized multiscale schemes. AP schemes are first studied in steady neutron transport
problems [23–26]. For unsteady problems, some AP schemes have also been proposed
[27, 28]. Recently, an AP method, the unified gas kinetic scheme (UGKS) was success-
fully developed for radiative transfer problems [29–31]. 1
Introduction Another asymptotic preserving
multiscale method, the discrete unified gas kinetic scheme, which was initially designed
for gas flows [32, 33], was also extended to solve the radiative heat transfer problems in
isotropic scattering media [1]. As the distribution function at a cell interface in DUGKS is
constructed from the characteristic numerical solution rather than the local integral one
in the UGKS, the DUGKS has a simpler structure and is more computational efficient. Furthermore, it can be shown that the DUGKS has the unified preserving properties such
that it can serve as an efficient multiscale method [34]. However, both the UGKS and
DUGKS have not considered the effects of the anisotropic scattering properties. In present work, we will extend the DUGKS for isotropic radiative transfer to problems
with anisotropic scattering effects. With the considering of the anisotropy, the radiative
transfer features of the forward scattering media and the backward scattering media can
be described clearly. The major difficulty of simulating the anisotropic problem stems
from the change of the anisotropic scattering phase function in the RTE. In the present
study, the anisotropic phase function will be calculated from the Legendre polynomial
expansions [35, 36]. The remainder of this paper is organized as follows. Section 2 introduces the anisotropic
scattering radiative transfer equation, and the DUGKS for RTE is described in Section 3. Some numerical tests are performed in Section 4. Finally, a brief summary is given in
Section 5. 2
Gray radiative transfer equation (9)
3
N
i
l
h G =
4π
I (x, s, t) d, G =
4π
I (x, s, t) d,
(8) (8)
4π
q =
4π
I (x, s, t) sd. (9) q =
4π
I (x, s, t) sd. q =
4π
I (x, s, t) sd. (9) (9) 2
Gray radiative transfer equation In the RTE, the anisotropic scattering characteristic of
the participating media is fully expressed by the scattering phase function (s′, s), which
satisfies the normalization condition, where σ is the Stefan-Boltzmann constant and T is the local temperature of the medium,
and (s′, s) is the scattering phase function, which describes the fraction of the radiative
energy scattered into the outgoing direction s from the incoming direction s′, and ′ is the
corresponding solid angle domain. In the RTE, the anisotropic scattering characteristic of
the participating media is fully expressed by the scattering phase function (s′, s), which
satisfies the normalization condition, 1
4π
4π
s′, s
d′ = 1. 1
4π
4π
s′, s
d′ = 1. (5) (5) Unlike the isotropic scattering problems where the scattering phase function is constant
( ≡1), the scattering phase function in anisotropic scattering problems changes accord-
ing to the scattering angle. In this study it is approximated by a finite series of Legendre
polynomials [17, 35, 36], i.e., (s′, s) = (cosψ) =
N
j=0
Cj(α1, α2)Pj(cosψ),
(6)
cosψ = μμ′ +
1 −μ21/2
1 −μ′21/2
cos(ϕ′ −ϕ),
(7) (s′, s) = (cosψ) =
N
j=0
Cj(α1, α2)Pj(cosψ),
(6) (6) cosψ = μμ′ +
1 −μ21/2
1 −μ′21/2
cos(ϕ′ −ϕ),
(7) (7) where ψ is the angle between incoming direction s′
μ′, ϕ′
and scattered direction
s (μ, ϕ). μ is the cosine of the zenith angle and ϕ is the azimuth angle of the direction s. Cj(α1, α2) is the angular distribution coefficients, where α1 = πD/υ and α2 = mπD/υ
with D being the diameter of radiative medium particle, υ is the wavelength of the inci-
dent radiation in the surrounding medium, and m is the complex index of refraction of
radiative medium particle relative to the surrounding medium. Pj is Legendre polynomi-
als of order j. For strongly anisotropic scattering media, the upper limit N should be big
enough to ensure the accuracy of the calculation results. In radiative transfer, the incident radiation energy G and heat flux q are two important
physical quantities, which are defined from the radiation intensity, G =
4π
I (x, s, t) d,
(8)
q =
4π
I (x, s, t) sd. 2
Gray radiative transfer equation Gray radiative transfer equation The gray radiative transfer equation with anisotropic scattering reads [37, 38] The gray radiative transfer equation with anisotropic scattering reads [37, 38] 1
c
∂I(x, s, t)
∂t
+ s · ∇I(x, s, t) = −βI(x, s, t) + βS (x, s, t) ,
(1) (1) S (x, s, t) = (1 −ω)Ib(x, t) + ω
4π
4π
I(x, s′, t)(s′, s)d′,
(2) (2) where I(x, s, t) is the distribution function of radiation intensity of photons, related to the
spatial position x, the direction of photon propagation s and time t. c is the light speed,
β is the extinction coefficient which is the inverse of the local photon MFP, i.e., β = 1/λ. S (x, s, t) is the source term of the RTE, and ω is the scattering albedo. The function Ib(x, t)
is the blackbody intensity. For equilibrium radiative problems, the blackbody intensity can Song et al. Advances in Aerodynamics (2020) 2:3 dynamics (2020) 2:3
Page 3 of 15 (2020) 2:3 Song et al. Advances in Aerodynamics (2020) 2:3 Page 3 of 15 Song et al. Advances in Aerodynamics be calculated according to energy conservation, be calculated according to energy conservation, Ib (x, t) = 1
4π
4π
I (x, s, t) d, Ib (x, t) = 1
4π
4π
I (x, s, t) d,
(3) (3) where is the solid angle domain of s. For radiative nonequilibrium problems, when the
temperature field of the medium is given, the blackbody intensity can be calculated by the
Stefan-Boltzmann law [37], i.e., Ib (x, t) = σT4 (x, t)
π
,
(4) Ib (x, t) = σT4 (x, t)
π
, (4) where σ is the Stefan-Boltzmann constant and T is the local temperature of the medium,
and (s′, s) is the scattering phase function, which describes the fraction of the radiative
energy scattered into the outgoing direction s from the incoming direction s′, and ′ is the
corresponding solid angle domain. 3.1
DUGKS with anisotropic scattering effects 3.1
DUGKS with anisotropic scattering effects In this section, the discrete unified gas kinetic scheme for gray radiative transfer equation
involving the anisotropic scattering effects (Eq. (1)) is constructed in detail. Similar to the
discretization approach in Ref [39], the solid angle space is discretized into M discrete
angles using the discrete ordinates method based on certain spherical quadratures, and Song et al. Advances in Aerodynamics (2020) 2:3 dynamics (2020) 2:3
Page (2020) 2:3 Song et al. Advances in Aerodynamics Page 4 of 15 correspondingly we obtain M discrete directions sk. With these discrete directions, the
RTE (1) can be expressed as 1
c
∂I (x, sk, t)
∂t
+ sk · ∇I (x, sk, t) = −βI (x, sk, t) + βS (x, sk, t) ,
(10)
S (x, sk, t) = (1 −ω) Ib (x, t) + ω
4π
M
m=1
[I (x, sm, t) (sm, sk) ωm] ,
(11) (10) (11) where k, m = 1, 2, ..., M, and ωm is the weight assigned to the discretized direction sm. Following Refs. [1, 32], integrating Eq. (10) on a control volume Vj centered at xj from
time tn to tn+1 = tn + △t, we can obtain I
xj, sk, tn+1
−I
xj, sk, tn
+ c△t
Vj
Fn+1/2 = cβ△t
2
S
xj, sk, tn+1
−I
xj, sk, tn+1
+ cβ△t
2
S
xj, sk, tn
−I
xj, sk, tn
, 2
2
(12) (12) where Fn+1/2 =
f
sk · nf
I
xf , sk, tn+1/2
△Sf ,
(13) Fn+1/2 =
f
sk · nf
I
xf , sk, tn+1/2
△Sf ,
(13) (13) is the flux across the cell interface, I
xj, sk, tn
denotes the cell averaged value for the dif-
fuse intensity at time tn with control volume of Vj located at xj along photon propagation
direction sk, nf is the outward unit normal vector at xf of an interface, and △Sf is the
corresponding interface area. The midpoint rule for the integration of the second term
on the left-hand of Eq. (12) and trapezoidal rule for the right-hand of Eq. (12) are used,
respectively. Two new distribution functions are introduced to remove the implicitness in
Eq. 3.1
DUGKS with anisotropic scattering effects (20)
¯I+
xf −skch, sk, tn
can be reconstructed by
¯I+
xf −skch, sk, tn
= ¯I+
xj, sk, tn
+
xf −skch −xj
· σj,
h
V
(21) ¯I
xf , sk, tn+1/2
= ¯I+
xf −skch, sk, tn
. (20) (20)
f
k
k
y
¯I+
xf −skch, sk, tn
= ¯I+
xj, sk, tn
+
xf −skch −xj
· σj,
xf −skch
∈Vj,
(21) (21) where σj is the slope of the distribution function ¯I+ in cell j. In the present study, the van
Leer limiter [40] is used to calculate the slope. The new distribution functions I, I+, ¯I, ¯I+ are all related to the original distribution
function I and the scattering phase function (cosψ). Their relations in the present work
can be finally obtained as I (x, s, t) =
4
4 + χ
¯I (x, s, t) +
χ
4 + χ (1 −ω) Ib (x, t)
+
χω
4π (4 + χ)
4π
I
x, s′, t
(s′, s)d′,
(22)
¯I+ (x, s, t) = 4 −χ
4 + 2χ
I (x, s, t) +
3χ
4 + 2χ (1 −ω) Ib (x, t)
+
ωχ
8π (2 + χ)
4π
I (x, s, t) + 2¯I+ (x, s, t)
(s′, s)d′,
(23)
I+ (x, s, t) = 4
3
¯I+ (x, s, t) −1
3
I (x, s, t) . (24) (22) (23) t) = 4
3
¯I+ (x, s, t) −1
3
I (x, s, t) . (24) (24) 3
3
In the isotropic scattering condition ( ≡1), Eqs. (22) and (23) can be solved explicitly
[1]. However, in anisotropic case, due to the complexity of the phase function ((s′, s)),
Eqs. (22) and (23) can not be solved explicitly. Here a simple iterative method is employed
as follows. 3.1
DUGKS with anisotropic scattering effects (12), I (x, s, t) = I (x, s, t) + χ
2 [I (x, s, t) −S (x, s, t)] ,
(14) (14) I+ (x, s, t) = I (x, s, t) −χ
2 [I (x, s, t) −S (x, s, t)] ,
(15)
where χ = cβ△t. Then Eq. (12) can be rewritten as (15) I
xj, sk, tn+1
= I+
xj, sk, tn
−c△t
Vj
Fn+1/2. (16) I
xj, sk, tn+1
= I+
xj, sk, tn
−c△t
Vj
Fn+1/2. (16) (16) In order to evaluate the cell interface flux at the half time-step Fn+1/2, we integrate
Eq. (10) along the characteristic line with a half time step, I
xf , sk, tn+1/2
−I
xf −skch, sk, tn
= cβh
2
S
xf , sk, tn+1/2
−I
xf , sk, tn+1/2
+ cβh
2
S
xf −skch, sk, tn
−I
xf −skch, sk, tn
,
(17) (17) where h = △t/2, and the trapezoidal rule is again used to evaluate the right-hand term
of Eq. (10). Another two new distribution functions are also introduced to remove the
implicitness in Eq. (17), where h = △t/2, and the trapezoidal rule is again used to evaluate the right-hand term
of Eq. (10). Another two new distribution functions are also introduced to remove the
implicitness in Eq. (17), ¯I (x, s, t) = I (x, s, t) + χ
4 [I (x, s, t) −S (x, s, t)] ,
(18)
¯I+ (x, s, t) = I (x, s, t) −χ
4 [I (x, s, t) −S (x, s, t)] . (19) (18) 4
¯I+ (x, s, t) = I (x, s, t) −χ
4 [I (x, s, t) −S (x, s, t)] . (19) ¯I+ (x, s, t) = I (x, s, t) −χ
4 [I (x, s, t) −S (x, s, t)] . (19) (19) Song et al. Advances in Aerodynamics (2020) 2:3
Page 5 of 15 erodynamics (2020) 2:3
Page 5 (2020) 2:3 Song et al. Advances in Aerodynamics Page 5 of 15 Substituting Eqs. (18) and (19) into Eq. (17), we can obtain ¯I
xf , sk, tn+1/2
= ¯I+
xf −skch, sk, tn
. 3.3
Algorithm In summary, the main procedure of the DUGKS from time step tn to tn+1 can be
summarized as follows: 1. Calculate the microflux Fn+1/2 at cell interface xf and at time tn+1/2. (a)
Calculate ¯I+ from I at each cell center with the iterative method
according to Eq. (23);
(b)
Reconstruct the slope σj of ¯I+ in each cell center;
(c)
Reconstruct the distribution function ¯I+ at xf −skch according to Eq. (21);
(d)
Calculate the distribution function ¯I at cell interface at time tn+1/2
according to Eq. (20);
(e)
Calculate the original distribution function I at cell interface and at time
tn+1/2 with the iterative method according to Eq. (25);
(f)
Calculate the microflux Fn+1/2 through each cell interface from
I
xf , sk, tn+1/2
according to Eq. (13). (a)
Calculate ¯I+ from I at each cell center with the iterative method
according to Eq. (23); (b)
Reconstruct the slope σj of ¯I+ in each cell center; (c)
Reconstruct the distribution function ¯I+ at xf −skch according to Eq. (21); (d)
Calculate the distribution function ¯I at cell interface at time tn+1/2
according to Eq. (20); (e)
Calculate the original distribution function I at cell interface and at time
tn+1/2 with the iterative method according to Eq. (25); (f)
Calculate the microflux Fn+1/2 through each cell interface from
I
xf , sk, tn+1/2
according to Eq. (13). 2. Calculate I+ at cell center and at time tn according to Eq. (24). 3. Update the cell averaged I in each cell from tn to tn+1 according to Eq. (16). When the transformed intensity distribution is known, the local incident radiation
energy can be calculated based on Eq. (14) G (x, t) =
2
2 + χ (1 −ω)
M
k=1
ωk I (x, sk, t) +
χ (1 −ω)
2 + χ (1 −ω)4πIb (x, t) ,
(29) (29) and the net radiative heat flux can be calculated based on Eqs. (14) and (19), and the net radiative heat flux can be calculated based on Eqs. (14) and (19), q (x, t) = 1
3
M
k=1
ωksk
I (x, sk, t) + 2¯I+ (x, sk, t)
. (30) (30) 3.1
DUGKS with anisotropic scattering effects First, the iteration procedure for the calculation of the original distribution
function I
xf , sk, tn+1/2
is Il+1
xf , sk, tn+1/2
=
4
4 + χ
¯I
xf , sk, tn+1/2
+
χ
4 + χ (1 −ω)Ibl
xf , tn+1/2
+
χω
4π (4 + χ)
M
m=1
N
j=0
ωm
Il
xf , sm, tn+1/2
CjPj(cosψ)
,
(25) (25) where l is the iteration index, and where l is the iteration index, and Ibl
xf , tn+1/2
= 1
4π
M
k=1
ωkIl
xf , sk, tn+1/2
. (26) (26) The iteration stops as the intensity is converged, i.e., | Il+1 −Il |< ϵ, where ϵ is a small
number which is set to be 10−4 in our simulations. Eq. (23) is also solved iteratively in
a similar way. We found that the number of iterations is less than 10 in almost all the
numerical tests in the present work. But for more complex problems, more advanced
acceleration technique should be employed. Finally, we note that with the use of the trapezoidal rule in Eqs. (12) and (17), the present
DUGKS is a semi-implicit scheme and the time step t is not limited by the scattering,
which is determined by the Courant-Friedrichs-Lewy (CFL) condition [41], t = α x
c ,
(27) t = α x
c , (27) where 0 < α < 1 is the CFL number, and x is the minimal grid spacing. where 0 < α < 1 is the CFL number, and x is the minimal grid spacing. Page 6 of 15 (2020) 2:3 Song et al. Advances in Aerodynamics 3.2
Boundary conditions In the present work, diffusely emitting and reflecting boundaries are considered. When
the wall is black, a photon is absorbed as it hits the wall, and a new photon in thermal
equilibrium with boundary temperature is emitted into the domain. When the wall is gray,
some of the incident photons are absorbed and the rest are reflected diffusively back to
the domain, depending on the reflectivity of the wall. The general boundary condition for
Eq. (1) can be expressed as I (xw, s, t) = εwIb (xw) + ρw
π
nw·s′<0
nw · s′
I
xw, s′, t
d′,
(28) (28) where εw is the diffuse emissivity, ρw is the diffuse reflectivity, and nw is the unit inner
normal vector at the boundary. Ib (xw) is the blackbody radiation intensity at the boundary
surface having a specified temperature. This boundary condition is implemented in the
DUGKS straightforwardly by replacing the s with each discrete angle sk, and evaluating
the integral with the numerical quadrature. 4
Numerical examples In this section, three radiative transfer problems in anisotropic scattering media are sim-
ulated to validate the proposed DUGKS, including the radiative transfer in a slab with
different wall temperatures, radiative transfer in a square domain with a hot wall, and dynamics (2020) 2:3
Pag Song et al. Advances in Aerodynamics (2020) 2:3 Song et al. Advances in Aerodynamics Page 7 of 15 radiative transfer in a square domain with collimated incidence. In each case, Cartesian
coordinates is used to discretize the physical space, and the Gauss-Legendre quadrature
[42] is used for angular discretization, where μ ∈[ −1, 1] and ϕ ∈[ 0, 2π] are the cosine
of zenith and azimuth angle, respectively. The CFL number is taken to be α = 0.5. For
steady problems, the system is regarded as converged as E < 10−6, where radiative transfer in a square domain with collimated incidence. In each case, Cartesian
coordinates is used to discretize the physical space, and the Gauss-Legendre quadrature
[42] is used for angular discretization, where μ ∈[ −1, 1] and ϕ ∈[ 0, 2π] are the cosine
of zenith and azimuth angle, respectively. The CFL number is taken to be α = 0.5. For
steady problems, the system is regarded as converged as E < 10−6, where E =
i,j |Gn
i,j −Gn+1000
i,j
|
i,j |Gn
i,j|
. (31) E =
i,j |Gn
i,j −Gn+1000
i,j
|
i,j |Gn
i,j|
. (31) 4.1
Radiation in a slab with different wall temperatures
In the first case, the DUGKS is applied to the radiative heat transfer in a slab with
thickness L filled with anisotropic absorbing-scattering media, as shown in Fig. 1. The
temperatures on the boundaries located at x = 0 and x = L are maintained at T0 and
T1, respectively, where T1 > T0. The angular space is discretized into 40 control angles. The physical space is discretized into Nx = 21 uniform cells. Two anisotropic media
composed of different particle clouds are considered, where the radiative properties are
summarized in Table 1. The scattering phase function is expressed by N-th Legendre
polynomial expansions obtained from Eq. (6). Figure 2 shows the non-dimensional radi-
ation energy b at different optical thickness τ = βL, where b = (G −G0)/(G1 −G0)
with G0 = 4σT4
0 and G1 = 4σT4
1. 4.2
Radiation in a square domain with a hot wall 4.2
Radiation in a square domain with a hot wall
In this subsection, we consider a square with the side length of L enclosed by four bound-
aries, as shown in Fig. 3. The bottom wall is kept hot with a non-dimensional temperature
of T1 = 1, while the other walls and the media are kept cold with a non-dimensional
temperature of T0 = 0. A grid size of Nx × Ny = 26 × 26 is used for physical space discretization. The
direction cosine of zenith angle μ
∈[ −1, 1] is discretized with Nμ
=
16 points,
while the azimuth angle ϕ ∈[ 0, 2π] is discretized into Nϕ = 16 points. The scat-
tering albedo is set to be ω
= 1.0. Four different kinds of anisotropic scattering
media and the isotropic scattering medium are considered. Table 2 shows the expan-
sion coefficients Cj and the asymmetry factors Cj/3 of the phase function for different
scattering media [17]. The anisotropic and isotropic scattering phase functions vary-
ing with the scattering angle are shown in Fig. 4. It can be observed that the phase
function for the anisotropic scattering media changes dramatically with the change of
the scattering angle, which will significantly influence the energy transfer in practical
problems. Figure 5 shows the net radiative heat flux in the y-direction along the vertical
centerline at x = L/2. The net hot surface radiative heat flux is shown in Fig. 6. For comparison, we also present the solutions of DOM [17] for this problem. It can
be seen the DUGKS results agree quite well with the DOM results for five differ-
ent media. From Fig. 5 and Fig. 6, the effects of the anisotropic scattering media
can be seen clearly. The forward scattering media transport more radiation heat
into the forward direction than the isotropic medium, while the backward scatter-
ing media transport less radiation heat into the forward direction than the isotropic
medium. The effect of the wall reflectivity on the radiative heat transfer is also examined with the
medium F2. Figure 7 shows the net radiative heat flux along the centerline of the enclosure The effect of the wall reflectivity on the radiative heat transfer is also examined with the
medium F2. Figure 7 shows the net radiative heat flux along the centerline of the enclosure Fig. 2 Nondimensional temperature distribution for anisotropic media between isothermal plates. 4
Numerical examples It can be seen that the results are in good agreement
with the exact results for τ = 0.1, 1, 5 [43]. When τ is large, the incident radiation energy
G can be solved by the limit diffusion equation such that the distribution of G is linear,
where the optical thick cases for τ = 21, 40, 100, 1000 agree well with that. It is noted that
the results are still satisfied even as x/λ = τ/Nx ≥1. These results confirm the capa-
bility of the present DUGKS method in simulating anisotropic heat transfer process for
one-dimensional problems. Fig. 1 Schematic of one-dimensional radiation heat transfer Fig. 1 Schematic of one-dimensional radiation heat transfer Page 8 of 15 Song et al. Advances in Aerodynamics (2020) 2:3 Table 1 Radiative properties of anisotropic absorbing scattering particle clouds
media
D
m
β
ω
orders N
A
5
2-i
0.01904
0.5634
26
B
5
2
0.06420
1
27 Table 1 Radiative properties of anisotropic absorbing scattering particle clouds
media
D
m
β
ω
orders N
A
5
2-i
0.01904
0.5634
26
B
5
2
0.06420
1
27 Table 1 Radiative properties of anisotropic absorbing scattering particle clouds 4.2
Radiation in a square domain with a hot wall 9, the side length of the square is L. All walls
and the interior domain are kept cold with the temperature T0. A collimate beam (Ic = π)
is incident through the top boundary. The collimate beam is normal to the top boundary. The discretization of the physical space is Nx × Ny = 26 × 26. The solid angle is dis-
cretized with Nμ × Nϕ = 16 × 16. The scattering albedo is taken to be ω = 1.0. The
parameters of phase function for anisotropic scattering media are shown in Table 2. In
this test, with the collimated incidence from the top wall the radiative transfer equation
now can be expressed as follows [18], Fig. 4 Scattering phase function Fig. 4 Scattering phase function 4.2
Radiation in a square domain with a hot wall Reference
data are taken from [43] (a) medium A, (b) medium B Fig. 2 Nondimensional temperature distribution for anisotropic media between isothermal plates. Reference
data are taken from [43] (a) medium A, (b) medium B Song et al. Advances in Aerodynamics (2020) 2:3
Page 9 of 15 Page 9 of 15 Song et al. Advances in Aerodynamics (2020) 2:3 Song et al. Advances in Aerodynamics Fig. 3 Schematic of two-dimensional radiation heat transfer with hot wall Fig. 3 Schematic of two-dimensional radiation heat transfer with hot wall at different wall reflectivity. It is noted that with the increasing of ρ, the radiative heat
flux qy decreases significantly, which is caused by the increasing of the radiative flux that
reflects from the walls. The net radiative heat flux along the centerline qy for different optical thickness with the
medium F2 is also shown in Fig. 8. The optical thickness range from τ = 0.01 to τ = 100
are considered. The DUGKS results agree well with the DOM results [17] from τ = 0.01
to τ = 10, and have the reasonable results when x/λ = τ/Ny ≥1. Furthermore, in the
literature [17], when the optical thickness is larger than 2.5, the DOM scheme should use
finer mesh (52 × 52) to eliminate the error. The DUGKS does not have this problem, as
shown in Fig. 8. These results again confirm the capability of the present DUGKS method
in simulating 2D anisotropic radiative scattering problems. Table 2 Expansion coefficients for the phase function, Cj
j
isotropy
F1
F2
B1
B2
0
1.00000
1.00000
1.00000
1.00000
1.00000
1
2.53602
2.00917
-0.56524
-1.20000
2
3.56549
1.56339
0.29783
0.50000
3
3.97976
0.67407
0.08571
4
4.00292
0.22215
0.01003
5
3.66401
0.04725
0.00063
6
3.01601
0.00671
7
2.23304
0.00068
8
1.30251
0.00005
9
0.53463
10
0.20136
11
0.05480
12
0.01099
Cj/3
0.00000
0.84534
0.66972
-0.18841
-0.40000
Note: F represents forward scattering media and B represents backward scattering media Song et al. Advances in Aerodynamics (2020) 2:3
Page 10 of 15 Page 10 of 15 Song et al. Advances in Aerodynamics Fig. 4 Scattering phase function
4.3
Radiation in a square domain with collimated incidence
To further test the capability of the present DUGKS method in simulating radiative trans-
fer problem with anisotropic scattering media, we now apply it to 2D square problems
with collimated incidence. As shown in Fig. 4.3
Radiation in a square domain with collimated incidence To further test the capability of the present DUGKS method in simulating radiative trans-
fer problem with anisotropic scattering media, we now apply it to 2D square problems
with collimated incidence. As shown in Fig. 9, the side length of the square is L. All walls
and the interior domain are kept cold with the temperature T0. A collimate beam (Ic = π)
is incident through the top boundary. The collimate beam is normal to the top boundary. The discretization of the physical space is Nx × Ny = 26 × 26. The solid angle is dis-
cretized with Nμ × Nϕ = 16 × 16. The scattering albedo is taken to be ω = 1.0. The
parameters of phase function for anisotropic scattering media are shown in Table 2. In
this test, with the collimated incidence from the top wall the radiative transfer equation
now can be expressed as follows [18], Fig. 5 Effect of anisotropic media on the centerline nondimensional net radiative heat flux in the y-direction
(ρ = 0, ω = 1.0, τ = 1.0) Fig. 5 Effect of anisotropic media on the centerline nondimensional net radiative heat flux in the y-direction
(ρ = 0, ω = 1.0, τ = 1.0) Page 11 of 15 Song et al. Advances in Aerodynamics (2020) 2:3 (2020) 2:3 Song et al. Advances in Aerodynamics Fig. 6 Nondimensional net radiative heat flux at the hot wall (ρ = 0, ω = 1.0, τ = 1.0) Fig. 6 Nondimensional net radiative heat flux at the hot wall (ρ = 0, ω = 1.0, τ = 1.0) 1
c
∂I(x, s, t)
∂t
+ s · ∇I(x, s, t) = −βI(x, s, t) + βS (x, s, t)
+ βω
4π Ic(sc, s)exp
−
τ −τy
,
(32) (32) where sc is incident angle of the beam, and τy = βy. where sc is incident angle of the beam, and τy = βy. The walls energy losses for the different phase functions are illustrated in Fig. 10. Fig. 10a shows the reflected components of the radiative flux along the top wall, and the
transmitted components of the radiative flux along the bottom wall are shown in Fig. 10b. It is observed that the DUGKS results agree well with the DOM results [18] in all cases. them agree well with the DOM solutions [18]. These tests clearly show that the DUGKS
is an accurate solver for radiative transfer with anisotropic scattering media. them agree well with the DOM solutions [18]. These tests clearly show that the DUGKS
is an accurate solver for radiative transfer with anisotropic scattering media. them agree well with the DOM solutions [18]. These tests clearly show that the DUGKS
is an accurate solver for radiative transfer with anisotropic scattering media. 4.3
Radiation in a square domain with collimated incidence From these figures, the same conclusions as in section 4.2 that backward scattering media
reflect more radiative energy while forward scattering media transmit more energy can
also be obtained. The side walls energy losses for the different phase functions are shown
in Fig. 10c. The net radiative heat flux along the centerline qy for different anisotropic scattering
media and different optical thickness are shown in Fig. 11 and Fig. 12, respectively. All of Fig. 7 Effect of wall reflectivity on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ω = 1.0, τ = 1.0) Fig. 7 Effect of wall reflectivity on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ω = 1.0, τ = 1.0) Song et al. Advances in Aerodynamics (2020) 2:3 Page 12 of 15 (2020) 2:3 Song et al. Advances in Aerodynamics Fig. 8 Effect of optical thickness on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ρ = 0, ω = 1.0) Fig. 8 Effect of optical thickness on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ρ = 0, ω = 1.0) Funding Funding
The National Key R&D Program of China (No. 2018YFE0180900) and the Fundamental Research Funds for the Central
Universities (No. 2019kfyXMBZ040). Funding
The National Key R&D Program of China (No. 2018YFE0180900) and the Fundamental Research Funds for the Central
Universities (No. 2019kfyXMBZ040). Competing interests Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. 5
Summary In present work, we developed a discrete unified gas kinetic scheme for radiative trans-
fer with anisotropic scattering media based on the radiative transfer equation. Due to
the complex anisotropic scattering phase function which is calculated by the Legendre
polynomial expansion, a simple and efficient iterative approach is employed. The present
DUGKS has been validated by a set of radiative transport problems including the radiative
transfer in a slab with different wall temperatures, radiative transfer in a square domain
with a hot wall, and radiative transfer in a square domain with collimated incidence. All
results agree well with other different numerical schemes. In these cases, even if the mesh Fig. 9 Schematic of two-dimensional radiation heat transfer with collimated incidence Fig. 9 Schematic of two-dimensional radiation heat transfer with collimated incidence Song et al. Advances in Aerodynamics (2020) 2:3
Page 13 of 15 Song et al. Advances in Aerodynamics (2020) 2:3 Page 13 of 15 Song et al. Advances in Aerodynamics Fig. 10 Effect of anisotropic media along the walls energy losses (ρ = 0, ω = 1.0, τ = 1.0). a the reflected
flux loss on top wall, b the transmitted flux loss on bottom wall, c the flux loss on side walls Fig. 10 Effect of anisotropic media along the walls energy losses (ρ = 0, ω = 1.0, τ = 1.0). a the reflected
flux loss on top wall, b the transmitted flux loss on bottom wall, c the flux loss on side walls size is larger than the photon mean free path, the results predicted by the present scheme
are still reliable. As the scheme has the nice unified preserving properties and the mesh
size is not restricted by the photon mean free path, the present DUGKS will be an efficient
and accurate tool to describe the multiscale anisotropic radiative heat transfer. It will also
be easy to handle more complex radiative transfer problems with unstructured meshes,
due to its finite volume property. Fig. 11 Effect of anisotropic media on the centerline nondimensional net radiative heat flux in the
y-direction (ρ = 0, ω = 1.0, τ = 1.0) Fig. 11 Effect of anisotropic media on the centerline nondimensional net radiative heat flux in the
y-direction (ρ = 0, ω = 1.0, τ = 1.0) Song et al. Advances in Aerodynamics (2020) 2:3
Page 14 of 15 Song et al. Acknowledgments
h
k Acknowledgments
This work was supported by the National Key R&D Program of China (No. 2018YFE0180900) and the Fundamental
Research Funds for the Central Universities (No. 2019kfyXMBZ040). 5
Summary Advances in Aerodynamics (2020) 2:3 Page 14 of 15 (2020) 2:3 Song et al. Advances in Aerodynamics Fig. 12 Effect of optical thickness on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ρ = 0, ω = 1.0) Fig. 12 Effect of optical thickness on the centerline nondimensional net radiative heat flux in the y-direction
(Medium: F2, ρ = 0, ω = 1.0) Availability of data and materials All data generated or analyzed during this study are included in this published article. References References
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22. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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H157 cells were transfected with control (Ctrl), RARα/γ, RARβ, or RARα/γ plus
RARβ siRNAs for 72 h. Cellular total RNA was then extracted from these cells for
detection of the given RAR genes with real-time quantitative RT-PCR. The data are
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RARβ siRNAs for 72 h. Cellular total RNA was then extracted from these cells for
detection of the given RAR genes with real-time quantitative RT-PCR. The data are
means ± SDs of triplicate determinations. Fig. S3. Validation of knockdown efficiencies of RAR siRNAs used in the study. H157 cells were transfected with control (Ctrl), RARα/γ, RARβ, or RARα/γ plus
RARβ siRNAs for 72 h. Cellular total RNA was then extracted from these cells for
detection of the given RAR genes with real-time quantitative RT-PCR. The data are
means ± SDs of triplicate determinations. Dhandapani et al. Fig. S3
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Associação entre desordens musculoesqueléticas e gastos com cuidado à saúde entre pacientes do Sistema Único de Saúde
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ARTIGO ORIGINAL ARTIGO ORIGINAL Associação entre desordens musculoesqueléticas e gastos com
cuidado à saúde entre pacientes do Sistema Único de Saúde Jamile Sanches Codogno1,2, Bruna Camilo Turi1, Rômulo Araújo Fernandes1,2, Mariana Rotta Bonfim1,
Carlos Marcelo Pastre3, Henrique Luiz Monteiro1,4 Associa
Associa
Associa
Associa
Association betw
tion betw
tion betw
tion betw
tion between m
een m
een m
een m
een musculosk
usculosk
usculosk
usculosk
usculoskel-
el-
el-
el-
el-
etal disor
etal disor
etal disor
etal disor
etal disorder
der
der
der
ders and healthcar
s and healthcar
s and healthcar
s and healthcar
s and healthcare e
e e
e e
e e
e expen-
xpen-
xpen-
xpen-
xpen-
ditur
ditur
ditur
ditur
ditures among pa
es among pa
es among pa
es among pa
es among patients fr
tients fr
tients fr
tients fr
tients from the
om the
om the
om the
om the
Br
Br
Br
Br
Brazilian pub
azilian pub
azilian pub
azilian pub
azilian public healthcar
lic healthcar
lic healthcar
lic healthcar
lic healthcare system
e system
e system
e system
e system Associação entre desordens musculoesqueléticas e gastos com
cuidado à saúde entre pacientes do Sistema Único de Saúde ABSTRACT Study Design: Cross-sectional. Study Design: Cross sectional. Objectives: (i) To identify the prevalence of musculoskeletal disorders, (ii) to identify musculoskeletal
disorders correlates and (iii) to identify the existence of relationship between musculoskeletal disorders
and healthcare expenditures in adults attended to by the public healthcare system. Methods: Cross-
sectional study involving 963 adults (over 50 years of age and of both sexes) treated in basic healthcare
units (BHU). The participants answered questionnaires about self-reported morbidity, economic condition,
musculoskeletal symptoms, occupational activity behavior, physical therapy consultation and prescription
drugs purchased. Weight and height were obtained for body mass index computation. Healthcare expen-
ditures in the last 12 months were computed. Results: The prevalence of musculoskeletal symptoms was
65%. Patients diagnosed with musculoskeletal system/connective tissue diseases (OR= 4.11 [2.98 – 5.67])
and people who needed to lift loads at work (OR= 1.45 [1.27 – 1.66]) presented higher likelihood to report
the outcome. Moreover, occurrence of musculoskeletal symptoms in shoulders, low back, knees and
ankles/feet were positively related to increased prescription drugs used and medical consultations. Conclusion: There is a high occurrence of musculoskeletal symptoms in Brazilian patients, which is
affected by sex and occupational activity, and is positively related to increased healthcare expenditures. Key Words: Musculoskeletal Pain; Costs and Cost Analysis; Motor Activity; Public Health. 2. PhD. Professor. Department of Physical Education. UNESP,
Presidente Prudente, Brazil. 3. PhD. Professor. Department of Physical Therapy. UNESP, Pre-
sidente Prudente, Brazil. 4. PhD. Professor. Department of Physical Education. UNESP,
Bauru, Brazil. Conflict of interest: The authors declare that there is no conflict of interest. Medicina (Ribeirão Preto) 2015;48(2): 143-150 1. PhD. Graduate Program in Movement’s Sciences. UNESP –
Univ Estadual Paulista, Rio Claro, Brazil.
2. PhD. Professor. Department of Physical Education. UNESP,
Presidente Prudente, Brazil.
3. PhD. Professor. Department of Physical Therapy. UNESP, Pre-
sidente Prudente, Brazil.
4. PhD. Professor. Department of Physical Education. UNESP,
Bauru, Brazil. Conflict of interest: The authors declare that there is no conflict of interest. Sample This project was a cross-sectional study con-
ducted from August 2010 to December 2010 in the
city of Bauru. The study was previously approved by
the Ethics Committee Group from Sao Paulo State
University (UNESP), Bauru campus (Process number
1046/46/01/10) and all subjects signed a standard
written consent form. The sample size was estimated
based on the percentage of Brazilian population that
are covered only by the public health system (60%)8
and using parameters as 3.8% error (arbitrary because
there are no other similar studies), 5% statistical sig-
nificance and design effect of 50%. A sample size of
960 participants was estimated to be representative
(minimum of 192 in each Basic Healthcare Unit
[BHU]). The city was stratified into five geographi-
cal regions (south, west, north, east and center) and
the major BHU from each geographical region was
selected to the study (Municipality Secretariat cho-
sen the BHU). As inclusion criteria, the patients
should: i) be aged 50 years old or more; ii) have at
least one appointment in the last six months in the
BHU, iii) don’t have restriction to the practice of
physical activity. The medical records of all patients
who attended in the last six months were compiled
into a list and, at least, 250-500 patients were ran-
domly selected for an interview from each BHU. One of the most prevalent musculoskeletal dis-
orders among middle-aged adults is low back pain,
and hence is widely investigated1-3. In Brazil, where a
large percentage of the population is attended to by
the public healthcare system7,8,9, low back pain and
other musculoskeletal disorders are common reasons
for sick leave and disability pension3, which generate
high costs to government and significant healthcare
expenditure. So, due to the population ageing and changes
in the level of income in Brazil, the demand for health
care services and expenditures are expected to grow
substantially4. And in spite of the fact that muscu-
loskeletal disorders are so frequent, there is limited
knowledge about the percentage of patients affected
by these symptoms within public healthcare system
and its relationship with healthcare expenditures. The purposes of this study were (i) to identify
the prevalence of musculoskeletal disorders, (ii) to
identify musculoskeletal disorders correlates and (iii)
to identify the existence of relationship between mus-
culoskeletal disorders and healthcare expenditures
among adults attended to by the Brazilian public
healthcare system. Intr
Intr
Intr
Intr
Introduction
oduction
oduction
oduction
oduction Musculoskeletal disorders constitute a group of
complications which affect health perception1, mobil-
ity and quality of life. Therefore, preventative actions
targeting to identify its correlates constitute a relevant
action for health professionals.1,2,3 Previous studies
have identified some risk factors related to these dis-
eases, such as gender, age and occupational activity.4,5,6 Medicina (Ribeirão Preto) 2015;48(2): 143-150 Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. RESUMO Delineamento: transversal. Objetivos: (i) Identificar a ocorrência de desordens musculoesqueléticas, (ii) identificar seus determi-
nantes e (iii) suas relações com gastos com saúde em adultos atendidos pelo Sistema Único de Saúde. Métodos: Estudo transversal com 963 adultos, com 50 anos e de ambos os sexos tratados em unidades
básicas de saúde. Participantes responderam questionários sobre doenças, condição econômica, desor-
dens musculoesqueléticas, atividade ocupacional, consultas de fisioterapia e compra de medicamentos;
peso e estatura foram mensurados para calcular o índice de massa corporal. Custos com saúde foram
computados retroagindo 12 meses. Resultados: A prevalência de desordens musculoesqueléticas foi
65%, sendo maior em pacientes com doenças do tecido conjuntivo (OR=4,11 [2,98-5,67]) e pessoas que
levantam cargas no trabalho (OR=1,45 [1,27-1,66]). Além disso, a ocorrência de desordens musculoes-
queléticas nos ombros, região lombar, joelhos e tornozelos/pés foram relacionados a maiores gastos
com medicamentos e consultas médicas. Conclusão: A ocorrência de desordens musculoesqueléticas
foi elevada entre estes pacientes, é afetada por sexo e atividade ocupacional e positivamente relaciona-
da com gastos com saúde. Palavras-chave: Dor Musculoesquelética; Custos e Análise de Custo; Atividade Motora; Saúde Pública. Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Independent variables There is a previously validated version of this ques-
tionnaire for the Brazilian language.13,14 In all patients, the medical records and the self-
report were used to determine their diseases. The In-
ternational Statistical Classification of Diseases and
Related Health Problems (ICD-10, released in 2008)
was used both to identify and cluster diseases of the
musculoskeletal system and connective tissue (code
ICD M00-M99) (dichotomized in either presence or
absence of any disease related to ICD M00-M99). Economic condition was assessed through the ques-
tionnaire previously validated by the Brazilian Crite-
rion for Economic Classification - ABEP11, which
estimates the family income taking into account: edu-
cational level, number of home appliances, number
of cars in the house, as well the presence of domestic
servants. In this questionnaire, individuals are classi-
fied into five categories from A (highest socioeco-
nomic level) to E (lowest) and in our study the sam-
ple was stratifies as follow: High economic condition
(categories: A and B) and Low economic condition
(categories: C, D and E). Behaviors related to occupational activity and
being overweight Behavior related to occupational activ-
ity was assessed by four questions in section 1 of
Baecke’s Questionnaire15. Behavior at work was as-
sessed as follows: (i) “At work I sit”, (ii) “At work I
stand”, (iii) “At work I walk” and (iv) “At work I lift
heavy loads”. For each behavior the patient provided
a response based on the Likert scale (never, seldom,
sometimes, often or always [score 1, 2, 3, 4 and 5,
respectively]). Patients with no formal job received a
score of zero. To calculate body mass index (BMI [in
kg/m2]), body mass was measured with a Filizola elec-
tronic scale (precision 0.1 kg) (Filizola PL 150,
Filizola Ltda) and the height with a wall-mounted
stadiometer [precision 0.1 cm (Sanny®, São Paulo,
Brazil)] with the subjects in standing position, breath-
ing normally and with arms relaxed beside the trunk. The record was made at the end of a normal expira-
tion. All anthropometric measurements were made
following the recommendations proposed by Lohman
et al.16. Overweight and obesity were identified as
values ranging from 25 to 29.9kg/m2 and e”30kg/m2,
respectively.17 The medical records (self-report was used as
supplementary data) were used to identify both the
number of physical therapy consultations and prescrip-
tion drugs purchased in the month immediately prior
to the study (only prescription drugs used regularly
were taken into account: dichotomized into either pres-
ence or absence). Moreover, sex and age (either <65
years or e”65 years) were treated as potential
confounders. Sample After phone contact an interview was sched-
uled and, after data collection, the final sample size
was 963 patients. 144 Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Healthcare expenditures The procedures to estimate healthcare expen-
ditures are described in previous publication18. Briefly,
the period of time considered to assess expenditures
was one year prior to the interview. Expenditures due
were estimated including all items registered in the
medical records from each patient [laboratory tests
performed in private laboratories paid by BHU; medi-
cal consultations (e.g. dentists, gynecologist, obste-
trician, general practitioner and psychiatrist); prescrip-
tion drugs discharged]. A specific standard table, in-
cluding public healthcare reimbursement values, were
provided by BHU offices and used in order to com-
pute monetary values of laboratory tests and medical
specialist consultations. Invoices obtained from BHUs
were used to compute the dosage and the market prices
of medication used by patients. All expenditures were
computed in the Brazilian currency (Real) and con-
verted to US dollar using the average value of the
dollar against the Brazilian currency in the 12 months
of 2009. In the study, healthcare expenditures were
presented as: medical consultations, prescription drugs
and overall. Outcome The “standardized Nordic questionnaire for the
analysis of musculoskeletal symptoms” proposed by
Kuorinka et al.12 was used to assess the presence of
musculoskeletal symptoms (ache, pain, discomfort
and numbness) in different anatomical areas (neck,
shoulders, upper back, elbows, shoulders, low back,
wrists / hands, hips / thighs, knees and ankles / feet). The questionnaire provides four dichotomized ques-
tions (either yes or no) for each anatomical area: (i)
“Have you at any time during the last 12 months had
trouble in…”; (ii) “During the last 12 months have
you been prevented from carrying out normal activi-
ties (e.g. job, housework, hobbies) because of this
trouble in…”; (iii) “during the last 12 months have
you seen a physician for this condition…”; (iv) “Dur-
ing the last 7 days have you had trouble in…”. The
presence of the outcome was the positive response to
all four questions in any of the nine anatomical areas. 145 Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. R
RR
RResults
esults
esults
esults
esults The sample was composed of 963 patients of
both genders (707 women [73.4%]) and their age
ranged from 50 to 96 years (mean 64.7 years). The
prevalence of musculoskeletal symptoms was 65%
(95%CI: 61.9% – 68.1% [n= 626]), in addition, there
was a high occurrence of diseases of the musculoskel-
etal system and connective tissue in this sample
(72.1% [95%CI: 69.2% – 74.9%]). Twelve different
diseases were diagnosed in this group; those of higher
occurrence were as follows: low back pain (55.6%),
arthritis / osteoarthritis (38.1%), scoliosis (23%) and
osteoporosis (20.6%). Similarly, the prevalence of
overweight/obesity was high (80%). During the month
prior to the study, only 8% (95%CI: 6.2-9.7%) and
35% (IC95%: 31.9% - 38%) of the patients had physi-
cal therapy consultations and bought prescription
drugs, respectively (Table 1). For numerical variables, descriptive statistics
were composed of values of mean, median, 95% con-
fidence interval (95%CI), standard-deviation (SD) and
interquartile range. The Spearman correlation was used
to assess the relationship between the dependent and
several independent variables. Categorical data were
expressed as rates and its 95%CI. Chi-square test (÷2)
analyzed the existence of association (Yates’ correc-
tion was applied in 2 x 2 contingence tables), as well
as, binary logistic regression (presented as values of
odds ratio [OR] and its 95%CI [OR95%CI]) indicated
the magnitude of these associations. In binary logistic
regression, all independent variables with p-value
<0.05 in ÷2 were inserted simultaneously in the
multivariable model. Statistical procedures were per-
formed by the software BioEstat (release 5.0) and all
statistical analyzes were set at p-value < 0.05. Table 1. General characteristics of the analyzed patients (Brazil, n= 963). R
RR
RResults
esults
esults
esults
esults Descriptive Statistic (n= 963)
Variables
Mean ± SD (95%CI)
Median (IR)
K-S
Numerical
Age (years)
64.7±9.1 (64.1 – 65.3)
63.7 (13.6)
0.001
Body weight (kg)
72.8±15.5 (71.8 – 73.8)
71.3 (19.3)
0.001
Height (m)
1.57±0.08 (1.56 – 1.57)
1.56 (0.11)
0.001
BMI (kg/m2)
29.4±5.8 (29.1 – 29.8)
28.6 (7)
0.001
Categorical
n (% [95%CI])
Diseases (%)
ICD M00-M99
694 (72.1 [69.2 – 74.9])
Overweight / Obesity (%)
Normal
193 (20 [17.5 – 22.5])
Overweight
380 (39.5 [36.3 – 42.5])
Obesity
390 (40.5 [37.4 – 43.6])
Physical Therapy (%)
Yes
77 (08 [6.2 – 9.7])
No
886 (92 [90.2 – 93.7])
Prescription Drugs Purchased (%)
Yes
337 (35 [31.9 - 38])
No
626 (65 [61.9 - 68])
Economic condition (%)
High
162 (16.8 [14.4 – 19.1])
Low
801 (83.2 [80.8 – 85.5])
SD= standard-deviation; K-S= Kolmogorov-Smirnov’s test; IR= interquartile range; BMI= body mass index; ICD M00-M99= diseases of
the musculoskeletal system and connective tissue. SD= standard-deviation; K-S= Kolmogorov-Smirnov’s test; IR= interquartile range; BMI= body mass index; ICD M00-M99= diseases of
the musculoskeletal system and connective tissue. 146 Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. The self-report of musculoskeletal symptoms
was significantly associated with the female gender
(p-value= 0.001), overweight/obesity (p-value=
0.031), lower economic condition (p-value= 0.013),
physical therapy consultation (p-value= 0.004), pre-
scription drugs used (p-value= 0.029) and diagnosis
of some diseases of the musculoskeletal system and
connective tissue (p-value= 0.001). On the other hand,
age was not significantly associated (p-value= 0.279)
(Table 2). Spearman correlation analyzed the rela-
tionship between the score for occupational activity
related behavior (walk [rho= 0.003; p-value= 0.935]; sit position [rho= 0.026; p-value= 0.423]; standing
position [rho= 0.038; p-value= 0.238]; lift heavy load
walk [rho= 0.178; p-value= 0.001]) and the presence
of musculoskeletal symptoms. After adjustment (all independent variables
inserted simultaneously), the female gender (OR=
1.86 [OR95%CI: 1.53 – 2.57]), diagnosis of some dis-
ease of the musculoskeletal system and connective
tissue (OR= 4.35 [OR95%CI: 3.18 – 5.95]) and physi-
cal therapy consultation in the last month (OR= 2.01
[OR95%CI: 1.08 – 3.75]) still associated with muscu-
loskeletal symptoms. Patients who have to lift heavy Table 2. §= numerical variable (score ranging from 0 to 5); *= chi-square test with p-value < 0.05%; OR Crude= odds ratio without adjust; OR Adjusted=
odds ratio adjusted by all independent variables with chi-square p-value < 0.05%; 95%CI= 95% confidence interval; BMI= body mass
index; OW= overweight; OB= obesity; EC= economic condition; ICD M00-M99= diseases of the musculoskeletal system and connective
tissue R
RR
RResults
esults
esults
esults
esults Chi-square test and multivariate association between musculoskeletal symptoms and independent
variables among patients of the Brazilian Public Healthcare system (Brazil, n= 963). Outcome: musculoskeletal symptoms
Binary Logistic Regression
Independent Variables
%
OR Crude (OR95%CI)
OR Adjusted (OR95%CI)
Gender*
Male
48
1.00
1.00
Female
71.1
2.66 (1.98 – 3.57)
1.89 (1.35 – 2.64)
Age (years)
<65
66.7
1.00
—-
e”65
63.1
0.85 (0.65 – 1.11)
—-
EC*
High
56.2
0.63 (0.45 – 0.89)
0.79 (0.53 – 1.18)
Low
66.8
1.00
1.00
Prescription drugs purchased*
No
61.2
1.00
1.00
Yes
70.6
1.52 (1.15 – 2.00)
1.10 (0.80 – 1.52)
Physical Therapy*
No
63.7
1.00
1.00
Yes
80.5
2.36 (1.32 – 4.21)
2.00 (1.05 – 3.78)
ICDM00-M99*
No
37.2
1.00
1.00
Yes
75.8
5.29 (3.91 – 7.15)
4.11 (2.98 – 5.67)
BMI*
Normal
64.2
1.00
—-
OW
59.5
0.81 (0.57 – 1.16)
—-
OB
70.8
1.34 (0.93 – 1.94)
—-
Sit at work§
—-
—-
0.95 (0.82 – 1.09)
Standing at work§
—-
—-
1.06 (0.85 – 1.32)
Walk at work§
—-
—-
0.82 (0.65 – 1.03)
Lift heavy load§
—-
—-
1.45 (1.27 – 1.66)
§= numerical variable (score ranging from 0 to 5); *= chi-square test with p-value < 0.05%; OR Crude= odds ratio without adjust; OR Adjusted=
odds ratio adjusted by all independent variables with chi-square p-value < 0.05%; 95%CI= 95% confidence interval; BMI= body mass
index; OW= overweight; OB= obesity; EC= economic condition; ICD M00-M99= diseases of the musculoskeletal system and connective
tissue. Table 2. Chi-square test and multivariate association between musculoskeletal symptoms and independent
variables among patients of the Brazilian Public Healthcare system (Brazil, n= 963). §= numerical variable (score ranging from 0 to 5); *= chi-square test with p-value < 0.05%; OR Crude= odds ratio without adjust; OR Adjusted=
odds ratio adjusted by all independent variables with chi-square p-value < 0.05%; 95%CI= 95% confidence interval; BMI= body mass
index; OW= overweight; OB= obesity; EC= economic condition; ICD M00-M99= diseases of the musculoskeletal system and connective
tissue. 147 Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. loads at work presented an increased occurrence of
musculoskeletal symptoms too (OR= 1.45 [OR95%CI:
1.27 – 1.66]). Discussion
Discussion
Discussion
Discussion
Discussion This cross-sectional study was developed with
patients of the Brazilian public healthcare system and
found elevated occurrences of musculoskeletal symp-
toms, which seems significant related to higher
healthcare expenditures. In this sample, lifting heavy loads at work was
related to the presence of the outcome. Musculoskel-
etal symptoms have a high burden among the disor-
ders classified as “occupational diseases”25 and its
presence has a relevant range according to different
types of occupational activity.27,28,29 It is noteworthy
to identify that a lower economic condition was asso-
ciated with musculoskeletal symptoms, but only in
crude analysis. In fact, occupational activities of
higher physical effort are observed more frequently
in groups of lower economic condition and, therefore,
the association observed in crude analysis could be
explained, at least in part, by this relationship between
physical effort at work and economic condition. Agreeing with such idea, a recent study found asso-
ciation between stress level and several musculoskel-
etal outcomes, with higher ratio of musculoskeletal
disorders among workers with high demand job.30 There was a high occurrence of musculoskel-
etal symptoms among the analyzed patients (65%). A
large research in US showed that low back pain
(24.3%) and neck pain (10.5%) are the highest preva-
lent musculoskeletal symptoms among agricultural
workers19. In Taiwan, a nationwide study found that
37% of the workers had musculoskeletal disorders20. An explanation for the highest prevalence of muscu-
loskeletal symptoms found in our study may be fact
of the target population been located within Basic
Healthcare Units, which nevertheless should be
considered a relevant concern by public health ad-
ministers, because worldwide this outcome is re-
lated to absenteeism / lower productivity, increased
healthcare costs and a lower quality of life gener-
ated by pain.21-24 In our study, musculoskeletal symptoms were
more frequent among women and this finding is simi-
lar to previous data.6 A large variety of hypothesis
could be postulated to explain this result. Firstly, in
modern society, women have double the activities (job It was found a positive relationship between
musculoskeletal symptoms and higher healthcare ex-
penditures. In fact, low back pain is an outcome widely
observed among adult population1,2,3 and its relation- tween musculoskeletal symptoms in different anatomical regions and healthcare
ts. Table 3. Relationship between musculoskeletal symptoms in different anatomical regions and healthcare
expenditures among adults. Table 3. Relationship between musculoskeletal symptoms in different anatomical regions and healthcare
expenditures among adults. R
RR
RResults
esults
esults
esults
esults ship with increased prescription drugs discharged it
is not a surprise, because adults with low back pain
have the medication use as a tool to decrease pain
and to possibility the maintenance of daily activities,
such as occupational activities. On the other hand,
surprisingly, our findings identified that musculoskel-
etal symptoms in lower limb articulations were posi-
tively related to prescription drugs discharged and
medical consultations. Apparently, this finding could
be supported by the negative effect of these symp-
toms over human gait, which is a fundamental human
action related to most human behaviors. Musculoskeletal symptoms in shoulders, low
back, knees and ankles/feet were positively related to
increased expenditures related to prescription drugs
discharged and medical consultation (Table 3). Discussion
Discussion
Discussion
Discussion
Discussion Upper
Wrists/
Low
Hips/
Ankles/
Healthcare
Neck
Shoulders
back
Elbows
hands
back
Thighs
Knees
Feet
Expenditures
Rho
rho
rho
rho
rho
rho
rho
rho
rho
Consultations
0.010
0.025
0.028
-0.008
-0.001
0.039
0.050
0.020
0.072*
Medication
0.013
0.077*
0.033
0.054
0.004
0.067*
0.037
0.094*
0.126*
Overall
0.007
0.045
0.022
0.005
0.003
0.040
0.040
0.035
0.089*
rho= Spearman hank order correlation; *= p-value < 0.05. 148 Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br 4. Kilsztajn S, Rossbach A, Câmara MB, Carmo MS. Serviços
de saúde, gastos e envelhecimento da população brasileira. Rev bras estud popul. 2003;20:93-108. and housework activities) and this condition could
expose the female gender to physical overloads1. Moreover, the above mentioned double activities
could act synergistically with gender differences re-
lated to females (e.g. lower muscle mass and higher
body fatness)1,31 and, therefore, could explain our find-
ings. 5. Picoloto D and Silveira ED. Prevalence of musculoskeletal
symptoms and associated factors among metal industry work-
ers in Canoas - RS. Ciênc Saúde Coletiva. 2008; 13:507-16. 6. Pinheiro FA, Tróccoli, BT and Paz MG. Psychosocial predic-
tors of musculoskeletal symptoms: the relevance of medi-
ated and moderated relationships. Psicol Reflex Crit. 2006;
19:142-150. g
The association between musculoskeletal symp-
toms with physical therapy consultation and diagno-
sis of diseases of the musculoskeletal system and con-
nective tissue gave expected results. On the other hand,
although there was a high occurrence of musculoskel-
etal symptoms, only a few patients had physical
therapy consultations. Low rates of physical therapy
consultations constitute a pattern previously observed
in the Brazilian population9, but it is noteworthy that
high medication use constitutes another one21. Appar-
ently, in the Brazilian public healthcare system, the
use of medication has been a tool widely adopted to
treat musculoskeletal symptoms, instead of non-phar-
macological actions.23 The concerns described above
indicate that the promotion of non-pharmacological
actions related to lower medication use and muscu-
loskeletal symptoms (such as physical activity prac-
tice) are necessary among these patients. 7. Bastos GA, Duca GF, Hallal PC, Santos IS. Utilization of medi-
cal services in the public health system in the Southern Bra-
zil. Rev Saúde Pública. 2011; 45:475-54. 8. Kilsztajn S, Silva DF, Camara MB, Ferreira VS. Level of pri-
vate health insurance coverage and regional distribution of
public health expenditure. Saude Soc. 2001; 10:35-41. 9. Discussion
Discussion
Discussion
Discussion
Discussion Siqueira FV, Facchini LA and Hallal PC. Epidemiology of physi-
otherapy utilization among adults and elderly. Rev Saúde
Pública. 2005; 39:663-8. 10. Sistema Único de Saúde. [http://sigtap.datasus.gov.br/tabe-
la-unificada/app/sec/consultarServicoClassificacao.jsp]. 11. Associação Brasileira de Empresas de Pesquisa. Critério de
classificação econômica Brasil. [http://www.abep.org/novo/
Content.aspx?ContentID=302]. 12. Kuorinka I, Jonsson B, Kilbom A, et al. Standardized Nordic
questionnaires for the analysis of musculoskeletal symptoms. Appl Ergon. 1987; 18:233-7. 13. Pinheiro FA, Troccoli BT and Carvalho CV. Validity of the Nor-
dic Musculoskeletal Questionnaire as morbidity measurement
tool. Rev Saúde Pública. 2002; 36:307-12. 14. Barros EN and Alexandre NM. Cross-cultural adaptation of
the Nordic musculoskeletal questionnaire. Int Nurs Rev. 2003;
50:101-8. Limitations should be recognized. In fact, the
cross-sectional design constitutes the main limitation
due to non-possibility of causality statements. There-
fore, prospective studies analyzing this issue in pa-
tients of the public healthcare system are necessary. 15. Baecke JA, Burema J and Frijters JE. A short questionnaire
for the measurement of habitual physical activity in epide-
miological studies. Am J Clin Nutr. 1982; 36:936-42. 16. Lohman TG. Anthropometric Standardization Reference
Manual. Champaign: Human Kinetics Books, 1988. In summary, our findings identified an elevated
occurrence of musculoskeletal symptoms among the
patients of the Brazilian public healthcare system,
which affects negatively healthcare expenditures. In
addition, our results identified that women and peo-
ple with an occupational activity with higher physi-
cal effort are groups at an increased risk of present-
ing musculoskeletal symptoms. 17. World Health Organization. Obesity, Preventing and Manag-
ing the Global Epidemic: Report of the WHO Consultation on
Obesity. World Health Organization [http://www.who.int/nu-
trition/publications/ obesity_executive_summary.pdf]. 18 Codogno JS, Fernandes RA, Sarti FM, Freitas Junior IF,
Monteiro HL. The burden of physical activity on type 2 diabe-
tes public healthcare expenditures among adults: a retrospec-
tive study. BMC Public Health. 2011; 11:275. 19. Lee S-JJ, Tak S, Alterman T, Calvert GM. Prevalence of Mus-
culoskeletal Symptoms Among Agricultural Workers in the
United States: An Analysis of the National Health Interview
Survey, 2004-2008. J Agromedicine. 2014; 19:268–80. R
RR
RRef
ef
ef
ef
efer
er
er
er
erences
ences
ences
ences
ences 20. Guo HR, Chang YC, Yeh WY, Chen CW, Guo YL. Prevalence
of musculoskeletal disorder among workers in Taiwan: a na-
tionwide study. J Occup Health. 2004;46:26-36. 1. Ferreira GD, Silva MC, Rombaldi AJ, Wrege ED, Siqueira FV,
Hallal PC. Prevalence and associated factors of back pain in
adults from southern Brazil: a population-based study. Rev Bras
Fisioter. 2011; 15:31-6. 21. Brooks PM. The burden of musculoskeletal disease – a glo-
bal perspective. Clin Rheumatol. 2006; 25:778-781 22. Woolf AD and Pfleger B. Burden of major musculoskeletal
conditions. Bull World Health Organ. 2003; 81:646-56. 2. Almeida IC, Sá KN, Silva M, Baptista A, Matos MA, Lessa I. Chronic low back pain prevalence in the population of the city
of Salvador. Rev Bras Ortop. 2008; 43:96-102. 23 Mata MS, Costa FA, Souza TO, Mata AN, Pontes JF. Pain and
functionality in primary health care. Ciênc Saúde Coletiva. 2011; 16:221-30 3. Meziat Filho N, Silva GA. Disability pension from back pain
among social security beneficiaries, Brazil. Rev Saude Publica. 2011;45:494-502. 149 Medicina (Ribeirão Preto) 2015;48(2):143-50
http://revista.fmrp.usp.br Codogno JS, Turi BC, Fernandes RA, Bonfim MR, Pastre CM,
Monteiro HL. Musculoskeletal disorders and expenditures. 24. Cimmino MA, Ferrone C and Cutolo M. Epidemiology of chronic
musculoskeletal pain. Baillieres best pract. res., Clin. rheumatol. 2011; 25:173-83. 29. Brandão AG, Horta BL and Tomasi E. Signs of musculoskel-
etal disorders in bank workers from the city of Pelotas and
region: prevalence and associated factors. Rev Bras
Epidemiol. 2005; 8:295-305. 25 Reis RJ, Pinheiro TM, Navarro A, Martin MM. Profile of occu-
pational disease outpatients and the presence of repetitive
strain injury. Rev Saúde Pública. 2000; 34:292-8. 30. Gerr F, Fethke NB, Anton D, Merlino L, Rosecrance J, Marcus
M, Jones MP. A prospective study of musculoskeletal out-
comes among manufacturing workers: II. Effects of psycho-
social stress and work organization factors. Hum Factors. 2014;56:178-90. 26. Gurgueira GP, Alexandre NM and Corrêa Filho HR. Self-re-
ported musculoskeletal symptoms among nursing personnel. Rev Latinoam Enferm. 2003; 11:608-13. 31. Maciel ACC, Fernandes MB and Medeiros LS. Prevalence
and factors associated with pain symptoms in professionals
of the textile industry. Rev Bras Epidemiol. 2006; 9:94-102. 27. Branco JC, Guido-e-Silva F and Giusti PH. Prevalence of
musculoskeletal symptoms in the faculty of public schools
and the private school. Fisioter Mov. 2011; 24:307-14. 28. Carvalho AJ and Alexandre NM. Musculoskeletal symptoms
in elementary school teachers. Rev Bras Fisioter. 2006; 10:35-
41. 150
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https://openalex.org/W2905655078
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https://hal-pasteur.archives-ouvertes.fr/pasteur-02175109/document
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English
| null |
DNABarcodeCompatibility: an R-package for optimizing DNA-barcode combinations in multiplex sequencing experiments
|
Bioinformatics
| 2,018
|
cc-by
| 1,853
|
To cite this version: Céline Trébeau, Jacques Boutet de Monvel, Fabienne Wong Jun Tai, Christine Petit, Raphael Etour-
nay. DNABarcodeCompatibility: an R-package for optimizing DNA-barcode combinations in mul-
tiplex sequencing experiments. Bioinformatics, 2019, 10.1093/bioinformatics/bty1030. pasteur-
02175109 Céline Trébeau, Jacques Boutet de Monvel, Fabienne Wong Jun Tai, Christine
Petit, Raphael Etournay Céline Trébeau, Jacques Boutet de Monvel, Fabienne Wong Jun Tai, Christine
Petit, Raphael Etournay DNABarcodeCompatibility: an R-package for
optimizing DNA-barcode combinations in multiplex
sequencing experiments Céline Trébeau, Jacques Boutet de Monvel, Fabienne Wong Jun Tai, Christine
Petit, Raphael Etournay Distributed under a Creative Commons Attribution 4.0 International License Ce´line Tre´beau1,2,3, Jacques Boutet de Monvel1,2,3,
Fabienne Wong Jun Tai1,2,3, Christine Petit1,2,3,4,5
and Raphae¨l Etournay
1,2,3,* 1Unite´ de Ge´ne´tique et Physiologie de l’Audition, De´partement Neuroscience, Institut Pasteur, 75015 Paris, France,
2UMRS 1120, Institut National de la Sante´ et de la Recherche Me´dicale, 75015 Paris, France, 3Sorbonne Universite´,
75006 Paris, France, 4Colle`ge de France, 75005 Paris, France. 5Institut de la Vision, Paris, France and 5Institut de la
Vision, 75012 Paris, France *To whom correspondence should be addressed. Associate Editor: Alfonso Valencia Received on July 9, 2018; revised on November 19, 2018; editorial decision on December 8, 2018; accepted on December 17, 2018 Abstract Summary: Using adequate DNA barcodes is essential to unambiguously identify each DNA library
within a multiplexed set of libraries sequenced using next-generation sequencers. We introduce
DNABarcodeCompatibility, an R-package that allows one to design single or dual-barcoding multi-
plex experiments by imposing desired constraints on the barcodes (including sequencer chemistry,
barcode pairwise minimal distance and nucleotide content), while optimizing barcode frequency
usage, thereby allowing one to both facilitate the demultiplexing step and spare expensive library-
preparation kits. The package comes with a user-friendly interface and a web app developed in
Java and Shiny (https://dnabarcodecompatibility.pasteur.fr), respectively, with the aim to help
bridge the expertise of core facilities with the experimental needs of non-experienced users. Availability and implementation: DNABarcodeCompatibility can be easily extended to fulfil specif-
ic project needs. The source codes of the R-package and its user interfaces are publicly available
along with documentation at [https://github.com/comoto-pasteur-fr] under the GPL-2 licence. Contact: raphael.etournay@pasteur.fr g
Availability and implementation: DNABarcodeCompatibility can be easily extended to fulfil specif-
ic project needs. The source codes of the R-package and its user interfaces are publicly available
along with documentation at [https://github.com/comoto-pasteur-fr] under the GPL-2 licence. Contact: raphael.etournay@pasteur.fr p
y
p
Supplementary information: Supplementary data are available at Bioinformatics online. HAL Id: pasteur-02175109
https://pasteur.hal.science/pasteur-02175109v1
Submitted on 5 Jul 2019 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Downloaded from https://academic.oup.com/bioinformatics/advance-article-abstract/doi/10.1093/bioinformatics/bty1030/5255876 by guest on 15 February 2019 Data and text mining 2.1 Algorithm (DNABarcodeCompatibility R-package) 2.1 Algorithm (DNABarcodeCompatibility R-package) 2.1 Algorithm (DNABarcodeCompatibility R package)
We describe here only the main steps and ideas. For details see the
Supplementary Material. The inputs of the algorithm are a list of
n distinct barcodes {i1, . . . , in}, the number N of required libraries,
and the multiplex level k to be used for the experiment (so that
N ¼ak, where a is the number of lanes of the flow cells). Step 1. This step consists of identifying a set of barcode combina-
tions that are compatible with the constraints set for the experiment
(including pairwise barcode distance, nucleotide content, and se-
quencer chemistry). Given the number n of barcodes and the multi-
plex level k, the total number of barcode combinations (compatible
or not) reads
n
k
. If this number is not too large (Supplementary
File S1), the algorithm will perform an exhaustive search and return
all compatible combinations of k barcodes. Otherwise, it will pro-
ceed sequentially by picking up barcode combinations at random,
and stopping when a set of Ncomp distinct compatible combinations
has been generated, that is large enough for the next step, entropy
maximization, to be effective. Fig. 1. Graphical user interface: dual-barcoding example. Left panel: input (1–6). Right panel: graphical output of the results (7–8). The user selects the platform
(1), loads the barcode datasets (2), selects the number of libraries (3) and the
multiplex level (4). Barcodes can be filtered according to their chemical proper-
ties and desired pairwise distance (5). They can also be manually unchecked
(6). Results are shown in text mode (7), and graphically (8) properties according to either the Hamming, SeqLev or phaseshift dis-
tance (Bystrykh, 2012; Buschmann, 2017). Next, the application seeks
an optimized set of compatible barcodes given the number of libraries
and multiplex level. Finally, the interface also allows one to visualize
how barcoded libraries are distributed among lanes of flow cells. Step 2. We then use a Shannon entropy maximization approach
to select the N/k compatible combinations to be used in the experi-
ment out of the Ncomp combinations found in Step 1. Namely, these
combinations are chosen in such a way that the resulting distribu-
tion of barcodes has maximum entropy S¼P
i fi log fi, where
f1,. . ., fn denote the frequencies of the various barcodes occurring in
the selection. 3 Conclusion DNABarcodeCompatibility provides a rational optimized design of
multiplex sequencing experiments integrating barcode constraints
from various NGS platforms including (i) barcode chemical properties,
(ii) barcode pairwise distance, (iii) Illumina barcode-combination rules
and (iv) barcode frequency usage. Controlling these barcode con-
straints is not only critical to ensure proper demultiplexing, but also to
optimize consumable usage and reduce costs. The major strength of
the package lies on its generic features that are applicable to wide
range of multiplex pooling design on most NGS platforms. This
should be useful notably for spatially and temporally resolved tran-
scriptomics
(see
Supplementary
File
S3
for
a
comparison
of
DNABarcodeCompatibility with other existing software). It can be shown (Supplementary File S2) that the maximum value
of the entropy that can be attained for a selection of a library of N
barcodes among n, with possible repetitions, is given by equation 1: Smax ¼ n r
ð
Þ bN
nc
N log
bN
nc
N
r dN
ne
N log
dN
ne
N
(1) (1) 1 Introduction 2017; Somervuo et al., 2018; Varet and Coppe´e, 2018) do not provide
any integrated tool to automatically refine sets of compatible barcodes
given all the above-mentioned constraints. Yet, a rational and general
approach allowing to optimize the design of multiplexed NGS experi-
ments under various constraints is highly desirable. To this end, we
developed DNABarcodeCompatibility, a generic R-package allowing
one to generate potentially large libraries of barcode combinations
compatible with most sets of multiplexing constraints and optimized to
achieve least-heterogeneity in terms of barcode usage. It is accompa-
nied by a user-friendly interface designed with Illumina barcode-
combination rules in mind, while allowing the user to apply constraints Inherent to next-generation sequencing (NGS) techniques is the re-
quirement to provide high enough sequence complexity of barcodes for
the design of multiplex experiments, in order to allow for proper
demultiplexing, i.e. identifying back each individual sample from a
pooled library. In general, finding optimal combinations of compatible
barcodes under given experimental constraints (single or dual barcod-
ing, sequencer chemistry, DNA composition, barcode pairwise distance
and error correction properties, availability in the laboratory) can be-
come challenging for users. Existing software such as DNABarcodes,
checkMyIndex, Illumina Experiment Manager, Barcosel (Buschmann, V
C The Author(s) 2018. Published by Oxford University Press. V
C The Author(s) 2018. Published by Oxford University Press. 1
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com C.Tre´beau et al. 2 specific to other NGS platforms on the distance between barcodes and
on the nucleotide composition. Fig. 1. Graphical user interface: dual-barcoding example. Left panel: input (1–6). Right panel: graphical output of the results (7–8). The user selects the platform
(1), loads the barcode datasets (2), selects the number of libraries (3) and the
multiplex level (4). Barcodes can be filtered according to their chemical proper-
ties and desired pairwise distance (5). They can also be manually unchecked
(6). Results are shown in text mode (7), and graphically (8) specific to other NGS platforms on the distance between barcodes and
on the nucleotide composition. Funding This work was supported by the European Research Council [ERC-2011-
ADG_294570]; LabEx LIFESENSES [ANR-10-LABX-65]; BNP Paribas
Foundation; and LHW-Stiftung. This work was supported by the European Research Council [ERC-2011-
ADG_294570]; LabEx LIFESENSES [ANR-10-LABX-65]; BNP Paribas
Foundation; and LHW-Stiftung. Conflict of Interest: none declared. 2.1 Algorithm (DNABarcodeCompatibility R-package) This ensures that this distribution is as uniform as pos-
sible given the constraints imposed by the particular set of compat-
ible
combinations
at
hand,
thereby
optimally
balancing
the
consumable usage to spare library-preparation kits. Acknowledgements in which r denotes the rest of the division of N by n, anddN=ne
(bN/nc) stands for the upper (lower) integer part of N=n. This ex-
pression reduces to log (min (N,)), that is the entropy of the uniform
distribution, if either N < n or n divides N (r ¼ 0). In our algorithm,
we use the Smax value as a stopping criterion to perform a random-
ized greedy search for a selection whose index distribution has max-
imum entropy. Simulations suggest that this algorithm is near
optimal, in the sense that with high probability it finds a solution
whose entropy is maximum or very close to maximum. In most
cases, the solution found is much improved in terms of reducing the
heterogeneity of barcode frequencies, compared to a randomly
chosen (non-optimized) selection (see Supplementary File S1). We thank Boris Goure´vitch, Hugo Varet for useful discussions, Gilles
Tre´beau for the Java frontend deployment and Jean-Pierre Hardelin for critic-
al reading of the manuscript. 2 Results Downloaded from https://academic.oup.com/bioinformatics/advance-article-abstract/d 2.2 Graphical user interface Buschmann,T. (2017) DNABarcodes: an R package for the systematic con-
struction of DNA sample tags. Bioinformatics, 33, 920–922. The DNABarcodeCompatibility user interface harbours two panels
for input and output, respectively (Fig. 1) Once barcodes are loaded
into the interface, the user can easily manipulate the list of barcodes
by clicking on them. The interface provides tools to visualize and fil-
ter barcodes according to their chemical properties (GC content, i.e. the number of G or C nucleotides divided by the barcode length,
and homopolymer length at most 2) and pairwise separation Bystrykh,L.V. (2012) Generalized DNA barcode design based on Hamming
codes. PLoS One, 7, e36852. Somervuo,P. et al. (2018) BARCOSEL: a tool for selecting an optimal barcode
set for high-throughput sequencing. BMC Bioinformatics, 19, 257. Varet,H. and Coppe´e,J.-Y. (2018) checkMyIndex: a web-based R/Shiny inter-
face for choosing compatible sequencing indexes. Bioinformatics.
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https://aip.scitation.org/doi/pdf/10.1063/5.0106866
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English
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Acceleration of hollow carbon nanospheres by gas leakage: An efficient nanomotor
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AIP advances
| 2,022
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cc-by
| 6,004
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RESEARCH ARTICLE | SEPTEMBER 02 2022
Acceleration of hollow carbon nanospheres by gas leakage:
An efficient nanomotor AIP Advances 12, 095204 (2022)
https://doi.org/10.1063/5.0106866 RESEARCH ARTICLE | SEPTEMBER 02 2022 I. INTRODUCTION van der Waals potential energy in the carbon nanotube can be up
to 1000 m/s.17 To further improve the nanomotor’s performance,
great effort has been devoted toward searching for new nanomotor
systems.18 Nanomotors can transform various energies into mechanical
energy, and they are promising candidates for application in many
fields, such as drug transportation,1–3 environmental governance,4–6
and sensing and monitoring.7–9 Due to their important role in driv-
ing nanodevices, great effort has been devoted toward searching for
new nanomotor systems. Nanomotors are usually driven by physi-
cal or chemical energy. For example, the electric field can drive the
rotation motion of the carbon nanotube.10 The self-electrophoresis
motion drives striped nanorods to move directionally.11 The tem-
perature gradient can cause the movement of cargoes along the
carbon nanotube.12 The bubbles generated by the chemical reac-
tion can provide the driving force for the nanomotor.13,14 The
catalytically pneumatic behavior can drive the micromotor.15 Since the discovery of C60 in 1985 by Kroto et al.,19 the hollow
carbon nanosphere (HCNS) has become a popular nanomaterial in
recent decades due to its low mass density, high porosity, and large
surface area. The diameter of C60 is 7 Å, which is a small value. The
diameter of the HCNS has been considerably increased in recent
years. The HCNS with a large diameter of 30 nm has been synthe-
sized by the arc discharge method.20 Using the template technique
based on silica spheres, Liu et al. produced carbon shells with a
diameter of 400 nm and a thickness of 4 nm, which has a similar
configuration to the HCNS.21 i
Due to the hollow structure, the HCNS of a large diameter is
mechanically unstable. Some ripples can be formed on the surface of
the HCNS, as a result of buckling instability.20 To enhance its sta-
bility, the HCNS is filled with Au, Fe3O4, or MnOx.15,22 It is also
possible that the HCNS can be filled with some gases, considering
that the HCNS is typically synthesized under a gas environment. N2 gas and Ar gas are used during the growth of the HCNS in the A key parameter to characterize the performance of the
nanomotor is the resultant speed of mechanic motion. ABSTRACT Nanomotors serve as nanoscale engines by converting various energies into mechanical energy. In addition to the huge number of existing
nanomotors, we propose a simple nanomotor based on the hollow carbon nanosphere (i.e., fullerene) that is full of gas. We investigate the
acceleration of the nanosphere by leakage of gas through a nanopore by molecular dynamics simulations. The nanosphere can be driven to a
high speed of 100 m/s under proper simulation conditions, which can be further tuned by temperature, gas density, and pore diameter. We
observe rotation of the pore direction during the acceleration process for a nanosphere of different pore diameters. The acceleration process
can be well described by the Meshchersky theory. We also simulate the deceleration process of the nanosphere due to the damping force of
the gas, which can be analyzed in terms of the kinetic motion of gas molecules. The nanomotor proposed in this work shall be realizable in
experiments and may be useful in driving the mechanic motion of fullerenes. 24 October 2024 04:01:07 © 2022 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0106866 Articles You May Be Interested In Monitoring mechanical motion of carbon nanotube based nanomotor by optical absorption spectrum Appl. Phys. Lett. (December 2016) Ultrafast nanotube based diffusiophoresis nanomotors Ultrafast nanotube based diffusiophoresis nanomotors Appl. Phys. Lett. (February 2010) Molecular dynamics simulation of Y-type nanomotors with different angles in aqueous solution
AIP Advances (November 2019) 24 October 2024 04:01:07 24 October 2024 04:01:07 ARTICLE AIP Advances scitation.org/journal/adv Yi Dong,
Yu Li,
Zheng-Rong Guo, and Jin-Wu Jianga) Yi Dong,
Yu Li,
Zheng-Rong Guo, and Jin-Wu Jianga) AFFILIATIONS Shanghai Key Laboratory of Mechanics in Energy Engineering, Shanghai Institute of Applied Mathematics and Mechanics,
School of Mechanics and Engineering Science, Shanghai University, Shanghai 200072, People’s Republic of China a)Author to whom correspondence should be addressed: jwjiang5918@hotmail.com Acceleration of hollow carbon nanospheres
by gas leakage: An efficient nanomotor Cite as: AIP Advances 12, 095204 (2022); doi: 10.1063/5.010686
Submitted: 30 June 2022 • Accepted: 9 August 2022 • Published Online: 2 September 2022 I. INTRODUCTION The veloc-
ity of a nanoparticle that moves in the carbon nanotube caused by
temperature gradients is on the order of 10 m/s.16 The velocity of
the cargo that moves along the carbon nanotube driven by ther-
mal gradients is about 100 m/s.12 The velocity of C60 driven by AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866
© Author(s) 2022 AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-1 © Author(s) 2022 ARTICLE AIP Advances AIP Advances scitation.org/journal/adv TABLE I. Potential parameters for nitrogen (N), oxygen (O), and carbon (C). N–N
O–O
C–N
C–O
N–O
σ (Å)
3.262
3.119
3.347
3.276
3.191
ε(eV)
0.0030
0.0026
0.0037
0.0035
0.0028
r0 (Å)
1.120
1.160
K (eV/Å2)
66.393
48.845 TABLE I. Potential parameters for nitrogen (N), oxygen (O), and carbon (C). arc discharge process at a high temperature of 2400 ○C.20 N2 is also
used in the growth of the carbon shell.21 The gas captured in the
HCNS may leak through some point defects, which shall generate
some driving force on the HCNS in the backward direction. In this paper, we perform molecular dynamics (MD) simula-
tions to investigate the kinetic behavior of the HCNS filled with gas. The leakage of the gas through a nanopore can drive the HCNS
to accelerate to a high speed of 100 m/s. The acceleration phe-
nomenon can be described by the Meshchersky theory. We consider
several effects on the acceleration process, including temperature,
gas density, and pore diameter. It is interesting that the acceleration
is sensitive to the pore diameter. The direction of the pore will be
rotated during the leakage of the gas, which is harmful for the accel-
eration. We also study the deceleration process of the HCNS due to
the damping force of the gas, where the MD simulation results can
be well explained by the analytic formula. We simulate the real growth process of coating a nanoscale
spherical particle with graphene. We first place a small graphene
layer (hexagon carbon ring) on the spherical nanoparticle where
carbon atoms are continuously added to the edge until the particle is
fully coated by graphene. Compared with a regular fullerene (such as
C60), which contains only 7 pentagons, the large fullerene contains
more pentagons and heptagons to keep its shape spherical, as shown
in Fig. 1. III. ACCELERATION PROCESS The HCNS will first be accelerated when the gases diffuse into
the outside region through the nanopore. After the leakage of all gas
molecules, the HCNS will start to decelerate by the damping force of
the gas. We investigate the acceleration of the HCNS driven by the
gas in this section. II. STRUCTURE AND SIMULATION DETAILS g
MD simulations are performed using the open source
code Large-scale Atomic/Molecular Massively Parallel Simulator
(LAMMPS).23 Visualization is achieved by OVITO.24 The Newton
equations of motion are integrated using a velocity Verlet algorithm
with a time step of 0.001 ps. The interactions between carbon atoms
in the HCNS are described by the Adaptive Intermolecular Reac-
tive Empirical Bond Order (AIREBO) potential,25 which consists of
the short-ranged Brenner potential26 and the long-ranged Lennard-
Jones (LJ) potential.27 The intermolecular interactions of N2 and
O2 molecules are described by the LJ potential. The potential para-
meters ϵ and σ are obtained from the Universal Force Field (UFF)
model.28 The cutoff distance for LJ interactions is 10.0 Å. The inter-
actions between two atoms within a gas molecule are described by
the harmonic bond potential, We perform MD simulations to study the kinetic behavior of
the HCNS with a nanoscale pore filled with gases, as illustrated in
Fig. 1. The gases are N2 and O2 at a ratio of 4:1. The gases diffuse
into the outside region. As a result of the momentum conservation,
the HCNS will be accelerated to move in the opposite direction. FIG. 1. Snapshot of structures. Carbon atoms are in gray. Nitrogen molecules are
in blue. Oxygen molecules are in red. (a) The nanoporous HCNS is accelerated by
the gas leakage. (b) The HCNS is decelerated by the gas damping force. Eb = K(r −r0)2,
(1) (1) where K is the force constant and r0 is the equilibrium distance,
which are obtained from the UFF model. The interactions between
different atoms are described by the LJ potential, where the potential
parameters are evaluated by the Lorentz–Berthelot mixing rule.28,29
The potential parameters are listed in Table I. A. Simulation results (4) (4) In this work, the characteristic time τ is obtained by fitting the time
dependent gas leakage ratio to Eq. (3). Figure 2(a) illustrates that
the leakage process can be well described by Eq. (3) with the fitting
characteristic time τ = 123.21 ps. A. Simulation results A simulation box of a large dimension of 5400 × 5400 × 5400 Å3
is chosen. This is to ensure that the pressure outside the HCNS
is almost zero and the inverse diffusion of the gas from the out-
side region into the inner space can be ignored. The nonperiodic
boundary condition is applied to all directions because the peri-
odic boundary will cause the collision between the HCNS and the
gas flow that leaked out. The simulation is performed in two steps. FIG. 1. Snapshot of structures. Carbon atoms are in gray. Nitrogen molecules are
in blue. Oxygen molecules are in red. (a) The nanoporous HCNS is accelerated by
the gas leakage. (b) The HCNS is decelerated by the gas damping force. AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866
© Author(s) 2022 12, 095204-2 12, 095204-2 © Author(s) 2022 AIP Advances scitation.org/journal/adv where N is the number of leaked gas molecules, N0 is the total
number of gas molecules. D is the diffusion coefficient, Ap is the
area of the nanopore, and V is the volume of the HCNS. We thus
obtain First, all gas molecules are constrained inside the HCNS, and the
whole system is thermalized for 100 ps within the NVT ensemble at
500 K. Then a nanopore is created in the HCNS, after which the
system evolves within the NVE ensemble. The gas molecules will
leak through the nanopore, and the HCNS is accelerated. The tra-
jectory of the second step is used to analyze the acceleration process
of the HCNS. N(t)
N0
= 1 −e−t
τ ,
(3) (3) Figure 2(a) shows the time dependence of the percentage of the
leaked gas molecules. Here, the HCNS has a large diameter of 112 Å,
and the pore diameter is 60 Å. There are 4000 gas molecules inside
the HCNS. We find that the percentage of the leaked gas molecules
increases exponentially. The characteristic time for the leaking pro-
cess is 123.21 ps. The velocity for the HCNS is shown in Fig. 2(b). The velocity increases with time and reaches a saturate value of
2.47 Å/ps. We will introduce the theoretical formula for the leakage
phenomenon next. where τ, the characteristic time for the gas leakage process, is where τ, the characteristic time for the gas leakage process, is τ =
V
DAp
. B. Theoretical analysis The HCNS and internal gas molecules can be regarded as a vari-
able mass system due to the gas leakage, so the velocity of the HCNS
is described by the Meshchersky equation, According to Fick’s law,30 the gas diffusion through the
nanopore in the HCNS is governed by the following equation: mdv
dt = FR + vr dm
dt ,
(5) dN
dt = DAp N0 −N
V
,
(2) dN
dt = DAp N0 −N
V
,
(2)
FIG. 2. Gas leakage process. (a) The variation in the gas leakage ratio with time. (b) The variation in the HCNS’s velocity with time. mdv
dt = FR + vr dm
dt ,
(5)
FIG. 3. Temperature effect on the velocity. (a) The dependence of velocity on tem-
perature. The lines are the theoretical results from Eq. (6). (b) The characteristic
time of the gas leakage process at various temperatures. (2) (5) FIG. 3. Temperature effect on the velocity. (a) The dependence of velocity on tem-
perature. The lines are the theoretical results from Eq. (6). (b) The characteristic
time of the gas leakage process at various temperatures. 24 October 2024 04:01:07 24 October 2024 04:01:07 FIG. 3. Temperature effect on the velocity. (a) The dependence of velocity on tem-
perature. The lines are the theoretical results from Eq. (6). (b) The characteristic
time of the gas leakage process at various temperatures. FIG. 2. Gas leakage process. (a) The variation in the gas leakage ratio with time. (b) The variation in the HCNS’s velocity with time. AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-3 © Author(s) 2022 ARTICLE AIP Advances scitation.org/journal/adv where m is the total mass. v is the velocity of the HCNS, and vr is the
velocity of the gas molecules relative to the HCNS. FR is the external
force, which is zero in this work. The time dependence of the velocity
of the HCNS during the acceleration process is dependence of the velocity of the HCNS. The velocity increases with
the increase in temperature. There are two reasons for this tem-
perature dependence. First, the diffusion coefficient increases with
increasing temperature.30 According to Eq. (4), the characteristic
time will be reduced, which is confirmed by Fig. 3(b). Second, the
relative velocity vr increases with increasing temperature. We find
that the MD results can be well described by Eq. (6). B. Theoretical analysis v = vr ln
mball + mgas
mball + mgase−t
τ ,
(6) (6) We investigate the effect of gas density on the velocity of the
HCNS. Figure 4(a) shows that the velocity increases with increas-
ing gas density. In particular, the final value of the velocity increases
with the increase in the gas density, as shown in Fig. 4(b), which can
be described by Eq. (6) in the long time limit. We note that the gas
density has only a limited effect on the characteristic time.ii where mball is the mass of the HCNS and mgas is the mass of the
total gas molecules. Figure 2(b) shows that Eq. (6) can provide an
accurate description of the time dependence of the HCNS’s veloc-
ity with the fitting parameter vr = 5.41 Å/ps. It should be pointed
out that the relative velocity is actually the root-mean-square of the
thermal vibration velocity,31 vr =
√
3RT
M . However, the fitted value
for vr is smaller than the theoretical value. It is because the gas
molecules outside the simulation box will be deleted due to the
nonperiodic boundary condition, which results in a decrease in the
temperature. When the time approaches infinity, we can get the final velocity
of the HCNS as vmax = vr ln mball + mgas
mball
. (7) (7) C. Discussion We find that vmax is related to mgas
mball . We find that vmax is related to mgas
mball . We find that the acceleration process is affected by temperature,
gas density, and pore diameter. Figure 3(a) shows the temperature i
mball
Finally, we explore the effect of pore diameter on the velocity
of the HCNS. Figure 5(a) displays that the velocity increases faster FIG. 4. Gas density effect on the velocity. (a) The dependence of velocity on gas
density. The lines are the theoretical results from Eq. (6). (b) The final velocity for
different gas densities. FIG. 5. Pore diameter effect on (a) the velocity and (b) the acceleration angle. The
inset in (b) displays the definition of the acceleration angle. FIG. 4. Gas density effect on the velocity. (a) The dependence of velocity on gas
density. The lines are the theoretical results from Eq. (6). (b) The final velocity for
different gas densities. FIG. 4. Gas density effect on the velocity. (a) The dependence of velocity on gas
density. The lines are the theoretical results from Eq. (6). (b) The final velocity for
different gas densities. FIG. 5. Pore diameter effect on (a) the velocity and (b) the acceleration angle. The
inset in (b) displays the definition of the acceleration angle. AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-4 © Author(s) 2022 ARTICLE
scitation.org/journal/adv
FIG. 7. Correlation between the final velocity and the average acceleration angle
for the HCNS with different pore diameters. scitation.org/journal/adv AIP Advances scitation.org/journal/adv (i.e., larger acceleration) for the pore with a larger diameter because
of the faster gas leaking process. The final velocity varies with the
pore diameter randomly. There is no obvious relation between the
final velocity and the pore diameter. Furthermore, the velocity of
the HCNS with dp = 50 Å is decreased after 600 ps. However, the
final velocity should be independent of the pore diameter accord-
ing to Eq. (6), so the final velocity for all pore diameters should be
the same.ii We find that these abnormal phenomena for the final velocity
in Fig. 5(a) are closely related to the acceleration angle. The acceler-
ation angle is defined to be the angle between the HCNS’s velocity
and the normal direction of the pore, as illustrated in the inset of
Fig. 5(b). Figure 5(b) shows that the acceleration angle is almost 180○
at the initial stage for all HCNSs. C. Discussion The acceleration angle starts to
deviate from 180○after about 200 ps as a result of the rotation of
the HCNS. The rotation effect is harmful for the acceleration effi-
ciency because the reverse force from the gas leakage is not along
the moving direction of the HCNS. In particular, the HCNS will be
decelerated by the leaking gas if the acceleration angle is less than
90○. For the HCNS with dp = 50 Å, the acceleration angle becomes
smaller than 90○after 600 ps, which is the reason for the reduction in
the velocity, shown in Fig. 5(a). It should be noted that the accelera-
tion angle for the HCNS with dp = 70 Å is also smaller than 90○after
about 600 ps, but the velocity is not reduced as shown in Fig. 5(a). It
is because the gas leakage process has been completed after a short
time of 300 ps for large pore diameters. As a result, the rotation
of the HCNS has no effect on the acceleration process any more
after 300 ps. FIG. 7. Correlation between the final velocity and the average acceleration angle
for the HCNS with different pore diameters. FIG. 7. Correlation between the final velocity and the average acceleration angle
for the HCNS with different pore diameters. the same for these five simulation samples. However, Fig. 6(b) dis-
plays obvious different final velocities for these five samples, which
is due to the different acceleration angles, as shown in Fig. 6(c). In
particular, for sample 2, the acceleration angle is less than 90○after
900 ps, leading the reduction in the velocity after 900 ps shown in
Fig. 6(b). Figure 6(d) shows a direct relation between the accelera-i the same for these five simulation samples. However, Fig. 6(b) dis-
plays obvious different final velocities for these five samples, which
is due to the different acceleration angles, as shown in Fig. 6(c). In
particular, for sample 2, the acceleration angle is less than 90○after
900 ps, leading the reduction in the velocity after 900 ps shown in
Fig. 6(b). Figure 6(d) shows a direct relation between the accelera-
tion angle and the final velocity, where the x-axis is the average of
the acceleration angle during the leakage process. It shows that the
final velocity is larger for a larger acceleration angle. C. Discussion simulate the deceleration process of the HCNS surrounded by
molecules, as shown in Fig. 1(b). There is no nanopore in the
NS here. The diameter of the HCNS is 50 Å. The gas density is
×10−3 amu/Å3. The dimension of the simulation box is set to
700 × 100 × 100 Å3. The HCNS and the gas are thermalized
in the NVT ensemble for 100 ps at 300 K. Then we set the initial
city of the HCNS to be 2 Å/ps along the x-direction. The periodic
ndary condition is applied to this simulation. The HCNS will
lerate due to the collision with the gas molecules (i.e., the gas
ping force), as shown in Fig. 8. The deceleration is caused by the damping force of the gas. m the microscopic point of view, the damping force is generated
he collision between gas molecules and the HCNS.31 For a static
ture surrounded by gas molecules, the collision-induced force
he surface is
F = 1
3ρAv2
rms,
(8)
re ρ is the gas density, A is the area, and vrms is the root-mean-
re velocity of the gas molecule. For a static structure, the collision-induced force is balanced
e the forces on the front and back sides counteract with each
r. However, if the structure is traveling at a speed of v, then the
sion-induced force on the front side is larger than the force on
ack side, with a force difference of
8. Variation in the HCNS’s velocity with time during the deceleration process. FIG. 9. Dependence of the damping coefficient on (a) gas density, (b) contact area,
and (c) temperature. The lines are the theoretical results from Eq. (10). C. Discussion Figure 7 further
shows that the correlation between the final velocity and the average
acceleration angle is also valid for other HCNSs with different pore
diameters.i 24 October 2024 04:01:07 There is some randomness in the rotation of the HCNS, which
leads to the randomly distributed acceleration angle for the HCNS
with different pore diameters. This is the origin of the divergent
final velocity shown in Fig. 5(a). To further illustrate the randomness
effects, we perform five independent MD simulations with differ-
ent initial velocity distributions for the HCNS with dp = 40 Å, as
shown in Fig. 6. Figure 6(a) shows that the gas leakage process is We compare the final velocity of the HCNS obtained by the gas
leakage in this work with the velocity obtained by other nanomo-
tors. The velocity of the HCNS is about 2 Å/ps (i.e., 200 m/s) in FIG. 6. Random rotation effect on a
velocity of 40 Å of the HCNS with a pore
diameter. The time dependence of (a)
the gas leakage ratio, (b) the velocity,
and (c) the acceleration angle. (d) The
relationship between the final velocity
and the average acceleration angle. AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866
12, 095204-5
© Author(s) 2022 FIG. 6. Random rotation effect on a
velocity of 40 Å of the HCNS with a pore
diameter. The time dependence of (a)
the gas leakage ratio, (b) the velocity,
and (c) the acceleration angle. (d) The
relationship between the final velocity
and the average acceleration angle. AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-5 12, 095204-5 © Author(s) 2022 AIP Advances scitation.org/journal/adv ΔF = 4
3ρA
√
3RT
M v ≡γv,
(9) this work. The velocity of the nanoparticle moving in the carbon
nanotube caused by the temperature gradient is on the order of
10 m/s.16 The velocity of cargoes along the carbon nanotube driven
by the thermal gradient is about 100 m/s.12 The velocity of C60
driven by the van der Waals force in the carbon nanotube is about
1000 m/s.17 The final velocity realized in this work is comparable
with that in other methods. (9) where R is the universal gas constant, T is the temperature of gas,
and M is the molar mass of gas. Equation (9) gives the origin of the
damping force. The damping coefficient is DECELERATION PROCESS
We now explore the deceleration of the HCNS in this section. IV. DECELERATION PROCESS FIG. 9. Dependence of the damping coefficient on (a) gas density, (b) contact area,
and (c) temperature. The lines are the theoretical results from Eq. (10). 12, 095204-6 We now explore the deceleration of the HCNS in this section. We simulate the deceleration process of the HCNS surrounded by
gas molecules, as shown in Fig. 1(b). There is no nanopore in the
HCNS here. The diameter of the HCNS is 50 Å. The gas density is
2.1 ×10−3 amu/Å3. The dimension of the simulation box is set to
be 2700 × 100 × 100 Å3. The HCNS and the gas are thermalized
within the NVT ensemble for 100 ps at 300 K. Then we set the initial
velocity of the HCNS to be 2 Å/ps along the x-direction. The periodic
boundary condition is applied to this simulation. The HCNS will
decelerate due to the collision with the gas molecules (i.e., the gas
damping force), as shown in Fig. 8. The deceleration is caused by the damping force of the gas. From the microscopic point of view, the damping force is generated
by the collision between gas molecules and the HCNS.31 For a static
structure surrounded by gas molecules, the collision-induced force
on the surface is 24 October 2024 04:01:07 24 October 2024 04:01:07 F = 1
3ρAv2
rms,
(8) (8) where ρ is the gas density, A is the area, and vrms is the root-mean-
square velocity of the gas molecule. For a static structure, the collision-induced force is balanced
since the forces on the front and back sides counteract with each
other. However, if the structure is traveling at a speed of v, then the
collision-induced force on the front side is larger than the force on
the back side, with a force difference of FIG. 8. Variation in the HCNS’s velocity with time during the deceleration process. FIG. 9. Dependence of the damping coefficient on (a) gas density, (b) contact area,
and (c) temperature. The lines are the theoretical results from Eq. (10). FIG. 9. Dependence of the damping coefficient on (a) gas density, (b) contact area,
and (c) temperature. The lines are the theoretical results from Eq. (10). FIG. 8. Variation in the HCNS’s velocity with time during the deceleration process. Author Contributions Finally, we discuss possible experiments for the numerical pro-
posal in this work. The diameter of the HCNS in our simulation is
112 Å. This diameter is achievable in the current experiments. There
have been several experimental methods to grow the HCNS. The dia-
meter of C60 is 7 Å.19 The diameter of the HCNS has been increased
considerably in recent years. The HCNS with a large diameter of
30 nm has been synthesized by the arc discharge approach.20 Using
the template technique based on the silica sphere, Liu et al. produced
a carbon shell with a diameter of 400 nm and thickness of 4 nm,
which has a similar configuration to the HCNS.21 Yi Dong: Data curation (equal); Formal analysis (equal); Visual-
ization (equal); Writing – original draft (equal). Yu Li: Writing –
review & editing (equal). Zheng-Rong Guo: Resources (equal). Jin-
Wu Jiang: Formal analysis (equal); Methodology (equal); Writing –
review & editing (equal). VI. CONCLUSION where v is the velocity of the HCNS and m is the mass of the HCNS. We have In conclusion, we perform MD simulations to investigate the
acceleration of the HCNS driven by gas leakage. We find that the
HCNS can reach a speed as high as 100 m/s under proper conditions,
which is comparable with that obtained from other nanomotors. The acceleration process of the HCNS can be affected by temper-
ature, gas density, and pore diameter. The direction of the nanopore
will rotate during the acceleration process, which induces harmful
effects on the final velocity of the HCNS. The numerical results can
be explained by the Meshchersky theory. We also explore the decel-
eration of the HCNS due to the damping force of the gas. An analytic
damping force is derived to explain the MD simulation results. v = v0e−γ
m t,
(12) (12) where v0 is the initial velocity. In this work, the numerical value of
γ can be obtained by fitting the velocity v from MD simulations to
Eq. (12). For example, the damping coefficient is γ = 36.29 amu/ps
for the structure shown in Fig. 8.i Based on the above-mentioned analysis, we find that the damp-
ing coefficient should be related to the gas density, the contact area of
the HCNS and gas, and the temperature. We will verify the analytic
expression in Eq. (10) by MD simulations.i IV. DECELERATION PROCESS AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-6 © Author(s) 2022 ARTICLE scitation.org/journal/adv γ = 4
3ρA
√
3RT
M ,
(10) To save the simulation cost, a large gas pressure has been used
in this work, which is from 93 to 373 atm. However, this gas pressure
is realizable in the laboratory. For example, Lou et al. prepared the
HCNS in an autoclave at 1000 atm.32 (10) where A is the cross-sectional area. From the above-mentioned investigations on the experimental
setup, we are aware that the numerical models proposed in this work
should be able to be realized in the experiment. According to Newton’s second law, the velocity of the HCNS is
governed by the following equation: mdv
dt = −γv,
(11) mdv
dt = −γv,
(11) (11) The authors have no conflicts to disclose. The authors have no conflicts to disclose. DATA AVAILABILITY The data that support the findings of this study are available
from the corresponding author upon reasonable request. i
At the same time, some functional nanoparticles are inserted
into the HCNS to upgrade its performance. For example, the HCNS
can be filled with Au, Fe3O4, or MnOx.15,22 It is also possible that the
HCNS may be filled with some gases, considering that the HCNS is
typically synthesized under the gas environment. The preparation of
C60 is performed in He gas.19 N2 gas and Ar gas are used during the
growth of the HCNS in the arc discharge process at a high tempera-
ture of 2400 ○C.20 N2 is also used in the growth of the carbon shell.21
As a result, it is possible for the current experiment to construct the
HCNS filled by gases, as proposed in this work. ACKNOWLEDGMENTS Figures 9(a) and 9(b) show that the damping coefficient
increases linearly with increasing gas density and contact area,
respectively, which is consistent with the analytic expression in
Eq. (10). The damping coefficient increases with increasing tem-
perature, as shown in Fig. 9(c). It is obvious that MD simulation
results are comparable with the theoretical results, which verifies the
validity of Eq. (10). It should be noted that we perform six inde-
pendent MD simulations for each data in these set of simulations. The average of these six results is used as the value for the damping
coefficient, while the corresponding root mean square gives the error
bar in Fig. 9. This work was supported by the National Natural Science
Foundation of China (Grant Nos. 11822206 and 12072182), the
Innovation Program of the Shanghai Municipal Education Commis-
sion (Grant No. 2017-01-07-00-09-E00019), and the Key Research
Project of Zhejiang Laboratory (Grant No. 2021PE0AC02). The first
author thanks Yu Li and Yi-Xuan Xue for their help in this work. 24 October 2024 04:01:07 1D. Patra, S. Sengupta, W. Duan, H. Zhang, R. Pavlick, and A. Sen, Nanoscale 5,
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© Author(s) 2022 AIP Advances 12, 095204 (2022); doi: 10.1063/5.0106866 12, 095204-8 12, 095204-8 © Author(s) 2022
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0111701&type=printable
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CDO, an Hh-Coreceptor, Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling
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PloS one
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Introduction The significance of Hh signaling in carcinogenesis was also
explored in the proliferation of small cell lung cancer (SCLC),
which is a highly aggressive lung cancer constituting about 20–
25% of all lung cancers [21]. Inhibition of the activity of Hh
signaling using SMO antagonist, cyclopamine resulted in the
serious growth reduction in SCLC cell lines [21,23,25,26]. Whereas, it was initially suggested that Hh signaling is less
associated with NSCLC, the most dominant type of lung cancer
and the most lethal malignancy. However, several evidences have
recently indicated that NSCLC is dependent on Hh signaling
activity in proliferation as well [27,28,29,30]. Hh signaling pathway is one of essential signaling pathways,
which is implicated in embryonic development, morphogenesis
and proliferation [1,2,3,4,5,6]. The molecular mechanism how to
regulate Hh signaling is still under investigation. Generally, once
Hh ligand binds to its primary receptor, Patched 1 (PTCH1),
Smoothened (SMO) is released from PTCH1-mediated inhibition
and migrates to primary cilium. Stimulation of SMO triggers
sequential signal transduction that activates the transcription
factors of GLI family. The active form of GLI protein is
translocated into the nucleus and regulates the expression of
downstream target genes, including PTCH1 and GLI1 [1,7,8]. y
p
[
]
Although the major receptor against Hh is PTCH1, there are
additional co-receptors assisting the Hh signaling positively, such
as CDO, BOC and GAS1 [31,32,33,34,35]. Hh signaling is
involved in various cellular and developmental processes, and
consequently tight regulations are absolutely required for the
signaling to recognize and control micro-variation in cellular
environment. Meanwhile, many lung cancer cell lines are
producing various levels of Hh ligand [25,30]. Even if a low level
of Hh ligand is seen in some lung cancer cells, these cells reveal the
increase in Hh target gene expression implying upregulation of Hh
signaling. Under these circumstances, the presence of Hh co-
receptors may contribute to the amplification of the weak
extracellular cue in cancer cells in addition to the fine adjustment
of Hh signaling during embryogenesis. g
g
g
The loss of Hh signaling during embryonic development is
associated with several genetic disorders including holoprosence-
phaly, which is the most common malformation of the forebrain
[9,10,11]. In contrast, constitutive activation of Hh signaling has
been known to be involved in initiation and progression of several
cancers in skin (sporadic basal cell carcinoma, BCC), brain
(medulloblastoma), muscle (rhabdomyosarcoma, RMS), gastroin-
testinal tract, prostate, pancreas, and lung [12,13,14,15,16,
17,18,19,20,21,22,23]. CDO, an Hh-Coreceptor, Mediates Lung Cancer Cell
Proliferation and Tumorigenicity through Hedgehog
Signaling un Leem, Hye-Lim Ha, Ju-Hyeon Bae, Kwan-Hyuck Baek, Jong-Sun Kang* Young-Eun Leem, Hye-Lim Ha, Ju-Hyeon Bae, Kwan-Hyuck Baek, Jong-Sun Kang*
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, 440–746, Republic of Korea Young-Eun Leem, Hye-Lim Ha, Ju-Hyeon Bae, Kwan-Hyuck Baek, Jong-Sun Kang
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suw ology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, 440–746, Republic of Korea epartment of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon, Abstract Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in
genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer
development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate
cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer
(NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along
with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated
that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO
is required for proliferation and survival of lung cancer cells via Hh signaling. itor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America Editor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America Received June 2, 2014; Accepted October 1, 2014; Published November 4, 2014 2014 Leem et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, w
use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files. Funding: This work was funded by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (2012R1A1A2005448 to YEL)
and (NRF-2011-0017315 to JSK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E
il k
j
@ kk
d Competing Interests: The authors have declared that no competing interests exist. * Email: kangj01@skku.edu November 2014 | Volume 9 | Issue 11 | e111701 Total RNA preparation and real-time qRT-PCR development
and
neuronal
di embryonic
development
and
neuronal
differentiation
[36,37,38,39]. Structural analysis demonstrated that the fibronec-
tin repeats in the extracellular domain of CDO is critical for Hh
binding [39]. The positive regulation of Hh signaling pathway by
CDO was initially identified in Drosophila. It has been known that
ihog (ortholog of CDO in Drosophila) together with boi (ortholog
of BOC in Drosophila) is important to the activation of Hh
signaling in the developing wing imaginal disc [40,41]. In mice,
Cdo deficiency exhibits multiple congenital defects, including
holoprosencephaly, which is the defect commonly, associated with
mutations in Hh signaling [39,42]. Furthermore, CDO is involved
in Hh-mediated ventral neural patterning of the mammalian
neural tube [31] and as well as in Hh-mediated cell proliferation in
cerebellar granule neuron progenitors (CGNPs) [43]. Total RNA preparation and real time qRT PCR
Total RNA was isolated using easyBLUE total RNA extraction
kit
(iNtRON
Biotechnology
Inc.,
Korea),
and
cDNA
was
synthesized using PrimeScript RT reagent kit (TAKARA, Japan)
according to manufacturer’s instructions. Real-time qRT-PCR
was performed using SYBR Premix ExTaq kit (TAKARA, Japan)
and Thermal Cycler Dice real time system (TP800, TAKARA,
Japan) according to manufacturer’s instructions. The forward (F)
and reverse (R) primers used in this study are listed below. SHH F,
59-CCAAAAAGCTGACCCCTTTA-39, R, 59-GCTCCGGTG-
TTTTCTTCATC-39; PTCH1 F, 59-CAGCACTGGAAAAC-
TCGTCA-39, R, 59-TCTGATGAACCACCTCCACA-39; GLI1
F, 59-AAGGGGTTTCTATCCTTCCAGA-39, R, 59-TCCTTT-
ATTATCAGGAAACAGTGTCA-39; CDO F, 59-GGGAAATA-
CATCTGCGAAGC-39, R, 59-CTGAGCAGCATCAGGAAG-
TG-39; 18S rRNA F, 59-GTAACCCGTTGAACCCCATT-39,
R, 59-CCATCCAATCGGTAGTAGCG-39. Relative quantifica-
tion of a gene expression was described as fold of relative changes
in mRNA levels compared to a control, using the 22DDCt equation,
[DDCt = DCt (target gene)-DCt (reference gene)]. 18S rRNA was
used as a reference gene. In addition to the role of CDO in organ development and
cellular differentiation, the association of CDO to Hh signaling,
which is implicated in tumorigenesis intrigued us to elucidate a
role of CDO in lung cancer cell proliferation. In the present study
we sought to uncover the contribution of CDO to Hh signaling in
NSCLCs and additionally to proliferation and tumorigenicity in
lung tumor cells. We found that the level of CDO was higher in
NSCLC cells, and that the inhibition of CDO expression
downregulated Hh signaling, and reduced proliferation and
tumorigenesis in human lung cancer cells. In addition, CDO
expression was observed in advanced stage of NSCLC tissues. Flow cytometry assay of apoptosis Apoptosis was measured by using ApoScan Annexin V FITC
apoptosis detection kit (BioBud, Korea) and BD FACS CANTO II
flow cytometer (BD Biosciences) according to manufacturer’s
instructions. Cell cultures and reagents Cell cultures and reagents
BEAS-2B and NSCLC cell lines, A549, H1299, H460 and
H520 were kindly provided by Prof. D. H. Kim (Sungkyunkwan
University, Samsung Biomedical Research Institute, Korea) [44]. The culture of BEAS-2B followed ATCC’s guidelines. NSCLC
cells were cultured in a humidified incubator with 5% CO2 in
RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum
(FBS)
supplemented
with
penicillin
and
streptomycin. For
measuring mRNA/protein expression or cell death in siCDO-
transfected cells, the medium was switched to RPMI-1640
medium with 0.5% FBS one day after transfection, and the
transfected cells were cultured for 2 days further. 50 mM of
SANT1 (S4572, Sigma-Aldrich) dissolved in DMSO or the
corresponding volume of the vehicle DMSO was treated to cells
grown in RPMI-1640 medium containing 0.5% FBS at indicated
time point. RNA interference experiments Each NSCLC cell line was transfected with siCDO using
Lipfectamine
RNAiMAX
reagent
(Invitrogen)
according
to
manufacturer’s instructions. The following sequences were used
as siRNA against CDO. siCDO #1, Sense, 59-GGAUCUUG-
GACCCUUAUGUUU-39, Antisense, 59- ACAUAAGGGUC-
CAAGAUCCUU-39. siCDO #2, Sense, 59-CUUCAAAGUC-
GAAUAUAAAUU-39, Antisense, 59- UUUAUAUUCGACU-
UUGAAGUU9. For in vivo tumorigenicity assay, A549 cells that
stably express small hairpin RNA (shRNA) against CDO were
prepared by transfecting with pSuper-puro-shCDO. pSuper-puro-
shCDO vector was reported previously [42]. Cell proliferation and viability assay Cells were seeded at a concentration of 56103 or 16104 cells
per 96-well and cultured in RPMI-1640 containing 0.5% FBS. Trypsinized cells were stained with trypan blue (TB) solution, and
the non-stained viable cells were counted. For MTT assay, 20 ml
of 5 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetra-
zolium bromide) was added to 56103 or 16104 cells per 96-well. After 4 hour-incubation at 37 uC, the culture soup was switched to
200 ml DMSO. The absorbance was measured at 595 nm. For
BrdU assay, cells were grown with 10 mM bromodeoxyuridine
(BrdU; Sigma-Aldrich Corp.) for 1 hr. Fixed cells by 4% PFA were
probed with anti-BrdU antibody (Chemicon international Inc.). Fluorescence was detected by Alexa Fluor 488 goat anti-mouse
antibody (Molecular Probe). Total RNA preparation and real-time qRT-PCR Taken together, all data suggest that CDO, as a co-receptor for
Hh, is crucial for cancer cell proliferation and tumorigenicity, and
therefore it can be considered as a good candidate for anticancer
therapeutic applications. Western blot Western blot analyses were performed as described previously
[37]. Primary antibodies used in this study are listed as below;
Cleaved Caspase3 (#9664, Cell Signaling), Caspase9 (sc-81663,
Santa Cruz), Cdk2 (sc-6248, Santa Cruz), Cyclin D1 (sc-246,
Santa Cruz), Cyclin E (sc25303, Santa Cruz), GAPDH (Ab-
FRONTIER, Korea), p21 (sc-6246), p27 (ab7961, Abcam). Introduction The link of Hh pathway to carcinogenesis
was initially reported in Gorlin syndrome in which the mutation in
the PTCH1 gene is responsible for the cancer incidence [24]. Moreover, the aberrant upregulation of Hh signaling through the
loss of PTCH1 or the gain-of-function mutation in SMO was
extensively studied in BCC and medulloblastoma [13,14]. Among those co-receptors, CDO is a transmembrane protein
belonging to the immunoglobulin (Ig)/fibronectin type III (FNIII)
superfamily and plays an important role in muscle differentiation, PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e111701 1 November 2014 | Volume 9 | Issue 11 | e111701 A Role of CDO in Lung Cancer Cell Proliferation November 2014 | Volume 9 | Issue 11 | e111701 Hh components, which are involved in lung cancer cell
proliferation were inhibited by CDO depletion in NSCLCs proliferation were inhibited by CDO depletion in NSCLCs
Previous literatures have shown that CDO acts as an accessory
receptor for Hh ligand and positively regulates Hh signaling
[39,48]. The close relevance of the activation of Hh signaling to
cancer cell proliferation prompted us first to test CDO expression
in NSCLC cells. To do so, total RNAs were isolated from A549,
H1299, H460 and H520 NSCLC cell lines as well as from BEAS-
2B human non-cancerous lung cell, and used for analyzing CDO
expression by doing real-time qRT-PCR. The result showed that
CDO was expressed higher in A549, H1299 and H520 cells than
BEAS-2B however H460 revealed relatively lower CDO level
(Figure 1A). In addition, we investigated the expression of Hh
signaling components in NSCLC cells by performing real-time
qRT-PCR. The higher expression of SHH was seen in all four
NSCLC cell lines than in the non-cancerous lung cell. Likewise,
the downstream target genes of Hh signaling, PTCH1 and GLI1
were expressed higher in those NSCLC cells (Figure 1A). A549
showed relatively lower level of Hh ligand, compared with other
cell lines, however revealed the increase in Hh target gene
expression (PTCH1 and GLI1). In H520, CDO, SHH and
PTCH1 were expressed comparatively higher than any other cell
lines but GLI1 expression was fairly low. The decrease in cell proliferation and viability in the CDO-
depleted NSCLC cells could be due to reduced cell cycle
progression and/or enhanced cell death. To verify the expression
levels of cell cycle regulators/repressors, A549 cells transfected
with the scrambled or siCDO were examined for immunoblotting. As a result, the protein levels of cell cycle regulators (Cyclin D1,
Cyclin E, and Cdk2) were declined, while the expressions of Cdk
inhibitors (p27 and p21) were elevated in siCDO-treated A549
cells compared with the scrambled-treated cells (Figure 2C). These
data indicate that the inhibition of CDO expression seems to cause
cell cycle arrest in NSCLC cells. We first determined the inhibitory effect of SANT1 on Hh
signaling by examining the expression of Hh signaling target genes
after SANT1 treatment. SANT1 binds directly to SMO, impeding
the signaling. Total RNAs isolated from DMSO- or SANT1-
treated A549, H1299 and H460 were used for analyzing PTCH1
and GLI1 expressions by real-time qRT-PCR. As predicted, the Then, we asked whether CDO depletion was also associated
with cell death. Mice and immunohistochemistry
G12D LSL-K-ras G12D mice were provided by Dr. Tyler Jacks (MIT
Cancer Center) [46,47]. The paraffin sections from LSL-K-ras
G12D mice were immunoreacted with 2B3 (mouse ascites against
CDO) and anti-GLI1 antibody (1:100; ab49314, Abcam), and
detected using immunofluorescence. Confocal microscopy was
performed at SKKU School of Medicine-Microscopy Shared
Resource Facility with Zeiss LSM-510 Meta confocal microscope. All animal studies were reviewed and approved by the Interna-
tional Animal Care and Use Committee (IACUC) of SKKU
School of Medicine (SUSM). SUSM is an Association for
Assessment
and
Accreditation
of
Laboratory
Animal
Care
(AAALAC) international accredited facility and abides by the
Institute for Laboratory Animal Research (ILAR) guide. CDO depletion led to an inhibition of proliferation and an
induction of apoptosis in NSCLCs Statistical analyses were performed using Student’s t test. Data
were presented as means 6SD from at least three independent
experiments and considered significant when p values were ,0.05
(*) or ,0.01(**). As shown in Figure 1B and C, we observed that proliferation of
NSCLC cells was quite sensitive to SANT1. Thus, we decided to
determine the effect of the down-regulation of Hh signaling by
CDO knockdown on cancer cell proliferation. To do so, we
evaluated the rate of cellular proliferation of A549, H1299, H460
and H520 under CDO-depleted condition by performing TB cell
counting and MTT assay (Figure 2A and D). The result showed
that CDO knockdown in all tested NSCLC cells considerably
diminished
cell
number
and
viability,
compared
with
the
scrambled control. However the extent of the proliferation
reduction was not stronger than that in SANT1 treatment
(compare Figure 2A and D to Figure 1B and C, respectively). The slope of the graphs for cell number and viability in CDO-
depleted cancer cells still kept rising at longer time point day 5,
compared with that in SANT1 treatment even though the levels
were absolutely much lower than those in the scrambled control
cells. To assess the effect of CDO depletion on proliferation
further, we performed BrdU incorporation assay. The result
showed that CDO knockdown in NSCLC cells led to 20–40% of
decrease in cell proliferation (Figure 2B). In vivo tumorigenicity assay In vivo tumorigenicity assay 56106 of the control (pSuper-puro)- or pSuper-puro-shCDO-
transfected A549 cells were subcutaneously injected into the flanks
of 6- to 8-week-old female nude mice. Tumor volume was
measured every 4 days, and calculated as described [45]. All
animal studies were reviewed and approved by the International
Animal Care and Use Committee (IACUC) of SKKU School of
Medicine (SUSM). SUSM is an Association for Assessment and
Accreditation of Laboratory Animal Care (AAALAC) internation-
al accredited facility and abides by the Institute for Laboratory
Animal Research (ILAR) guide. q
g
g
y
g
p
Next, we asked whether CDO contributed to the activity of Hh
signaling in NSCLCs. To determine that, we transfected three
different NSCLC cell lines, A549, H1299 and H460 with siCDO
or the scrambled siRNA and evaluated the effect of CDO
deficiency on the expressions of Hh signaling components by real-
time qRT-PCR. The expression of CDO was substantially
reduced with siCDO in all cell lines (Figure 1D). To deplete
CDO expression, we utilized two different sequences of siRNA,
siCDO #1 and #2. Both siCDOs were showing the similar CDO
depletion and the results (Data not shown), and we are presenting
the data of siCDO #1. The levels of the Hh signaling components,
SHH, PTCH1, and GLI1 were significantly reduced in CDO-
depleted lung cancer cells compared with the scrambled control
(Figure 1E). These data denote that CDO acts as a positive
regulator for Hh signaling in NSCLC cells, irrespective of its
expression level. A Role of CDO in Lung Cancer Cell Proliferation addition of SANT1 resulted in reduction of PTCH1 and GLI1
expressions (Figure S1 in File S1). In order to determine the effect
of Hh signaling on the proliferation of NSCLCs, we inhibited Hh
signaling in A549, H1299, H460 and H520 by using SANT-1. Then, TB cell counting and MTT assay were performed to
analyze cell proliferation and viability. The data showed that the
addition of 50 mM SANT1 resulted in significant reduction in cell
growth and viability of four individual NSCLC cells (Figure 1B
and C). The effect of SANT1-mediated inhibition was stronger at
late time points. These data indicate that the tested NSCLC cells
require Hh signaling activity for retaining their proliferation. addition of SANT1 resulted in reduction of PTCH1 and GLI1
expressions (Figure S1 in File S1). In order to determine the effect
of Hh signaling on the proliferation of NSCLCs, we inhibited Hh
signaling in A549, H1299, H460 and H520 by using SANT-1. Then, TB cell counting and MTT assay were performed to
analyze cell proliferation and viability. The data showed that the
addition of 50 mM SANT1 resulted in significant reduction in cell
growth and viability of four individual NSCLC cells (Figure 1B
and C). The effect of SANT1-mediated inhibition was stronger at
late time points. These data indicate that the tested NSCLC cells
require Hh signaling activity for retaining their proliferation. Colony forming assay Five thousand of cells were gently blended with 10% FBS
RPMI-1640 medium containing 0.35% agarose. The mixture
overlaid on the 0.5% bottom agarose with 10% FBS medium in 6-
well dishes. The plated cells were incubated at 37uC in humidified
incubator for 3 weeks with feeding of medium every second day. The colony formation was visualized with 0.01% crystal violet and
analyzed with a dissecting microscope. The quantification of
colony number per field was determined by using ImageJ. Each
experiment was performed in triplicate and repeated twice. November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 2 A Role of CDO in Lung Cancer Cell Proliferation November 2014 | Volume 9 | Issue 11 | e111701 Hh components, which are involved in lung cancer cell
proliferation were inhibited by CDO depletion in NSCLCs Immunoblotting against cleaved forms of caspase-
9 and -3 showed that these apoptotic factors were detected November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 3 A Role of CDO in Lung Cancer Cell Proliferation Figure 1. Hh signaling, which is associated with lung cancer cell proliferation was inhibited by CDO depletion. A. Real-time qRT-PCR
for the expression levels of CDO and Hh signaling components (SHH, PTCH1 and GLI1) in BEAS-2B and NSCLC cell lines (A549, H1299, H460 and H520). Each expression level was normalized to the level of 18S rRNA. The relative amount of each component in NSCLCs was determined as the amount of
each in BEAS-2B was set to 1.0. B. The number of viable cells was determined by cell counting at indicated time in A549, H1299, H460 and H520
treated with or without 50 mM SANT1. C. Cell viability was assessed by MTT assay at indicated time in A549, H1299, H460 and H520 treated with or
without 50 mM SANT1. The absorbance was measured at 595 nm. D. RT-PCR for CDO in A549, H1299 and H460 transfected with the scrambled siRNA
or siCDO #1. E. Real-time qRT-PCR for Hh signaling components in A549, H1299 and H460 transfected with the scrambled siRNA or siCDO. The
expression level of each component was normalized to the level of 18S rRNA. The relative amount of each component in CDO-depleted NSCLCs was
determined as the amount of each in the control cells was set to 1.0 (red line). All the values represent means of at least triplicate determinations 61
SD. *p,0.05 and **p,0.01. doi:10.1371/journal.pone.0111701.g001 Figure 1. Hh signaling, which is associated with lung cancer cell proliferation was inhibited by CDO depletion. A. Real-time qRT-PCR
for the expression levels of CDO and Hh signaling components (SHH, PTCH1 and GLI1) in BEAS-2B and NSCLC cell lines (A549, H1299, H460 and H520). Each expression level was normalized to the level of 18S rRNA. The relative amount of each component in NSCLCs was determined as the amount of
each in BEAS-2B was set to 1.0. B. The number of viable cells was determined by cell counting at indicated time in A549, H1299, H460 and H520
treated with or without 50 mM SANT1. C. Cell viability was assessed by MTT assay at indicated time in A549, H1299, H460 and H520 treated with or
without 50 mM SANT1. Hh components, which are involved in lung cancer cell
proliferation were inhibited by CDO depletion in NSCLCs The absorbance was measured at 595 nm. D. RT-PCR for CDO in A549, H1299 and H460 transfected with the scrambled siRNA
or siCDO #1. E. Real-time qRT-PCR for Hh signaling components in A549, H1299 and H460 transfected with the scrambled siRNA or siCDO. The
expression level of each component was normalized to the level of 18S rRNA. The relative amount of each component in CDO-depleted NSCLCs was
determined as the amount of each in the control cells was set to 1.0 (red line). All the values represent means of at least triplicate determinations 61
SD. *p,0.05 and **p,0.01. doi:10.1371/journal.pone.0111701.g001 November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 4 A Role of CDO in Lung Cancer Cell Proliferation Figure 2. CDO depletion inhibited and promoted cell proliferation and apoptosis in NSCLC cells, respectively. A. The number of viable
cells was determined by cell counting at indicated time in the control- or CDO-depleted A549, H1299, H460 and H520. B. Cell proliferation of CDO
knockdown A549, H1299, H460 and H520 was determined by BrdU incorporation. C. Western blot analysis for the expression of cell cycle regulators
(Cyclin D1, Cyclin E and Cdk2), repressors (p27 and p21) and apoptotic markers (cleaved Caspase 3 and cleaved Caspase 9) in the control or CDO
knockdown A549 cells. GAPDH was used as a loading control. D. MTT assay for the cell viability of the control- or siCDO-transfected A549, H1299,
H460 and H520 at indicated time. The absorbance was measured at 595 nm. E. Annexin V-FITC apoptosis and flow cytometry analysis with A549,
H1299, H460 and H520 transfected with the scrambled siRNA or siCDO. All the values represent means of at least triplicate determinations 61 SD. *p,0.05 and **p,0.01. doi:10.1371/journal.pone.0111701.g002 Figure 2. CDO depletion inhibited and promoted cell proliferation and apoptosis in NSCLC cells, respectively. A. The number of viable
cells was determined by cell counting at indicated time in the control- or CDO-depleted A549, H1299, H460 and H520. B. Cell proliferation of CDO
knockdown A549, H1299, H460 and H520 was determined by BrdU incorporation. C. Western blot analysis for the expression of cell cycle regulators
(Cyclin D1, Cyclin E and Cdk2), repressors (p27 and p21) and apoptotic markers (cleaved Caspase 3 and cleaved Caspase 9) in the control or CDO
knockdown A549 cells. GAPDH was used as a loading control. D. Hh components, which are involved in lung cancer cell
proliferation were inhibited by CDO depletion in NSCLCs MTT assay for the cell viability of the control- or siCDO-transfected A549, H1299,
H460 and H520 at indicated time. The absorbance was measured at 595 nm. E. Annexin V-FITC apoptosis and flow cytometry analysis with A549,
H1299, H460 and H520 transfected with the scrambled siRNA or siCDO. All the values represent means of at least triplicate determinations 61 SD. *p,0.05 and **p,0.01. p
p
doi:10.1371/journal.pone.0111701.g002 reduction in clonogenicity in vitro when CDO level was
decreased. stronger
in
siCDO-transfected
A549
than
in
the
control
(Figure 2C). Consistently, flow cytometry profile for apoptosis
with A549, H1299, H460 and H520 exhibited that higher
percentage of annexin-V and PI double positive cells was found
in four individual CDO-depleted NSCLCs (Figure 2E). Therefore,
these data suggest that the decreased proliferation of NSCLCs
upon CDO depletion is due to increased cell death as well as
attenuated cell cycle progression. In order to support the data of in vitro tumorigenicity, we
prepared stable cell line of A549 expressing shRNA against CDO,
and subcutaneously injected the control or CDO-depleted cells to
the flanks of nude mice. The tumor size was periodically
measured. As shown Figure 3C, D, and E, a significant decrease
in tumor growth was observed in nude mice injected with CDO-
knockdown A549. Together, these data indicate that CDO is
required for the in vivo growth of NSCLC cells. CDO expression was detected with GLI1 together in
advanced stages of NSCLCs The diminished cell proliferation in CDO knockdown led us to
investigate the influence of CDO depletion on malignant features
of NSCLC cells. For that, we assessed the extent of colony
formation of siCDO-transfected A549, H1299, H460 and H520
cells in soft agar (Figure 3A and B). The result displayed a marked To determine CDO expression during lung cancer progression,
we took advantage of a NSCLC mouse model, LSL-K-rasG12D
mice [46,47]. After intranasal inhalation of Ad-Cre to induce November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 5 A Role of CDO in Lung Cancer Cell Proliferation Figure 3. Knockdown of CDO in NSCLCs showed the reduction of in vitro and in vivo tumorigenesis. A. Colony formation of the control or
CDO knockdown A549, H1299, H460 and H520 in soft agar. B. Quantitative analysis of the experiment described in A. Colony numbers per field were
determined by ImageJ. The values represent means of triplicate determinations 61 SD. *p,0.05 and ** p ,0.01. C. Representative tumor growth 50
days after subcutaneous injection of nude mice with the control or shCDO-treated A549 cells. D. The tumor volume of the nude mice with the control
A549 (n = 8) or CDO-depleted A549 (n = 10) described in C. Means are shown as error bars. E. Tumors from the nude mice injected with the control
A549 or CDO-depleted A549 cells shown in C. doi:10.1371/journal.pone.0111701.g003
A Role of CDO in Lung Cancer Cell Proliferation Figure 3. Knockdown of CDO in NSCLCs showed the reduction of in vitro and in vivo tumorigenesis. A. Colony formation of the control or
CDO knockdown A549, H1299, H460 and H520 in soft agar. B. Quantitative analysis of the experiment described in A. Colony numbers per field were
determined by ImageJ. The values represent means of triplicate determinations 61 SD. *p,0.05 and ** p ,0.01. C. Representative tumor growth 50
days after subcutaneous injection of nude mice with the control or shCDO-treated A549 cells. D. The tumor volume of the nude mice with the control
A549 (n = 8) or CDO-depleted A549 (n = 10) described in C. Means are shown as error bars. E. Tumors from the nude mice injected with the control
A549 or CDO-depleted A549 cells shown in C. Discussion triggers apoptosis. The role of CDO as a dependence receptor
basically conflicts with the function of CDO in cancer cells with
Hh expression, what we uncovered in this study. Therefore,
appropriate level of Hh ligand seems to be essential to determine
the function of CDO as a positive regulator of Hh signaling or a
dependence receptor. In fact, the significance of CDO in cancer cell proliferation was
somewhat uncovered and referred earlier. Hayashi et al. [49]
made an effort to screen out genes encoding transmembrane
proteins, which are highly expressed in prostate cancer cell lines. Among candidates, they focused on 83% of CDO-overexpressed
prostate cancer, and demonstrated that reducing CDO expression
induced apoptosis and inhibited cell invasion in prostate cancer. However, the molecular mechanism how CDO is involved in
proliferation of prostate cancer cells was not explained. In this
study, we revealed that the fine-tuning and up-regulation of Hh
signaling by CDO is quite required for cell proliferation in lung
cancer. We additionally observed that the mRNA level of CDO in
the tested lung cancer cell lines except H520 was higher than in
BEAS-2B, but it was not significant. Particularly CDO mRNA
level in A549 was about 1.7-fold higher than the basal level of it in
BEAS-2B. Even though the increase is not dramatically high, it is
worthy of notice if we consider that CDO expression is generally
found during development and embryogenesis, and hardly
detected in adult tissues [36]. The inhibition of Hh signaling by SANT1 in all NSCLC cells
showed more substantial reduction of cell proliferation and
viability at longer time point, compared to the inhibition by
CDO gene depletion (compare Figure 1B and C to Figure 2A and
D, respectively). The limited effect of CDO knockdown on the
inhibition of cell proliferation might be due to that other Hh co-
receptors, such as BOC and GAS1 are still expressed and active,
even though we cannot rule out the possibility of the transient
CDO depletion with siRNA. It has been demonstrated that all Hh
co-receptors, CDO, BOC, and GAS1 are collectively required for
full activation of Hh signaling. CDO, BOC and GAS1 individually
make a complex with PTCH1 and the formation of the complexes
is critical to Hh-mediated CGNP proliferation [43]. Based on this,
understanding the roles of BOC and GAS1 during Hh-mediated
tumorigenesis would be needed later. CDO expression was detected with GLI1 together in
advanced stages of NSCLCs doi:10.1371/journal.pone.0111701.g003 spontaneous lung tumorigenesis in LSL-K-rasG12D mice, we
assessed CDO expression in the lung tumor tissue showing four
individual grades of tumor progression by fluorescent immuno-
histochemistry. CDO expression was nearly undetectable in grade-
1, which is an early stage of tumor progression featuring atypical
adenomatous hyperplasia (AAH) or small adenoma (Figure S2 in
File S1). However, its expression became enriched as tumor
progression has been advanced. In grade-2, in which larger
adenomas with a little enlarged nuclei are detected, CDO
expression was observed in the cytoplasm of cells in the tumor
lesions, and this high expression was retained until grade-3
(Figure 4). As tumors progressed to be invasive adenocarcinomas
(grade-4), CDO expression was somewhat decreased and detected in cell membrane. The expression of CDO in the tumor lesions led
us to investigate the expression of Hh pathway components in the
tumor tissue. Confocal immunofluorescence images showed that
GLI1, one of Hh signaling factors was colocalized with CDO, and
additionally its expression was also highly observed in grade-2 and
-3 tumor lesions, not in grade-4 (Figure 4). The deficiency of GLI1
expression in grade-4 is corresponding to the results suggested by
several previous studies [23,29]. The results of these experiments
indicate that CDO expression may be correlated to the up-
regulation of Hh signaling in tumor lesions and CDO is also likely
related to the progression of lung tumor, not in tumor initiation. November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 6 A Role of CDO in Lung Cancer Cell Proliferation Figure 4. CDO expression was colocalized with GLI1 in high-grade of NSCLCs. Confocal immunofluorescence detection of CDO (red) and
GLI1 (green) in grade-2, -3, and -4 lung tumor tissues from LSL-K-ras G12D model. Cell nuclei were visualized by DAPI (blue) and the phase contrast
images are shown. Scale bar indicates 50 mm. doi:10.1371/journal.pone.0111701.g004 Figure 4. CDO expression was colocalized with GLI1 in high-grade of NSCLCs. Confocal immunofluorescence detection of CDO (red) and
GLI1 (green) in grade-2, -3, and -4 lung tumor tissues from LSL-K-ras G12D model. Cell nuclei were visualized by DAPI (blue) and the phase contrast
images are shown. Scale bar indicates 50 mm. doi:10.1371/journal.pone.0111701.g004 November 2014 | Volume 9 | Issue 11 | e111701 References Biochem Pharmacol 67: 805–814. 30. Yuan Z, Goetz JA, Singh S, Ogden SK, Petty WJ, et al. (2007) Frequent
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differentiation via activation of the p38 mitogen-activated protein kinase
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et al. (2003) Widespread requirement for Hedgehog ligand stimulation in growth
of digestive tract tumours. Nature 425: 846–851. 39. Bae GU, Domene S, Roessler E, Schachter K, Kang JS, et al. (2011) Mutations
in CDON, encoding a hedgehog receptor, result in holoprosencephaly and
defective interactions with other hedgehog receptors. Am J Hum Genet 89:
231–240. 18. Sheng T, Li C, Zhang X, Chi S, He N, et al. (2004) Activation of the hedgehog
pathway in advanced prostate cancer. Mol Cancer 3: 29. 19. Sanchez P, Hernandez AM, Stecca B, Kahler AJ, DeGueme AM, et al. (2004)
Inhibition of prostate cancer proliferation by interference with SONIC
HEDGEHOG-GLI1 signaling. Proc Natl Acad Sci U S A 101: 12561–12566. 40. Lum L, Yao S, Mozer B, Rovescalli A, Von Kessler D, et al. (2003) Identification
of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299: 2039–2045. 20. Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts DJ, et al. (2003)
Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425: 851–856. 41. Camp D, Currie K, Labbe A, van Meyel DJ, Charron F (2010) Ihog and Boi are
essential for Hedgehog signaling in Drosophila. Neural Dev 5: 28. 21. Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, et al. (2003)
Hedgehog signalling within airway epithelial progenitors and in small-cell lung
cancer. Nature 422: 313–317. 42. Zhang W, Kang JS, Cole F, Yi MJ, Krauss RS (2006) Cdo functions at multiple
points in the Sonic Hedgehog pathway, and Cdo-deficient mice accurately
model human holoprosencephaly. Dev Cell 10: 657–665. 22. Discussion Each expression was
normalized to the level of 18S rRNA, and the relative amount of
each normalized level in SANT1-treated cells was determined as
the amount of each in the DMSO-treated cells was set to 1.0 (red
line). All the values represent means of triplicate determinations
61 SD. Figure S2, CDO expression was not detected in grade-1 of
NSCLCs. Confocal immunofluorescence detection of CDO (red)
in grade-1 lung tumor tissues from LSL-K-ras G12D model. Cell
nuclei were visualized by DAPI (blue). Scale bar indicates 50 mm. (DOCX) So far, the efforts to find anticancer therapies have been
targeting mainly SMO, and indeed several SMO inhibitors have
been applying to the clinic [52]. The expression of CDO is
generally restricted in development and embryogenesis, not in
adult stage. Abnormal expression of CDO in the cancerous
condition is possibly associated with aberrant cell proliferation. As
a part of clinical trials, CDO could be considered as a potential
indicator for tumorigenesis and a therapeutic target. Author Contributions File S1
Figure S1, SANT1 inhibited the expression of PTCH1
and GLI1 in NSCLC cells. qRT-PCR for the expression levels of
PTCH1 and GLI1 in A549, H1299 and H460, which were treated File S1
Figure S1, SANT1 inhibited the expression of PTCH1
and GLI1 in NSCLC cells. qRT-PCR for the expression levels of
PTCH1 and GLI1 in A549, H1299 and H460, which were treated Conceived and designed the experiments: JSK YEL. Performed the
experiments: YEL HLH JHB. Analyzed the data: JSK YEL. Contributed
reagents/materials/analysis tools: KHB. Wrote the paper: JSK YEL. Conceived and designed the experiments: JSK YEL. Performed the
experiments: YEL HLH JHB. Analyzed the data: JSK YEL. Contributed
reagents/materials/analysis tools: KHB. Wrote the paper: JSK YEL. Discussion It was initially presumed that Hh signaling was rarely linked to
NSCLC because Hh and GLI1 were observed at a relatively lower
level in NSCLC. Since then, the data, including the present data,
of the inhibition of NSCLC proliferation by a SMO antagonist
suggested that the activation of Hh signaling via the low level of
Hh is certainly associated with tumorigenesis in NSCLC. Under
this circumstance CDO may play a critical role as a ligand sensor
for a little amount of Hh, supporting the main receptor, PTCH1
and after all induces the activation of Hh signaling. g
Detection of CDO expression in high-grade tumor lesion of
NSCLC mouse model, not in the lesion of grade-1, excited our
curiosity on the role of CDO-mediated Hh signaling in tumor
progression. It has been dominantly suggested that the up-
regulation of Hh signaling seems to be correlated with tumor
grade although it is controversial in some types of cancer. In
prostate cancer, the mRNA levels of SHH, PTCH1 and GLI1
were highly elevated in more advanced stages of the cancer, and
all those components were closely linked to Gleason score [51]. On
the other hand, Gialmanidis et al. [29] have immunohistochem-
ically analyzed paraffin-embedded lung tissue sections of 80
NSCLC patients. They found that activation of Hh signaling was
associated with tumor grade, and the high activation of the
signaling was mainly detected in well-differentiated and moder-
ately differentiated tumors. The robust expression of CDO in the
high-grade of tumor may lead to a possibility that CDO as a Meanwhile, the function of CDO in cell proliferation seems to
be ambivalent depending on Hh level. Delloye-Bourgeois et al. [50] recently suggested that, the Hh level is important for CDO to
function as a dependence receptor, which is closely related to the
induction of proapoptotic signal. According to them when Hh
ligand is fairly limited, caspase 3/9 are recruited to the caspase
cleavage site in the intercellular domain of CDO and evoke
proteolytic cleavage. This caspase-mediated cleavage eventually November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org 7 A Role of CDO in Lung Cancer Cell Proliferation positive regulator regulates and results in aberrant activation of
Hh signaling in tumor development and progression. with DMSO or 50 mM SANT1 for 2 days. g g
48. Allen BL, Song JY, Izzi L, Althaus IW, Kang JS, et al. (2011) Overlapping roles
and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH
pathway function. Dev Cell 20: 775–787. 47. Shin J, Lee JC, Baek KH (2014) A single extra copy of Dscr1 improves survival
of mice developing spontaneous lung tumors through suppression of tumor
angiogenesis. Cancer Lett 342: 70–81. 45. Park BH, Vogelstein B, Kinzler KW (2001) Genetic disruption of PPARdelta
decreases the tumorigenicity of human colon cancer cells. Proc Natl Acad
Sci U S A 98: 2598–2603. 46. DuPage M, Dooley AL, Jacks T (2009) Conditional mouse lung cancer models
using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4: 1064–
1072. 49. Hayashi T, Oue N, Sakamoto N, Anami K, Oo HZ, et al. (2011) Identification
of transmembrane protein in prostate cancer by the Escherichia coli ampicillin
secretion trap: expression of CDON is involved in tumor cell growth and
invasion. Pathobiology 78: 277–284. p
p
52. Scales SJ, de Sauvage FJ (2009) Mechanisms of Hedgehog pathway activation in
cancer and implications for therapy. Trends Pharmacol Sci 30: 303–312. 51. Gonnissen A, Isebaert S, Haustermans K (2013) Hedgehog signaling in prostate
cancer and its therapeutic implication. Int J Mol Sci 14: 13979–14007. 50. Delloye-Bourgeois C, Gibert B, Rama N, Delcros JG, Gadot N, et al. (2013)
Sonic Hedgehog promotes tumor cell survival by inhibiting CDON pro-
apoptotic activity. PLoS Biol 11: e1001623. References Watkins DN, Berman DM, Baylin SB (2003) Hedgehog signaling: progenitor
phenotype in small-cell lung cancer. Cell Cycle 2: 196–198. p
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y
43. Izzi L, Levesque M, Morin S, Laniel D, Wilkes BC, et al. (2011) Boc and Gas1
each form distinct Shh receptor complexes with Ptch1 and are required for Shh-
mediated cell proliferation. Dev Cell 20: 788–801. 23. Chi S, Huang S, Li C, Zhang X, He N, et al. (2006) Activation o 23. Chi S, Huang S, Li C, Zhang X, He N, et al. (2006) Activation of the hedgehog
pathway in a subset of lung cancers. Cancer Lett 244: 53–60. pathway in a subset of lung cancers. Cancer Lett 244: 53–60. 44. Hwang JA, Kim Y, Hong SH, Lee J, Cho YG, et al. (2013) Epigenetic
inactivation of heparan sulfate (glucosamine) 3-o-sulfotransferase 2 in lung
cancer and its role in tumorigenesis. PLoS One 8: e79634. 24. Unden AB, Holmberg E, Lundh-Rozell B, Stahle-Backdahl M, Zaphiropoulos
PG, et al. (1996) Mutations in the human homologue of Drosophila patched November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e111701 8 A Role of CDO in Lung Cancer Cell Proliferation A Role of CDO in Lung Cancer Cell Proliferation A Role of CDO in Lung Cancer Cell Proliferation 45. Park BH, Vogelstein B, Kinzler KW (2001) Genetic disruption of PPARdelta
decreases the tumorigenicity of human colon cancer cells. Proc Natl Acad
Sci U S A 98: 2598–2603. 46. DuPage M, Dooley AL, Jacks T (2009) Conditional mouse lung cancer models
using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4: 1064–
1072. 50. Delloye-Bourgeois C, Gibert B, Rama N, Delcros JG, Gadot N, et al. (2013)
Sonic Hedgehog promotes tumor cell survival by inhibiting CDON pro-
apoptotic activity. PLoS Biol 11: e1001623. 51. Gonnissen A, Isebaert S, Haustermans K (2013) Hedgehog signaling in prostate
cancer and its therapeutic implication. Int J Mol Sci 14: 13979–14007. g g
48. Allen BL, Song JY, Izzi L, Althaus IW, Kang JS, et al. (2011) Overlapping roles
and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH
pathway function. Dev Cell 20: 775–787. 52. Scales SJ, de Sauvage FJ (2009) Mechanisms of Hedgehog pathway activation in
cancer and implications for therapy. Trends Pharmacol Sci 30: 303–312. November 2014 | Volume 9 | Issue 11 | e111701 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9
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Benchmarking Meta-embeddings: What Works and What Does Not
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cc-by
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Abstract Following the hypothesis that different knowl-
edge sources may contain complementary seman-
tic information (Goikoetxea et al., 2016), meta-
embeddings (Yin and Schütze, 2016) aim to ob-
tain an ensemble of distinct word embeddings each
trained using different methods and resources to
produce a word representation with an improved
overall quality. In the last few years, several methods have
been proposed to build meta-embeddings. The
general aim was to obtain new representa-
tions integrating complementary knowledge
from different source pre-trained embeddings
thereby improving their overall quality. How-
ever, previous meta-embeddings have been
evaluated using a variety of methods and
datasets, which makes it difficult to draw
meaningful conclusions regarding the merits
of each approach. In this paper we propose
a unified common framework, including both
intrinsic and extrinsic tasks, for a fair and
objective meta-embeddings evaluation. Fur-
thermore, we present a new method to gen-
erate meta-embeddings, outperforming previ-
ous work on a large number of intrinsic evalu-
ation benchmarks. Our evaluation framework
also allows us to conclude that previous extrin-
sic evaluations of meta-embeddings have been
overestimated. The main challenge when generating meta-
embeddings is preserving the information encoded
in the source embeddings and many different meth-
ods have been proposed to deal with the task. Con-
catenation (Goikoetxea et al., 2016) and averaging
(Coates and Bollegala, 2018) are two very strong
baselines, but much complex methods based on lin-
ear transformations and supervised neural models
have also been proposed (Bollegala et al., 2018;
Bollegala and Bao, 2018; Yin and Schütze, 2016). When it comes to evaluating meta-embeddings,
there is no consensus on either evaluation tasks or
methodology. Meta-embeddings are evaluated in a
wide range of tasks (Schnabel et al., 2015; Bakarov,
2018), ranging from intrinsic (i.e. word similar-
ity, word analogy) to extrinsic tasks such as short
text classification (Bollegala and Bao, 2018; Bol-
legala et al., 2018), common-sense stories (Speer
et al., 2017), Named Entity Recognition (O’Neill
and Bollegala, 2020) or Semantic Textual Simi-
larity (García-Ferrero et al., 2020). Furthermore,
different evaluation methodologies have been ap-
plied. For example, Yin and Schütze (2016) dis-
card the words in the datasets which are not repre-
sented in the meta-embedding model, while Speer
and Lowry-Duda (2017) use various strategies to
minimize the number of out-of-vocabulary (OOV)
words. Abstract To make things more complicated, previous
meta-embeddings approaches require some ad-hoc
pre-processing to tune multiple filtering criteria and
parameters according to the source embeddings
used (Bollegala et al., 2018; Bollegala and Bao,
2018; Yin and Schütze, 2016), which has a signifi- Benchmarking Meta-embeddings: What Works and What Does Not Iker García-Ferrero
Rodrigo Agerri
German Rigau
HiTZ Basque Center for Language Technologies - Ixa NLP Group
University of the Basque Country UPV/EHU
{ iker.garciaf, rodrigo.agerri, german.rigau }@ehu.eus Findings of the Association for Computational Linguistics: EMNLP 2021, pages 3957–3972
November 7–11, 2021. ©2021 Association for Computational Linguistics 2
Related work An additional issue is that most previous work
has focused on combining word embeddings gen-
erated from similar sources and algorithms. For
instance, combining Word2vec CBOW (Mikolov
et al., 2013a) with GloVe (Pennington et al., 2014)
embeddings. We empirically show that, since these
embeddings encode very similar knowledge, com-
bining them does not produce a significant gain. Instead, the best meta-embeddings are obtained by
combining embeddings trained with different algo-
rithms and resources. For example, by leveraging
vectors induced from text corpora together with
other embeddings obtained from knowledge bases. Previous research has shown that word embed-
dings created using different methods and resources
present significant variations in quality. For in-
stance, Hill et al. (2014) show that word embed-
dings trained from monolingual or bilingual cor-
pora capture different nearest neighbours. The term meta-embedding was coined by Yin
and Schütze (2016). They showed how to combine
five different pre-trained word embeddings using
a small neural network for improving the accu-
racy of cross-domain part-of-speech (POS) tagging. Following this, Bollegala et al. (2018) propose an
unsupervised locally linear method for learning
meta-embeddings from a given set of pre-trained
source embeddings while Bollegala and Bao (2018)
apply three types of autoencoders for the purpose
of learning meta-embeddings. In this paper we present a new method to gen-
erate meta-embeddings that outperform previous
approaches on a large number of intrinsic bench-
marks. Other contributions include: Although word embeddings are mainly con-
structed by exploiting information from text cor-
pora only (Mikolov et al., 2013a; Pennington et al.,
2014; Mikolov et al., 2018), some approaches also
tried different methods to integrate the knowledge
encoded in lexical resources such as WordNet (Ha-
lawi et al., 2012; Bollegala et al., 2016; Goikoetxea
et al., 2016), PPDB (Faruqui et al., 2015) or Con-
ceptNet (Speer et al., 2017). Goikoetxea et al. (2016) show that simply concatenating word em-
beddings derived from text and WordNet out-
perform alternative methods such as retrofitting
(Faruqui et al., 2015) at the cost of increasing the di-
mensionality of the meta-embeddings. Coates and
Bollegala (2018) prove that averaging is in some
cases better than concatenation, with the additional
benefit of a reduced dimensionality. The most pop-
ular approach to address the dimensionality prob-
lem is to apply dimensionality reduction algorithms 1. We empirically demonstrate that our method
generates better meta-embeddings thanks to
decreasing the information loss during the em-
bedding combination. 1https://github.com/ikergarcia1996/
MetaVec 1
Introduction Word embeddings successfully capture lexical se-
mantic information about words based on co-
occurrence patterns extracted from large corpora
(Mikolov et al., 2013a; Pennington et al., 2014;
Mikolov et al., 2018) or knowledge bases (Bor-
des et al., 2011), with excellent results on sev-
eral tasks, including word similarity (Collobert and
Weston, 2008; Turian et al., 2010; Socher et al.,
2011), Semantic Textual Similarity (Shao, 2017),
or more recently, unsupervised machine translation
(Artetxe et al., 2019), inferring representations for
rare words (Schick and Schütze, 2020), unsuper-
vised word alignment (Jalili Sabet et al., 2020) or
knowledge base probes (Dufter et al., 2021). In
these tasks, word embeddings perform similarly
or better than transformer-based language models
such as BERT (Devlin et al., 2019), while requir-
ing a comparatively tiny amount of resources for
training and inference. 3957 cant effect on the final evaluation results. Summa-
rizing, this lack of consistency in evaluation tasks,
methodologies and ad-hoc hyper-parameter tun-
ing makes it very hard to objectively compare the
proposed methods. Thus, to the best of our knowl-
edge, and despite the existence of multiple works
addressing this task, a unified and comprehensive
evaluation of meta-embeddings has not been yet
carried out. In fact, the lack of such unified and
comprehensive evaluation framework has arguably
caused erroneous assumptions and an overestima-
tion in the performance of meta-embeddings for
extrinsic tasks. The rest of the paper is organized as follows. Section 2 presents the related work. Section 3 fo-
cuses on the evaluation frameworks used by pre-
vious works and presents our own proposal. In
Section 4 we describe our approach for creating
meta-embeddings, with Section 5 describing the
source word embeddings explored and reporting
our experimental results in Section 6. Finally, Sec-
tion 7 presents some concluding remarks and our
future work. Our code and meta-embeddings are
publicly available1. 2
Related work Our approach does not
rely on hyper-parameter tuning. 2. We generate meta-embeddings using a wide
range of source embeddings trained with very
different algorithms and resources. Our exper-
iments show that the best meta-embeddings
are obtained when combining embeddings
that encode complementary knowledge. 3. A unified and comprehensive benchmarking
framework to facilitate a fair and objective
evaluation of embeddings in both intrinsic and
extrinsic settings. 4. We report the largest meta-embedding extrin-
sic evaluation performed so far showing that
meta-embedding performance in these tasks
has been overestimated by previous work. 3958 Paper
Intrinsic Tasks
Extrinsic Tasks
(Kiela et al., 2018)
SST, SNLI, Image Caption (1)
(He et al., 2020)
SST2, SNLI, NER (1), POS(1), Semcor
(Bollegala and Bao, 2018)
Sim. (4), An. (3), Relation Classification (1)
Short Text Classification (4), Psycholinguis-
tic Score Prediction (2)
(O’Neill and Bollegala, 2020)
Sim. (6), An. (3)
POS (1), NER (1), Sentiment Analysis(1)
(Jawanpuria et al., 2020)
Sim. (6), An. (2)
(Doval et al., 2018)
Sim. (4), Bilingual dictionary induction (4),
hypernym discovery (1)
(Bollegala et al., 2018)
Sim. (6), An. (2), Relation Classification (1)
Short-text classification (2)
(Coates and Bollegala, 2018)
Sim. (5), An. (1)
(Yin and Schütze, 2016)
Sim. (5), An. (1)
POS (1)
(Li et al., 2020)
MT (3), Text Classification (5)
(Chen et al., 2020)
Sim. (5)
SNLI (1)
(Goikoetxea et al., 2016)
Sim. (4)
(Speer et al., 2017)
Sim. (5). SAT An. (1)
Common-Sense Stories (1)
(García-Ferrero et al., 2020)
Sim. (14)
STS (1), POS (1)
This work
Sim. (7), An. (3), Categorization (4)
CoLA, SST-2, MRPC, STS-B, QQP, MNLI,
QNLI, RTE , WNLI, AX
Table 1: Evaluation tasks used in previous works. Table 1: Evaluation tasks used in previous works. w OOV
w/o OOV
Default
82.7
82.7
Clean dataset
69.8
74.3
Lowercase embedding
39.5
80.7
Trim vocabulary
40.4
84.1
Table 2: FastText embeddings accuracy in the Google
Analogy dataset using different pre-processing ap-
proaches. w OOV
w/o OOV
Default
82.7
82.7
Clean dataset
69.8
74.3
Lowercase embedding
39.5
80.7
Trim vocabulary
40.4
84.1 the final meta-embedding. An ablation study con-
firms that these steps increase the performance of
the generated meta-embeddings in both intrinsic
and extrinsic tasks. Another recent research line tries to dynamically
generate meta-embeddings for specific tasks (He
et al., 2020; Kiela et al., 2018; O’Neill and Bolle-
gala, 2020). These methods extend already existing
algorithms to generate meta-embeddings by learn-
ing task specific weights. 2
Related work Instead, the focus of our
research is to generate the best general purpose
meta-embedding that can be applied to any task. Table 2: FastText embeddings accuracy in the Google
Analogy dataset using different pre-processing ap-
proaches. such as SVD (Yin and Schütze, 2016), PCA (Ghan-
nay et al., 2016) or DRA (Raunak, 2017). In this
line of work, Numberbatch (Speer et al., 2017)
claims to be the best meta-embedding model so
far, by combining knowledge from a variety of
embeddings obtained from different corpora and
knowledge bases such as ConceptNet. 2Trained in Common Crawl corpus with 600B tokens. 3https://github.com/kudkudak/word-
embeddings-benchmarks Word Corpus 4. Jiant5 provides a framework for extrinsic evalu-
ation of word representations using GLUE (Wang
et al., 2019b) and SuperGLUE (Wang et al., 2019a). We use the same bag-of-words configuration used
in the GLUE leaderboard for the Cbow baseline 6
and we evaluate the embeddings in all GLUE tasks
(CoLa (Warstadt et al., 2019) , SST-2 (Socher et al.,
2013), MRPC (Dolan and Brockett, 2005) , STS-B
(Cer et al., 2017), QQP 7, MNLI (Williams et al.,
2018), QNLI (Rajpurkar et al., 2016; Wang et al.,
2019b), RTE (Dagan et al., 2006; Bar Haim et al.,
2006; Giampiccolo et al., 2007; Bentivogli et al.,
2009), WNLI (Levesque et al., 2011), AX (Wang
et al., 2019b)). This lack of evaluation consistency led us to
propose a unified evaluation framework that en-
compasses a wide range of tasks and datasets to
evaluate meta-embeddings. In order to make the
evaluation as simple and unified as possible we
chose two already existing out of the box frame-
works: 4
Our Method Our meta-embedding generation approach con-
sists of two main steps: (i) pre-processing of the
source embeddings and (ii) generation of the meta-
embedding by averaging. Our method can combine
any number of word embeddings as long as there
is some common vocabulary shared between them. The resulting meta-embedding vocabulary will be
the union of the vocabularies of the source word
embeddings used. Word embeddings benchmarks3 (Jastrzebski
et al., 2017) provides scripts for evaluating word
embeddings in three intrinsic evaluation tasks:
(i) Word similarity (WS353 (Finkelstein et al.,
2001), MTurk (Halawi et al., 2012), RG65 (Ruben-
stein and Goodenough, 1965), RW (Pilehvar et al.,
2018), SimLex999 (Hill et al., 2015), MEN (Bruni
et al., 2014)); (ii) Word analogy (Google Analogy
(Mikolov et al., 2013a), MSR Analogy (Mikolov
et al., 2013c), SemEval2012 (Jurgens et al., 2012))
and, (iii) Word categorization (AP (Almuhareb
and Poesio, 2005), BLESS (Baroni and Lenci,
2011), Battig (Battig and Montague, 1969), ESSLI
(McRae et al., 2005)). We use the provided script
for evaluating embeddings on all the tasks without
lowercasing them. 4https://books.google.com/ngrams/info
5https://github.com/nyu-mll/jiant-v1-
legacy
6https://github.com/nyu-mll/jiant-
v1-legacy/blob/master/jiant/config/
superglue_bow.conf
7https://www.quora.com/q/quoradata/
First-Quora-Dataset-Release-Question-
Pairs 6https://github.com/nyu-mll/jiant-
v1-legacy/blob/master/jiant/config/
superglue_bow.conf
7 3
Evaluation Framework As it has been earlier mentioned, several methods
to generate meta-embeddings have been previously
proposed and evaluated on many different bench-
marks, as shown by Table 1. Moreover, add-hoc
decisions (not always explicitly mentioned) to eval-
uate the embeddings caused large variations in the
results. Let us consider, for example, the problem
of out-of-vocabulary (OOV) words. Methods such as MUSE (Lample et al., 2018)
and VecMap (Artetxe et al., 2018) project em-
beddings of two different languages to a shared
common space by means of a bilingual dictionary
(Mikolov et al., 2013b). This requires minimal
bilingual supervision while still leveraging large
amounts of monolingual corpora with very com-
petitive results (Artetxe et al., 2016, 2018). These
techniques are used by Doval et al. (2018); García-
Ferrero et al. (2020); Jawanpuria et al. (2020); He
et al. (2020) to generate meta-embeddings. This
usually involves mapping all the source embed-
dings to a common vector space followed by aver-
aging. We extend this idea by proposing a multiple
step algorithm that: (i) normalizes the source em-
beddings; (ii) maps them to the same vector space;
(iii) handles the OOV words; and (iv), generates Two popular techniques are used to address OOV
words. Table 2 shows the accuracy of FastText em-
beddings 2 in the Google Analogy dataset using the
two approaches. The first one uses the average of
all the embeddings as a representation for unknown
words (With OOV). The second approach simply
removes from the dataset the examples containing
unknown words (Without OOV). Additionally, the
dataset is usually pre-processed. A common ap-
proach lowercase all the words and removes non 3959 Word Corpus 4. 5 English characters (Clean dataset) to reduce the
number of unknown words. The words in the em-
bedding can also be lowercased (Lowercase em-
beddings). Another popular practice to evaluate
analogy consist of trimming the vocabulary of the
embedding to the k most popular words. As an
example, trimming the vocabulary to the 100,000
most popular English words also speeds up the
computations (Trim vocabulary). These changes
in the pre-processing of the very same embeddings
cause the results to vary from 39.5% accuracy to
84.1%. Obviously, without a common evaluation
framework the comparison between the different
embeddings and meta-embeddings cannot be ob-
jectively done. 4https://books.google.com/ngrams/info
5https://github.com/nyu-mll/jiant-v1-
legacy
6 7https://www.quora.com/q/quoradata/
First-Quora-Dataset-Release-Question-
Pairs 5
Word embeddings transformation. Orthogonality allows monolingual
invariance during the mapping, preserving vector
dot products between word vectors. Monolingual
invariance ensures that no information is lost dur-
ing the mapping step, which is desirable for our
aim of generating meta-embeddings. In our ex-
periments we align the source embedding by pro-
jecting them to the vector space of one particular
source embeddings involved in the construction of
the meta-embeddings. This section describes the source word embeddings
used to generate our meta-embeddings. We choose
these pre-trained embeddings for two main rea-
sons. They have been trained using very diverse
algorithms and resources, and they obtain good per-
formance on our evaluation framework when tested
individually. That is, they may encode high quality
complementary knowledge. Using Large text corpora, Word2Vec (W2V)
(Mikolov et al., 2013a) embeddings from Google
News (100 billion words). A GloVe (GV) (Pen-
nington et al., 2014) model the Common Crawl vec-
tors (640 billion words). As recommended by the
authors, we apply a l2 normalization to its variables. And the FastText (FT) (Mikolov et al., 2018) em-
beddings from Common Crawl (600 billion words). Using WordNet (Miller, 1992),
RWSGwn
(UKB) (Goikoetxea et al., 2015) combines random
walks over WordNet with the skip-gram model. We
have used the vectors trained using WordNet3.0
plus gloss relations. JOINTChyb (J) (Goikoetxea
et al., 2018) combines Random Walks over multi-
lingual WordNets and bilingual corpora as input for
a modified skip-gram model that forces equivalent
terms in different languages to come closer during
training. We used the English-Spanish bilingual
embeddings publicly available. Using Large text corpora, Word2Vec (W2V)
(Mikolov et al., 2013a) embeddings from Google
News (100 billion words). A GloVe (GV) (Pen-
nington et al., 2014) model the Common Crawl vec-
tors (640 billion words). As recommended by the
authors, we apply a l2 normalization to its variables. And the FastText (FT) (Mikolov et al., 2018) em-
beddings from Common Crawl (600 billion words). 3) OOV generation: Different word embed-
dings have different vocabularies. When combin-
ing two word embeddings we can distinguish two
sets of words. Those for which we have a repre-
sentation in both embeddings and those for which
one of the embeddings has no representation. We
call the latter "OOV words". We unify the vocab-
ulary of the source embeddings by creating new
approximate representations for the OOV words. The process is as follows. 8For computation efficiency we limit the maximum k to
50. In our experiments the optimal k is usually smaller than
20. 5
Word embeddings Given two source em-
beddings E1 and E2 where for a word W only E1
has a representation, we generate a new approxi-
mation for the OOV word in E2 by revising the
most similar words from the common vocabulary
of E1 and E2. First, using the cosine similarity as
distance metric, we select the k (ranging from 2 to
50) nearest neighbours of the word W in E1 that
also appear in the common vocabulary with E2.8
For each k, we calculate k candidate representa-
tions of the OOV word in E2 and E1 as a weighted
average of the selected k nearest neighbours in
their corresponding spaces. We use the cosine sim-
ilarity from the nearest neighbors in E1 to W as
weights. Finally, the selected representation of the
OOV word in E2 is the one corresponding to the
closest candidate to W in E1. Using the Paraphrase Database (PPDB) (Gan-
itkevitch et al., 2013), Attract Repel (AR) (Mrkši´c
et al., 2017) improves word embeddings by inject-
ing synonymy and antonym constraints extracted
from monolingual and cross-lingual lexical re-
sources. We used the English vocabulary from
the four-lingual (English, German, Italian, Rus-
sian) vector space. Paragram (P) (Wieting et al.,
2015) are pre-trained word vectors learned using
word paraphrase pairs from PPDB using a modi-
fication of the skip-gram objective function. The
hyper parameters were tuned using the wordsim-
353 dataset. The word embeddings of the default
model are initialized with Glove word vectors. 4.1
Word embeddings pre-processing Word embeddings generated with different sources
or techniques can result in very different vectors
spaces and vocabularies. Before aligning the vec-
tor spaces an harmonization pre-processing step is
needed. Thus, we translate, scale, rotate and match
the vocabularies of the source embeddings. Word embeddings generated with different sources
or techniques can result in very different vectors
spaces and vocabularies. Before aligning the vec-
tor spaces an harmonization pre-processing step is
needed. Thus, we translate, scale, rotate and match
the vocabularies of the source embeddings. 1) Mean Centering and scaling: Following
(Artetxe et al., 2018), we first normalize the length
of the source embeddings. We mean center each
dimension, and we normalize them again by length. This translates all the source embeddings to the
origin and scales them to have the same length. It should be taken into account that, for Word
analogy, smaller vocabularies usually obtain bet-
ter results. This particularly hurts the performance
of those meta-embeddings that were generated us-
ing many source embeddings resulting in a meta-
embedding with a vocabulary of more than 4 mil-
lion words. Thus, in order to ensure a fair evalua-
tion regardless of the number of words in the vocab-
ulary, we trim the vocabulary of all the embeddings
and meta-embeddings to the 200,000 most popular
English words according to the Google’s Trillion 2) Aligning the vector spaces: We align the
vector spaces of the source embeddings using
VecMap (Artetxe et al., 2016). VecMap learns
word embedding mappings using an orthogonal 3960 4.2
Meta-embedding generation In our experi-
ments, the best results are achieved when combin-
ing source embeddings generated using very dif-
ferent resources, such as text and knowledge bases. Table 3: Source embedding intrinsic evaluation results. Text
WN
PPDB
CN
Text
67.9
66.3
68.5
69.1
WN
66.3
50.9
62.5
65.4
PPDB
68.5
62.5
60.2
67.8
CN
69.1
65.4
67.8
- Table 4: Comparison of the average performance in the
intrinsic evaluation tasks for meta-embeddings gener-
ated using pairs of embeddings that encode knowledge
form the same or different sources. WN stands for
WordNet and CN for ConceptNet. Table 4: Comparison of the average performance in the
intrinsic evaluation tasks for meta-embeddings gener-
ated using pairs of embeddings that encode knowledge
form the same or different sources. WN stands for
WordNet and CN for ConceptNet. These combinations produce a meta-embedding
that encodes the complementary knowledge of the
source embeddings resulting in an improved per-
formance. Also note that the meta-embedding com-
bining text (FT) and PPDB (P), and also text (FT)
with ConceptNet (N) outperforms the results of
Numberbach (N) alone. showed no significant improvements over the cho-
sen ones. showed no significant improvements over the cho-
sen ones. We generate our best meta-embeddings combin-
ing the best source embeddings created using large
text corpora (FT), WordNet (J), PPDB (P) and Con-
ceptNet (Numberbatch) (hereinafter FJNP). This
combination maximizes the complementary knowl-
edge encoded in the meta-embedding. We com-
pare our method with 3 baselines using the same
source embeddings: (i) Concatenation: (CONC+)
Concatenation is a very strong baseline in meta-
embedding generation. It allows combining mul-
tiple embeddings without any information loss. However, this comes at a high cost, as the meta-
embedding dimensionaly is increased dramatically. We standardize the source embeddings using the
approach described in Section 4.1. (ii) AutoEn-
coders (Bollegala and Bao, 2018): Autoencoders
are an unsupervised learning method that first com-
press the input in a space of latent variables and
then reconstructs the input based on the informa-
tion encoded in these latent variables. It aims to
learn meta-embeddings by reconstructing multi-
ple source embeddings. This method comes in
three flavours, DAEME, CAEME and AAEME. We used the last one because it obtains the best
results. We applied the default parameters and
enabled the option to generate OOV word represen-
tations. (iii) Locally Linear Meta-Embedding 9We calculate the Spearman Correlation for the Se-
mEval2012 dataset and accuracy for GoogleAnalogy and MSR 6
Experiments We evaluate all the word embeddings in a wide
range of intrinsic and extrinsic evaluation tasks
which composed the evaluation framwework de-
scribed in Section 3. 4.2
Meta-embedding generation We combine the harmonized source embeddings
by averaging them. In our experiments we demon-
strate that, thanks to the pre-processing steps de-
scribed above, averaging source embeddings effec-
tively combines multiple source embeddings result-
ing in representations as good as the ones generated
by concatenation without increasing their dimen-
sionality. Using ConceptNet; Numberbatch (N) (Speer
et al., 2017) combines knowledge encoded in Con-
ceptNet, Word2vec, GloVe and OpenSubtitles 2016
using concatenation, dimensionality reduction and
a variation of retrofitting. Numberbatch version
19.08 is used. We also tested other embeddings such as ExtVec
(Komninos and Manandhar, 2016), LexSub (Arora
et al., 2020) or LexVec (Salle et al., 2016) but 3961 Embedding
AVG
C
WS
A
FT
67.8
71.4
73.6
58.5
GV
64.7
69.9
70.3
54.0
W2V
59.1
67.9
65.6
43.9
J
52.2
70.0
65.2
21.4
UKB
46.6
67.9
61.8
10.2
P
58.5
66.5
70.2
38.9
AR
48.5
59.7
63.6
22.2
N
68.1
73.6
75.2
55.4
T bl 3 S
b ddi
i t i
i
l
ti
lt intrinsic evaluation benchmark of different pairs
of source embeddings. For each source class type
(Text Corpora, WordNet, PPDB and ConceptNet),
we combine the best embeddings of each class with
the best embeddings of the other classes. Within
the same class we combine the first and second best
embeddings. The results show that, instead of using embed-
dings based on the same information type, combin-
ing embeddings of different classes obtains most
of the time better results. That is, two embeddings
generated using similar sources do not contain com-
plementary knowledge, and its combination does
not result in better performance. In our experi-
ments, the best results are achieved when combin-
ing source embeddings generated using very dif-
ferent resources, such as text and knowledge bases. These combinations produce a meta-embedding
that encodes the complementary knowledge of the
source embeddings resulting in an improved per-
formance. Also note that the meta-embedding com-
bining text (FT) and PPDB (P), and also text (FT)
with ConceptNet (N) outperforms the results of
Numberbach (N) alone. The results show that, instead of using embed-
dings based on the same information type, combin-
ing embeddings of different classes obtains most
of the time better results. That is, two embeddings
generated using similar sources do not contain com-
plementary knowledge, and its combination does
not result in better performance. 6.1
Intrinsic evaluation results First we evaluate the source embeddings that we
will later use for meta-embedding generation. Ta-
ble 3 shows the averaged results of the Categoriza-
tion (C), Word Similarity (WS) and Analogy (A)
datasets. We report the average cluster purity score
of the Categorization datasets, the average Spear-
man correlation in the WS datasets, and the average
score9 in the Word Analogy datasets. The results
shows that FastText achieve the best performance
on the Analogy datasets and Numberbatch on Cat-
egorization and Word Similarity. As expected, on
average Numberbatch obtains the best results on
the intrinsic evaluations tasks. We start generating meta-embeddings with our
proposed method combining pairs of source em-
beddings. Table 4 shows the average score in the 3962 FJNP
AVG
C
WS
A
CONC+
70.1
71.7
78.5
60.1
LLE
52.4
60.8
68.1
28.3
AAEME
67.6
71.2
75.0
56.6
Our Method
70.6
73.5
78.4
59.9
Table 5: Comparison of our meta-embedding method,
baselines and prior work in the intrinsic evaluation. (Devlin et al., 2019). Thus, word embeddings may
be better suited for other tasks such as unsuper-
vised machine translation (Artetxe et al., 2019),
inferring high-quality embeddings for rare words
(Schick and Schütze, 2020), unsupervised word
alignment (Jalili Sabet et al., 2020) or knowledge
base queries (Dufter et al., 2021). However, we can
use the GLUE benchmark as part of an objective
and unified framework to evaluate word embed-
dings. In this sense, future research can also use
exactly the same setting and methodology to evalu-
ate new word embeddings and meta-embeddings. (Devlin et al., 2019). Thus, word embeddings may
be better suited for other tasks such as unsuper-
vised machine translation (Artetxe et al., 2019),
inferring high-quality embeddings for rare words
(Schick and Schütze, 2020), unsupervised word
alignment (Jalili Sabet et al., 2020) or knowledge
base queries (Dufter et al., 2021). However, we can
use the GLUE benchmark as part of an objective
and unified framework to evaluate word embed-
dings. In this sense, future research can also use
exactly the same setting and methodology to evalu-
ate new word embeddings and meta-embeddings. Table 5: Comparison of our meta-embedding method,
baselines and prior work in the intrinsic evaluation. Learning (LLE) (Bollegala et al., 2018): This ap-
proach which consists of two steps. In the recon-
struction step the embeddings of each word are
represented by the linear weighted combination of
the embeddings of its nearest neighbours. 6.1
Intrinsic evaluation results In the
projection step the meta-embedding of each word
is computed such that the nearest neighbours in
the source embedding spaces are embedded closely
to each other in the meta-embedding space. We
tested this method with the same parameters used
in the original paper. Note that the code provided
by the authors generates meta-embeddings using
the intersection of the vocabulary of the source em-
beddings. This results in a small vocabulary that
severely hurts its performance in some tasks. Table 6 presents the results of the extrinsic eval-
uation. Interestingly, FastText achieves the best re-
sults, outperforming every single meta-embedding
in every task. In fact, Numberbatch and AAEME
fail on the extrinsic evaluation achieving very low
results compared with the source word embed-
dings. Previous research in meta-embedding genera-
tion has limited the extrinsic evaluation to very few
tasks that are formulated closely to the intrinsic
evaluation such as short text classification (Bol-
legala and Bao, 2018; Bollegala et al., 2018) or
common-sense stories (Speer et al., 2017). Other
approaches combine meta-embeddings with con-
textual representations with the aim of achieving
SOTA results for tasks such as STS or POS tagging
(García-Ferrero et al., 2020). While those previous
works assume that meta-embeddings might be help-
ful for such extrinsic evaluation tasks, our results
show that when evaluating on ten challenging tasks,
FastText is indeed a very strong baseline that is not
improved by any meta-embedding proposed up to
date. These results suggest that meta-embeddings
generated using complementary knowledge from
WordNet, ConceptNet or PPDB help to improve
performance for intrinsic tasks, but that this is not
the case for extrinsic evaluations using GLUE. Table 5 reports the results for our method and
the baselines. The overall performance of our
method is slightly better than concatenation (im-
proved with our standardization method), mostly
due to the good results in Categorization. In any
case, the most important point here is to notice that
our method, unlike concatenation (CONC+), does
not increase the final dimensionality of the meta-
embeddings. Furthermore, our technique clearly
outperforms the meta-embeddings generated by
Autoencodding and LLE and all the embeddings
listed in Table 3 including Numberbatch, which is
a meta-embedding. To the best of our knowledge,
these are the best results published using these in-
trinsic benchmarks. 6.2
Extrinsic evaluation results We perform an ablation study to determine which
steps of our method contribute the most. For the
ablation study we use the best meta-embedding in
the intrinsic and extrinsic evaluation tasks. We do
this by skipping a different step of the method each
time. For -OOV we do not apply the technique to
obtain representations for the OOV words, we just
average the available representations for a given
word. With -NORM we do not perform the nor-
malization steps to the source embeddings. For We compare our meta-embeddings with the same
source embeddings and baselines used in the in-
trinsic evaluation (subsection 6.1). We test the
same combination of embeddings that provides the
best results in the intrinsic evaluation (FJNP). For
brevity we report the GLUE Score calculated as
proposed by the authors (Wang et al., 2019b). We
are aware that, for the GLUE benchmark, (static)
word embeddings are outperformed by contextual
representations such as those obtained by BERT 3963 FT
GV
W2V
J
UKB
P
AR
N
FJNP
CONC+
LLE
AAEME
Our Method
60.5
43.4
59.6
58.2
56.1
58.2
52.1
53.4
52.4
48.5
53.2
58.2
Table 6: Comparison of the source embeddings, our meta embedding method, baselines and previous work perfor-
mance on GLUE benchmarks. GLUE score is reported. FJNP
AVG
C
WS
A
GLUE
Our method
70.6
73.5
78.4
59.9
58.2
-OOV
70.6
72.5
78.1
61.2
59.5
-NORM
67.5
73.9
77.0
51.7
55.3
-Vecmap
66.7
72.7
75.0
52.3
58.0
Table 7: Ablation studies on our standardization steps. -Vecmap the source embeddings are not mapped
to a common vector space. The results reported in
Table 7 show that the normalization and the map-
ping steps provide most of the performance. If we
average embeddings that have not been normalized
the difference in scale and the centroid of the vector
space can cause some embeddings to take higher
importance in the meta-embeddings. Averaging
word embeddings that have not been mapped to the
same vector space can cause vectors to cancel each
other. Table 7: Ablation studies on our standardization steps. with OOV : overlove, outlove, antilove, lovea-
holic, have_no_regrets, sometimes_good, won-
derful_feeling, strong_like, filial_love, lovedom,
propose_to_woman, lov?d, family_love, Love,
love_dearly, love_heart, Adore, buy_ring, LOVE,
deep_affection, being_in_love, sovietophile, love-
ful, Loving, mislove, lovemonger, arachnophile
without OOV : overlove, loveaholic, outlove,
antilove With respect OOV, the results are mixed. 6.2
Extrinsic evaluation results This
step increases the performance in the categoriza-
tion and word similarity tasks but it hurts the per-
formance on the analogy and extrinsic tasks. This
is caused by two factors. First, since all the em-
beddings have been normalized and mapped to the
same vectors space, the average of the available
representations is already a good approximation
for OOV words. If the source embeddings would
have a representation for the OOV words, it would
be close to the ones already available. Table 8: Nearest neighbors to the word love for the
FJNP meta-embedding with a cosine similarity > 0.85
applying or not the OOV generation algorithm. Table 8: Nearest neighbors to the word love for the
FJNP meta-embedding with a cosine similarity > 0.85
applying or not the OOV generation algorithm. also propose a comprehensive and unified evalua-
tion framework for evaluating meta-embeddings. This framework allows to fairly and objectively
compare different meta-embedding generation ap-
proaches using the same settings and methodology. Using this framework we demonstrate that
combining embeddings that encode the most
complementary knowledge produces better meta-
embeddings. In fact, the meta-embeddings that
encode in the same vector space the knowledge
from large text corpora, WordNet, PPDB and Con-
ceptNet achieve the best published results in the
intrinsic evaluation benchmarks. Interestingly, and
contrary to what previous research suggested, we
empirically demonstrate that when evaluating in a
large set of extrinsic tasks, meta-embeddings are
not helpful for improving the results of the source
embeddings. We plan to investigate the perfor-
mance of our approach in a cross-lingual setting
for under-resourced languages. We suspect that the
performance of under-resource language embed-
dings can be improved by combining them with
embeddings from a rich-resource language. also propose a comprehensive and unified evalua-
tion framework for evaluating meta-embeddings. This framework allows to fairly and objectively
compare different meta-embedding generation ap-
proaches using the same settings and methodology. Additionally, a larger vocabulary is not benefi-
cial for every task. Consider the example in Table 2
where a much larger vocabulary obtains worse re-
sults in the Word Analogy task. We demonstrate
this by counting the number of nearest neighbors
to love with a cosine similarity greater than 0.85
in the meta-embeddings. Table 8 shows the most
similar words when using and not using the OOV
algorithm (27 and only 4 words respectively). 6.2
Extrinsic evaluation results Gen-
erating a meta-embedding containing the union of
the vocabularies of all the source embeddings may
be useful for some tasks, such as word similarity. However, for tasks such as word analogy, reducing
the final vocabulary to the set of most common
words is the best approach. This framework allows to fairly and objectively
compare different meta-embedding generation ap-
proaches using the same settings and methodology. Using this framework we demonstrate that
combining embeddings that encode the most
complementary knowledge produces better meta-
embeddings. In fact, the meta-embeddings that
encode in the same vector space the knowledge
from large text corpora, WordNet, PPDB and Con-
ceptNet achieve the best published results in the
intrinsic evaluation benchmarks. Interestingly, and
contrary to what previous research suggested, we
empirically demonstrate that when evaluating in a
large set of extrinsic tasks, meta-embeddings are
not helpful for improving the results of the source
embeddings. We plan to investigate the perfor-
mance of our approach in a cross-lingual setting
for under-resourced languages. We suspect that the
performance of under-resource language embed-
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partially funded by the Spanish Ministry of Sci-
ence, Innovation and Universities (DeepReading
RTI2018-096846-B-C21, MCIU/AEI/FEDER, UE)
and DeepText (KK-2020/00088), funded by the
Basque Government. Iker García-Ferrero is sup-
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and negative sampling for improved word represen-
tations. References In Proceedings of the 54th Annual Meet-
ing of the Association for Computational Linguistics
(Volume 2: Short Papers), pages 419–424, Berlin,
Germany. Association for Computational Linguis-
tics. Alex Wang,
Yada Pruksachatkun,
Nikita Nangia,
Amanpreet Singh, Julian Michael, Felix Hill, Omer
Levy, and Samuel R. Bowman. 2019a. Superglue:
A stickier benchmark for general-purpose language
understanding systems. In Advances in Neural Infor-
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on Neural Information Processing Systems 2019,
NeurIPS 2019, December 8-14, 2019, Vancouver,
BC, Canada, pages 3261–3275. Timo Schick and Hinrich Schütze. 2020. BERTRAM:
Improved word embeddings have big impact on con-
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the 58th Annual Meeting of the Association for Com-
putational Linguistics, pages 3996–4007, Online. Association for Computational Linguistics. Tobias Schnabel, Igor Labutov, David Mimno, and
Thorsten Joachims. 2015. Evaluation methods for
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bon, Portugal. Association for Computational Lin-
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Alex Warstadt, Amanpreet Singh, and Samuel R. Bow-
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Livescu. 2015. From paraphrase database to compo-
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guistics: Human Language Technologies, Volume
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Louisiana. Association for Computational Linguis-
tics. Wenpeng Yin and Hinrich Schütze. 2016. Learning
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Annual Meeting of the Association for Computa-
tional Linguistics (Volume 1: Long Papers), pages
1351–1360, Berlin, Germany. Association for Com-
putational Linguistics. 3969 3969 A
Meta-embedding Generation
Algorithm Illustrated Figure 2: Step 1 Length normalization of the source
embeddings In this section we illustrate our meta-embedding
generation algorithm using two sample embeddings
with 3 dimension vectors and 1000 word vocabu-
lary sizes (Figure 1). The vocabularies of the two
embeddings have 791 common words, and each
embedding has 209 unique words for which the
other embeddings does not have a representation
(OOV words). The resulting meta-embedding vo-
cabulary will be the union of the vocabularies, 1197
words. Our approach to generate meta-embeddings
consists of two main steps (i) pre-processing of
the source embeddings and (ii) generation of the
meta-embedding by averaging. Figure 2: Step 1 Length normalization of the source
embeddings Figure 1: Step 0 Source embeddings at the start of the
embedding generation process Figure 3: Step 2 Mean centering of the source embed-
dings Figure 1: Step 0 Source embeddings at the start of the
embedding generation process Figure 3: Step 2 Mean centering of the source embed-
dings A.2
Meta-embedding generation We combine the harmonized source embeddings by
averaging them (Figure 7). We empirically demon-
strate that thanks to the pre-processing steps, av-
eraging source embeddings effectively combines
multiple source embeddings resulting in representa-
tions as good as the ones generated by embedding
concatenation without increasing its dimensional-
ity. Figure 5: Step 4 Alignment of the source embeddings
using VecMap 39
3) OOV generation: Different word embed-
dings have different vocabularies. When combin-
ing two word embeddings we can distinguish two
sets of words. Those for which we have a represen-
tation in both embeddings and those for which one
of the embeddings has no representation. We call
the latter "OOV words". We unify the vocabulary
of the source embeddings by creating new approxi-
mate representations for the OOV words (Figure 6). The process is as follows. Given two source embed-
dings E1 and E2 where for a word W only E1 has A.1
Word embeddings pre-processing Word embedding generated with different sources
or techniques can result in very different vectors
spaces and vocabularies. Before aligning the vec-
tor spaces an harmonization pre-processing step is
needed. Thus, we translate, scale, rotate and match
the vocabularies of the source embeddings. vector spaces of the source embeddings using
VecMap (Artetxe et al., 2016) (Figure 5). VecMap
learns word embedding mapping using an orthog-
onal transformation. Orthogonality allows mono-
lingual invariance during the mapping, preserving
vector dot products between word vectors. Mono-
lingual invariance ensures no information loss dur-
ing the mapping step, which is desirable for our
aim of generating meta-embeddings. In our ex-
periments we align the source embedding by pro-
jecting them to the vector space of one particular
source embeddings involved in the construction of 1) Mean Centering and scaling: Following
(Artetxe et al., 2018) we first length normalize the
source embeddings (Figure 2). We mean center
each dimension (Figure 3), and we length normal-
ize them again (Figure 4). This translates all the
source embeddings to the origin and scales them to
have the same length. 2) Aligning the vector spaces: We align the 3970 Figure 4: Step 3 Second length normalization of the
source embeddings a representation, we generate a new approximation
for the OOV word in E2 by revising the most simi-
lar words from the common vocabulary of E1 and
E2. First, using the cosine similarity as distance
metric, we select the k (ranging from 2 to 50) near-
est neighbours of the word W in E1 that also appear
in the common vocabulary with E2. For each k, we
calculate k candidate representations of the OOV
word in E2 and E1 as a weighted average of the
selected k nearest neighbours in their correspond-
ing spaces. We use the cosine similarity from the
nearest neighbors in E1 to W as weights. Finally,
the selected representation of the OOV word in E2
is the one corresponding to the closest candidate to
W in E1. Figure 6: Step 5 OOV generation algorithm Figure 4: Step 3 Second length normalization of the
source embeddings the meta-embeddings. Figure 5: Step 4 Alignment of the source embeddings
using VecMap the meta-embeddings. the meta-embeddings. Figure 6: Step 5 OOV generation algorithm B
Computing infrastructure We run all the experiments in a Linux system with
an Intel Xeon CPU E5-2620 V4 CPU, 1024GB of
RAM and an Nvidia Titan V GPU. To reproduce
the generation of the FJNP meta-embedding with
a reasonable run-time (less than 24 hours) we rec-
ommend using at least a quad-core CPU, 32GB of 3971 Figure 7: Step 6 Meta-embedding generation by aver-
aging Figure 7: Step 6 Meta-embedding generation by aver-
aging RAM and a 2GB GPU with CUDA support (GPU
is optional but highly recommended). The intrin-
sic evaluation framework can be run in less than
one hour in a system with enough primary memory
to load a full embedding/meta-embedding (8GB). The extrinsic evaluation framework will run in less
than 24 hours in a system with a reasonably mod-
ern CPU and enough primary memory to load the
full embedding/meta-embedding and the bag-of-
words model (8GB). The extrinsic evaluation can
be speed-up with an 8GB GPU with CUDA and
FP16 support. 3972
|
https://openalex.org/W4362431949
|
https://figshare.com/articles/journal_contribution/Supplementary_Tables_S1-S5_from_Prediction_of_Broad_Spectrum_Resistance_of_Tumors_towards_Anticancer_Drugs/22439841/1/files/39890679.pdf
|
English
| null |
Supplementary Tables S1-S5 from Prediction of Broad Spectrum Resistance of Tumors towards Anticancer Drugs
| null | 2,023
|
cc-by
| 2,634
|
sensitivity testing of 59 diverse tumors.
Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d.
76
89
106
105
86
88
95
n.d.
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d.
68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 sensitivity testing of 59 diverse tumors. Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d. 76
89
106
105
86
88
95
n.d. 100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d. 68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 87
100
94
87
74
99
104
110
88
83
82
77
97
121
125
106
105
102
107
129
84
79
26
24
1
95
102
92
97
46
81
103
87
97
89
109
81
98
109
103
98
75
81
81
74
76
68
71
80
16
96
95
97
89
97
59
88
70
77
44
73
100
112
104
96
92
102
85
104
96
116
75
103
100
125
113
109
105
134
100
105
91
99
98
98
89
84
97
80
114
109
96
95
111
123
88
96
92
100
107
86
81
98
59
80
78
86
86
57
106
113
123
106
70
102
31
75
94
30
24
97
97
124
102
119
82
109
89
111
102
115
107
81
11
n.d. 99
104
99
99
2
108
103
101
87
67
86
74
95
106
70
71
94
111
n.d. n.d. sensitivity testing of 59 diverse tumors.
Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d.
76
89
106
105
86
88
95
n.d.
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d.
68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 99
89
101
95
110
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
ls (radioactive counts per minute, cpm) 95
110
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
adioactive counts per minute, cpm) 87
100
94
87
74
99
104
110
88
83
82
77
97
121
125
106
105
102
107
129
84
79
26
24
95
102
92
97
46
81
103
87
97
89
109
81
98
109
103
98
75
81
81
74
76
68
71
80
16
96
95
97
89
97
59
88
70
77
44
73
100
112
104
96
92
102
85
104
96
116
75
103
100
125
113
109
105
134
100
105
91
99
98
98
89
84
97
80
114
109
96
95
111
123
88
96
92
100
107
86
81
98
59
80
78
86
86
57
106
113
123
106
70
102
31
75
94
30
24
97
97
124
102
119
82
109
89
111
102
115
107
81
11
n.d. 99
104
99
99
108
103
101
87
67
86
74
95
106
70
71
94
111
n.d. n.d. 99
89
101
95
110
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
cells (radioactive counts per minute, cpm) pp
y
gy
y
g
g
Nucleotide Incorporation Assay
(% of Control)
Doxo-
5-Fluoro-
Cyclophos-
No. sensitivity testing of 59 diverse tumors.
Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d.
76
89
106
105
86
88
95
n.d.
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d.
68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 Histology
rubicin
uracil
phamide
TdR*
1
Small cell lung cancer
71
68
14
194
2
Adenocarcinoma
108
99
86
43
3
Squamous cell carcinoma
72
104
61
53
4
Pleura mesothelioma
28
72
53
87
5
Squamous cell carcinoma
40
75
70
80
6
Alveolar cellular carcinoma
25
27
78
497
7
Adenocarcinoma
21
20
62
1360
8
Small cell lung cancer
71
107
70
80
9
Small cell lung cancer
28
44
21
113
10
Small cell lung cancer
35
28
13
362
11
Small cell lung cancer
34
57
24
204
12
Squamous cell carcinoma
47
69
43
99
13
Small cell lung cancer
9
51
31
1714
14
Small cell lung cancer
93
93
14
410
15
Squamous cell carcinoma
15
17
32
1049
16
Small cell lung cancer
14
81
77
71
17
Squamous cell carcinoma
76
70
56
52
18
Small cell lung cancer
97
20
97
13472
19
Small cell lung cancer
51
44
12
378
20
Small cell lung cancer
33
65
13
282
21
Squamous cell carcinoma
100
91
67
74
22
Squamous cell carcinoma
52
39
29
147
23
Small cell lung cancer
47
34
8
615
24
Small cell lung cancer
24
37
53
466
25
Squamous cell carcinoma
104
86
102
55
26
Adenocarcinoma
63
129
41
37
27
Polymorphic cellular
sarcoma of the lung
64
48
27
37
28
lymph node metastasis
64
64
69
62
29
Large cell carcinoma
101
92
119
36
30
Squamous cell carcinoma
73
112
90
30
31
Squamous cell carcinoma
26
61
69
125
32
Polymorphic cellular
sarcoma of the lung
88
56
71
41
33
n.a. 19
53
14
598
34
n.a. 50
72
36
182
35
n.a. 96
94
90
40
36
n.a. 45
84
30
223
37
n.a. 84
110
64
188
38
n.a. 49
94
43
192
* Incorporation of tritium-labeled thymidine in untreated control cells
(radioactive counts per minute, cpm)
n a
not available tivity testing in 21 samples of
IC50 Values (MTT
Assay)
Daunorubicin
Cytarabine
6-
Thioguanine
0,71
1,53
38,29
0,429
n.d. 8,42
0,224
0,101
4,64
0,353
0,443
n.d. sensitivity testing of 59 diverse tumors.
Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d.
76
89
106
105
86
88
95
n.d.
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d.
68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 0,293
0,418
3,22
0,158
0,049
1,562
0,434
1,406
7,2
0,26
1,25
2,055
0,25
0,349
24,104
0,29
0,511
9,215
0,355
0,117
11,28
0,287
0,088
15,08
0,189
2,5
4,437
0,242
2,5
14,16
0,23
0,502
8,05
0,09
0,526
9,94
0,011
0,129
7,1
0,002
0,0025
1,562
0,002
0,018
1,562
0,045
0,165
5,715
0,123
0,155
6,25 Supplementary Table IV: Correlation of 531 natural products derived from traditional
Chinese medicine with the rhodamine 123 accumulation as a measure of P-glycoprotein
function in 60 NCI cell lines. The log10 IC50 values for 18 of 531 compounds correlated with
rhodamine 123 accumulation (R<-0.2 or R>0.2 and p<0.05). Compound
R-Value
p-Value
Compound
R-Value
p-Value
NSC123390
0,01341 -0,20536
NSC146397
0,00598
0,22662
NSC157035
0,01583 -0,21674
NSC150446
0,00191
0,26574
NSC215254
0,01122 -0,23855
NSC163088
0,00152
0,29888
NSC59193
0,0132 -0,21002
NSC338259
0,00139
0,26963
NSC624807
0,00649 -0,22202
NSC51524
0,01974
0,20094
NSC657824
0,00881 -0,23162
NSC653384
0,02189
0,20574
NSC7833
0,0055 -0,25946
NSC655524
0,00164
0,30342
NSC93681
0,01053 -0,22754
NSC678106
0,00956
0,22865
NSC701813
0,01064
0,21156
NSC93137
0,00434
0,24108 Supplementary Table V: Correlation of natural products derived from traditional Chinese
medicine with the doubling time of 60 NCI cell lines. The log10 IC50 values for 162 of 531
compounds correlated with the cell doubling times (hrs) (R<-0.2 or R>0.2 and p<0.05). sensitivity testing of 59 diverse tumors.
Nucleotide Incorporation Assay
(% of Control)
5-Fluoro-
Metho-
Cyt-
Procar-
Pro-
Treni-
uracil
trexate
arabine
bazine
resid
mon
75
94
83
110
86
n.d.
76
89
106
105
86
88
95
n.d.
100
107
84
98
112
117
121
117
109
117
112
87
105
90
75
98
95
85
86
115
93
96
137
141
114
118
139
128
84
114
103
131
79
76
110
117
107
98
88
112
87
90
84
87
76
95
104
96
91
79
114
82
103
82
104
81
111
93
63
102
106
114
92
60
83
98
90
81
71
103
94
78
n.d.
68
63
72
97
109
98
91
82
94
62
94
97
92
51
111
91
140
97
130
66
74
96
91
89
91
72
91
79
104
93
112
86
95
104
110
113
101
108
103
65
129
89
116
79
70
72
75
78
90
73
69
81
110
97
108
87
86
111
90
84
80
75
65
97
82
103
66
76
84
105
100
99
95
80
97
126
102
92
100
129
124
82
83
94
89
94
103 Compound
R-Value
p-Value
Compound
R-Value
p-Value
Compound
R-Value
p-Value
NSC629738
-0,33445
0,000119
NSC215254
0,323
0,001000000
NSC616348
0,261
0,001580000
NSC401288
-0,30502
0,000402
NSC105130
0,321
0,000724606
NSC241011
0,261
0,002100000
NSC678098
-0,27964
0,000965
NSC1012
0,321
0,000148082
NSC285166
0,260
0,001820000
NSC11779
-0,30224
0,00136
NSC35607
0,320
0,000150050
NSC722658
0,257
0,003280000
NSC156218
-0,26993
0,00138
NSC673347
0,317
0,000192906
NSC637461
0,256
0,005990000
NSC678253
-0,2695
0,002
NSC638485
0,317
0,000220189
NSC301457
0,256
0,004330000
NSC678100
-0,23274
0,00527
NSC2039
0,312
0,000336755
NSC135075
0,253
0,004800000
NSC682335
-0,21369
0,00792
NSC658443
0,309
0,000275701
NSC637458
0,251
0,005450000
NSC61837
-0,23784
0,00855
NSC614554
0,306
0,000270273
NSC227279
0,251
0,002300000
NSC407286
-0,23895
0,00892
NSC89310
0,305
0,000992718
NSC624806
0,250
0,002530000
NSC268791
-0,23163
0,01083
NSC349447
0,305
0,000288113
NSC34533
0,250
0,002380000
NSC34579
-0,20353
0,02304
NSC638390
0,303
0,001850000
NSC128487
0,250
0,003270000
NSC88928
-0,20327
0,02318
NSC658442
0,302
0,000457409
NSC4203
0,250
0,003560000
NSC637460
0,301
0,000597213
NSC239072
0,248
0,002950000
Compound
R-Value
p-Value
NSC9219
0,300
0,001850000
NSC379695
0,246
0,002960000
NSC77830
0,515
0,000000003
NSC638492
0,297
0,002780000
NSC265203
0,246
0,007410000
NSC676426
0,499
0,000000009
NSC382027
0,297
0,000770724
NSC638494
0,244
0,003180000
NSC641259
0,494
0,000000271
NSC638488
0,296
0,001200000
NSC319730
0,244
0,006240000
NSC38628
0,490
0,000003219
NSC650721
0,295
0,001380000
NSC320951
0,243
0,003240000
NSC126771
0,486
0,000000020
NSC113497
0,292
0,000807818
NSC189793
0,243
0,006400000
NSC3578
0,485
0,000000022
NSC310618
0,292
0,000602646
NSC282159
0,241
0,009100000
NSC11905
0,481
0,000000027
NSC709882
0,291
0,000513054
NSC36413
0,239
0,007160000
NSC639365
0,424
0,000000851
NSC637459
0,289
0,001680000
NSC618315
0,239
0,004710000
NSC641295
0,413
0,000014317
NSC234714
0,287
0,002230000
NSC79093
0,235
0,004050000
NSC380212
0,398
0,000004302
NSC405647
0,287
0,002240000
NSC710343
0,233
0,004210000
NSC125973
0,388
0,000005830
NSC627050
0,286
0,000626331
NSC352330
0,232
0,004390000
NSC608832
0,387
0,000006133
NSC650935
0,285
0,001730000
NSC337581
0,231
0,004820000
NSC93135
0,385
0,000009624
NSC77036
0,285
0,002600000
NSC251677
0,231
0,004600000
NSC160879
0,385
0,000080946
NSC403883
0,283
0,000838574
NSC7534
0,231
0,011150000
NSC637462
0,383
0,000356390
NSC710207
0,281
0,000752133
NSC106193
0,230
0,004650000
NSC683483
0,377
0,000010260
NSC210760
0,280
0,002720000
NSC684425
0,229
0,006330000
NSC713074
0,374
0,000046337
NSC119754
0,280
0,000786471
NSC699122
0,227
0,006740000
NSC658441
0,371
0,000016393
NSC330927
0,278
0,000951012
NSC659648
0,226
0,005300000
NSC708929
0,368
0,000016425
NSC59270
0,277
0,000898332
NSC2382
0,226
0,009610000
NSC658444
0,358
0,000026838
NSC5366
0,276
0,001330000
NSC678250
0,225
0,008640000
NSC698796
0,357
0,000044533
NSC407290
0,275
0,001240000
NSC657835
0,224
0,006920000
NSC638487
0,352
0,000321618
NSC722659
0,272
0,001660000
NSC709754
0,223
0,005880000
NSC11979
0,352
0,000156893
NSC123390
0,270
0,001640000
NSC638401
0,223
0,016540000
NSC251699
0,350
0,000262212
NSC76023
0,270
0,001810000
NSC111556
0,222
0,006810000
NSC641258
0,348
0,000184041
NSC684913
0,269
0,001190000
NSC299933
0,221
0,014050000
NSC622154
0,341
0,000360773
NSC686518
0,268
0,001220000
NSC65860
0,221
0,011870000
NSC14574
0,339
0,000074296
NSC33669
0,268
0,001490000
NSC656177
0,220
0,007750000
NSC163133
0,338
0,000601966
NSC381082
0,268
0,002140000
NSC638486
0,220
0,022960000
NSC103336
0,338
0,000104329
NSC658445
0,267
0,001270000
NSC641484
0,218
0,007000000
NSC156215
0,337
0,000714103
NSC314035
0,267
0,001410000
NSC659997
0,217
0,007210000
NSC32982
0,335
0,000087965
NSC29228
0,266
0,001320000
NSC698793
0,215
0,007640000
NSC657827
0,333
0,000280840
NSC678028
0,266
0,001910000
NSC406034
0,214
0,007860000
NSC712571
0,327
0,000215706
NSC65820
0,266
0,002280000
NSC619679
0,213
0,010900000
NSC661746
0,326
0,000148981
NSC645318
0,263
0,003780000
NSC656280
0,210
0,008800000
NSC409664
0,325
0,000136739
NSC638499
0,263
0,009300000
NSC641241
0,210
0,011110000
NSC638260
0,323
0,000465014
NSC656178
0,262
0,001690000
NSC77037
0,210
0,008940000
NSC26647
0,210
0,008940000
NSC135070
0 206
0 010550000
|
https://openalex.org/W2889787114
|
https://www.repository.cam.ac.uk/bitstreams/99a9f7f6-4f79-4e98-a01f-e89a9757892a/download
|
English
| null |
Solvatochromic covalent organic frameworks
|
Nature communications
| 2,018
|
cc-by
| 7,817
|
ARTICLE ARTICLE Solvatochromic covalent organic frameworks
Laura Ascherl1 Emrys W Evans2 Matthias Hennemann3 Daniele Di Nuzzo
2 Alexander G Hufnag
DOI: 10.1038/s41467-018-06161-w
OPEN However,
functionality
arising from the combination of the well-defined porosity and the
semiconducting properties of the COF backbone is still under-
explored. A suitably designed COF, on the other hand, could constitute a
supramolecular periodic analogue of the aforementioned solva-
tochromic dyes, with the added benefit of being an insoluble,
chemically and photochemically stable material31,32. The modular
COF design allows for matched combinations of electron-rich
and -deficient building blocks, generating a periodic lattice of
covalently linked donor–acceptor pairs that promote charge-
transfer transitions33,34. For optimal performance and fast
response times, COFs can be grown as thin films with their pores
oriented vertically to the substrate, thus exposing their high
internal surface area to the analyte35,36. Taking particular advantage of their tuneable porosity and the
resulting capability of selectively hosting specific guest molecules,
a predestined ambit for COFs could be the sensing of ions or
molecules. The COF-based sensing materials reported thus far
are able to detect heavy metal ions23,24, pH changes26 or organic
explosives26,27 via fluorescence quenching. A more general scope
for application, however, would be the detection of water and
solvent vapours in the gas phase with the possibility of differ-
entiating between various substances. COFs featuring this kind
of nosing capability could be a powerful tool for detecting
harmful volatile organic compounds in workplace environments,
or for real-time monitoring of the water content of gas and
solvent streams in industrial processes. Such on-line analysis
would require an easy read-out possibility, preferably via a colour
change of the detector material, in combination with full rever-
sibility
over
multiple
cycles
and
sufficient
photochemical
stability. Here we present oriented thin films of tetrakis(4-aminophenyl)
pyrene-based COFs that show an ultrafast and fully reversible
solvatochromic response upon exposure to various polar and
non-polar vapours. The newly developed COFs derive their high
degree of crystallinity from geometric interlocking of the cova-
lently linked two-dimensional (2D) sheets into a synchronized
offset-stacked pattern10,37. The charge-transfer character of the
optical transitions and hence the sensitivity to changes in polarity
inside the COF pores can be tuned through the aldehyde coun-
terpart used for assembling the COF. In this context, the strong
donor–acceptor contrast realized between tetraphenylpyrene and
thieno[3,2-b]thiophene yields the most pronounced solvato-
chromism. Solvatochromic covalent organic frameworks
Laura Ascherl1 Emrys W Evans2 Matthias Hennemann3 Daniele Di Nuzzo
2 Alexander G Hufnag
DOI: 10.1038/s41467-018-06161-w
OPEN Covalent organic frameworks (COFs) are an emerging class of highly tuneable crystalline,
porous materials. Here we report the first COFs that change their electronic structure
reversibly depending on the surrounding atmosphere. These COFs can act as solid-state
supramolecular solvatochromic sensors that show a strong colour change when exposed to
humidity or solvent vapours, dependent on vapour concentration and solvent polarity. The
excellent accessibility of the pores in vertically oriented films results in ultrafast response
times below 200 ms, outperforming commercially available humidity sensors by more than
an order of magnitude. Employing a solvatochromic COF film as a vapour-sensitive light filter,
we demonstrate a fast humidity sensor with full reversibility and stability over at least 4000
cycles. Considering their immense chemical diversity and modular design, COFs with fine-
tuned solvatochromic properties could broaden the range of possible applications for these
materials in sensing and optoelectronics. 1 Department of Chemistry and Center for NanoScience (CeNS), University of Munich (LMU), Butenandtstraße 5-13, 81377 Munich, Germany. 2 Cavendish
Laboratory, University of Cambridge, Cambridge CB3 0HE, UK. 3 Computer-Chemie-Centrum, Department of Chemistry and Pharmacy, Friedrich-Alexander-
University Erlangen-Nürnberg (FAU), Nägelsbachstraße 25, 91052 Erlangen, Germany. Correspondence and requests for materials should be addressed to
T.B. (email: bein@lmu.de) or to F.A. (email: fa355@cam.ac.uk) 1 NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications TURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-01 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w W
h diphenyl-4-(2,4,6-triphenylpyridinium)phenolate,
betaine
30),
display the highest sensitivity in this context30. W
ith covalent organic frameworks (COFs) already being
in their early teens, the scientific community has
gained
a
profound
understanding
regarding
the
synthesis of highly porous, crystalline and stable frameworks1,2. If
these materials are to evolve from a purely academic research
field, however, one of the next major challenges will be realizing
COFs that can compete with established materials in practical
applications. The solvatochromic effect has mostly been exploited for
defining solvent polarity scales such as the ET(30) and the nor-
malized ETN scales through measuring solvent-dependent energy
shifts of the absorption onset31. For detecting target molecules in
a stream of gas or liquid, however, a dissolved molecular dye
would be highly impractical. COFs are formed via reversible cross-linking of rigid organic
building blocks, whereby boronate esters3–6, imines7–10 and
hydrazones11,12 represent the most prominent linkage motifs. Potential for application has mainly been demonstrated in the
fields of gas storage13–15, catalysis and photocatalysis16,17, and in
electronics
and
optoelectronics18–22. Solvatochromic covalent organic frameworks
Laura Ascherl1 Emrys W Evans2 Matthias Hennemann3 Daniele Di Nuzzo
2 Alexander G Hufnag
DOI: 10.1038/s41467-018-06161-w
OPEN Oriented thin films of this COF with the pores
extending vertically from the substrate exhibit millisecond
response times to changes in the surrounding atmosphere and
fully retain their structure and function over several thousand
cycles. Reversible colour changes of solvated organic molecules as a
function of the solvent polarity are known as solvatochromism. This effect occurs when the ground and excited states of a
molecule are of different polarity, thus rendering the energy of
intramolecular electronic transitions sensitive to changes in the
polarity of the surrounding medium29. Charge-transfer transi-
tions, as they occur in the archetypic Reichardt’s dye (2,6- NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w 800
c
1.0
30
30
×10
25
25
20
20
Experimental
Rietveld refinement
Bragg positions
Difference
15
2 Theta (°)
15
10
10
5
0.8
0.6
Normalised diffuse reflectance
0.4
0.2
2.6 nm
0.0
400
500
600
Wavelength (nm)
700
800
Wet
Py–TT COF (wet)
Py–TT COF
Py–TT COF (dry)
Dry
Adsorption
Desorption
600
Adsorbed volume (cm3 g–1)
400
200
0
0.0
0.2
0.4
0.6
Relative pressure, (p/p0)
Pore width/nm
0
0.0
0.1
0.2
ABET = 1960 m2 g–1
AConnolly = 2139 m2 g–1
C2/m
a = 3.63 ± 0.02 nm
b = 3.58 ± 0.02 nm
c = 0.39 ± 0.01 nm
= 75 ± 2°
Aaccessible = 1808 m2 g–1
Vpore, theor = 1.01 cm2 g–1
Vpore = 1.22 cm3 g–1
dV(d) (cm3 g–1 nm–1)
2
4
6
8
10
0.8
1.0
a
b
e
d
Fig. 2 Structure analysis and solvatochromism of the Py–TT COF bulk powder. a Experimental PXRD pattern (black dots) of the Py–TT COF powder. Rietveld refinement (red line) using the structure model displayed in b provides a very good fit to the experimental data with only minimal differences
between the experimental and the refined patterns (green line). Rwp = 4.9%, Rp = 10.5%. Bragg positions are indicated by blue ticks. Inset, magnified view
of the 2θ > 9° region. b Top view (left) and side view (right) of the corresponding unit cell reveal the pseudo-quadratic, offset-stacked structure that is
typical for pyrene-based COFs. Crystallographic data are available as Supplementary Data 1. The structure has a Connolly surface of 2139 m2 g−1, an
accessible surface area of 1808 m2 g−1 and a pore volume of 1.01 cm3 g−1. c Nitrogen sorption isotherm of the Py–TT COF recorded at 77 K. Inset, QSDFT
calculation using an equilibrium model yields a very narrow pore size distribution with a maximum at 2.1 nm. d High-resolution TEM image showing the
large crystal domains of the Py–TT COF. Scale bar: 40 nm. Inset, magnified view onto a COF crystallite visualizing the pseudo-quadratic arrangement of the
COF pores with a periodicity of 2.6 ± 0.1 nm. Scale bar: 20 nm. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w e Diffuse reflectance spectra of the dry (orange) and water vapour-saturated (brown) Py–TT
COF powder showing a strong solvatochromic red-shift 30
30
×10
25
25
20
20
Experimental
Rietveld refinement
Bragg positions
Difference
15
2 Theta (°)
15
10
10
5
Py–TT COF
a AConnolly = 2139 m2 g–1
C2/m
a = 3.63 ± 0.02 nm
b = 3.58 ± 0.02 nm
c = 0.39 ± 0.01 nm
= 75 ± 2°
Aaccessible = 1808 m2 g–1
Vpore, theor = 1.01 cm2 g–1
b b a 2.6 nm
d 800
c
Adsorption
Desorption
600
Adsorbed volume (cm3 g–1)
400
200
0
0.0
0.2
0.4
0.6
Relative pressure, (p/p0)
Pore width/nm
0
0.0
0.1
0.2
ABET = 1960 m2 g–1
Vpore = 1.22 cm3 g–1
dV(d) (cm3 g–1 nm–1)
2
4
6
8
10
0.8
1.0 1.0
0.8
0.6
Normalised diffuse reflectance
0.4
0.2
0.0
400
500
600
Wavelength (nm)
700
800
Wet
Py–TT COF (wet)
Py–TT COF (dry)
Dry
e d e e c Fig. 2 Structure analysis and solvatochromism of the Py–TT COF bulk powder. a Experimental PXRD pattern (black dots) of the Py–TT COF powder. Rietveld refinement (red line) using the structure model displayed in b provides a very good fit to the experimental data with only minimal differences
between the experimental and the refined patterns (green line). Rwp = 4.9%, Rp = 10.5%. Bragg positions are indicated by blue ticks. Inset, magnified view
of the 2θ > 9° region. b Top view (left) and side view (right) of the corresponding unit cell reveal the pseudo-quadratic, offset-stacked structure that is
typical for pyrene-based COFs. Crystallographic data are available as Supplementary Data 1. The structure has a Connolly surface of 2139 m2 g−1, an
accessible surface area of 1808 m2 g−1 and a pore volume of 1.01 cm3 g−1. c Nitrogen sorption isotherm of the Py–TT COF recorded at 77 K. Inset, QSDFT
calculation using an equilibrium model yields a very narrow pore size distribution with a maximum at 2.1 nm. d High-resolution TEM image showing the
large crystal domains of the Py–TT COF. Scale bar: 40 nm. Inset, magnified view onto a COF crystallite visualizing the pseudo-quadratic arrangement of the
COF pores with a periodicity of 2.6 ± 0.1 nm. Scale bar: 20 nm. e Diffuse reflectance spectra of the dry (orange) and water vapour-saturated (brown) Py–TT
COF powder showing a strong solvatochromic red-shift solvatochromic COFs. Results COF design. We selected the electron-rich 1,3,6,8-tetrakis(4-
aminophenyl)pyrene, Py(NH2)4, as a basis for constructing COF design. We selected the electron-rich 1,3,6,8-tetrakis(4-
aminophenyl)pyrene, Py(NH2)4, as a basis for constructing Py–Py COF
Py(NH2)4
Py(CHO)4
Bridge
Bridge
Bridge
N
N
N
N
N
N
N
N
O
O
O
O
O
O
N
N
N
N
O
H2N
H2N
NH2
NH2
O
Bridge
1P(CHO)2
or
TT(CHO)2
S
S
Py–1P COF
or
Py–TT COF
Fig. 1 Synthesis of the imine-linked COFs. Combining the tetraphenylpyrene tetraamine Py(NH2)4 with the tetradentate pyrene aldehyde Py(CHO)4 in a 1:1
molar ratio yields the microporous Py–Py COF (left), whereas the combination of Py(NH2)4 with linear dialdehydes in a 1:2 molar ratio produces the
mesoporous Py–TT and Py–1P COFs, respectively (right) Bridge Fig. 1 Synthesis of the imine-linked COFs. Combining the tetraphenylpyrene tetraamine Py(NH2)4 with the tetradentate pyrene aldehyde Py(CHO)4 in a 1:1
molar ratio yields the microporous Py–Py COF (left), whereas the combination of Py(NH2)4 with linear dialdehydes in a 1:2 molar ratio produces the
mesoporous Py–TT and Py–1P COFs, respectively (right) Fig. 1 Synthesis of the imine-linked COFs. Combining the tetraphenylpyrene tetraamine Py(NH2)4 with the tetradentate pyrene aldehyde Py(CHO)4 in a 1:1
molar ratio yields the microporous Py–Py COF (left), whereas the combination of Py(NH2)4 with linear dialdehydes in a 1:2 molar ratio produces the
mesoporous Py–TT and Py–1P COFs, respectively (right) NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications 2 TURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w Transmission ultraviolet–visible
(UV–Vis) spectra recorded at different H2O relative pressures
reveal the appearance of an absorption band in the 520–640 nm
region and a simultaneous decrease in absorption across the
440–500 nm and 280–380 nm spectral regions (Fig. 3b). Plotting
the change in absorbance ΔA enables us to identify a strong
humidity-induced absorption band with a maximum at 545 nm
that is accompanied by two bleach bands with minima at 345 and
470 nm (Fig. 3c). The COF film exhibits the highest sensitivity
towards humidity changes between H2O relative pressures of 0.64
and 0.79. Above this, the absorption change saturates, possibly
due to condensation in the COF pores. The Py–TT COF was found to respond in a similar way to a
range of organic solvents (Fig. 3d). The magnitude of the colour
change upon exposure to a saturated atmosphere increases hereby
monotonically with the ETN polarity of the respective solvent
(Supplementary Figure 5)31. The Py–TT COF thus represents a
solid-state supramolecular analogue to the commercially available
molecular solvatochromic dyes with the added benefit of being
sensitive even to vapours diluted in a carrier gas. COF thin films. We anticipated that growing the solvatochromic
COF as an oriented film with the pores extending from the
substrate surface would greatly facilitate the diffusion of guest
molecules into and out of the framework and thus strongly
accelerate the response to changes in the surrounding atmo-
sphere. Supported COF thin films would moreover simplify
handling, improve re-usability and facilitate the read-out proce-
dure in sensing applications (see below). COF thin films. We anticipated that growing the solvatochromic
COF as an oriented film with the pores extending from the
substrate surface would greatly facilitate the diffusion of guest
molecules into and out of the framework and thus strongly
accelerate the response to changes in the surrounding atmo-
sphere. Supported COF thin films would moreover simplify
handling, improve re-usability and facilitate the read-out proce-
dure in sensing applications (see below). g
Oriented films of the Py–TT COF display a very fast response
towards step changes between dry and H2O-saturated gas streams
(Fig. 3e). For a 360 nm thick film, we observe a response time
(τrise) of 0.21 s for the absorption increase upon change from a
dry to humid atmosphere, while the transition to the dry state is
even faster with τfall = 0.15 s. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w High-resolution TEM confirms the pseudo-quadratic
geometry of the COF with a periodicity of 2.6 ± 0.1 nm, in
excellent agreement with the pore-to-pore repeat distance of 2.5
nm in the refined structure model. In accordance with the GIWAXS results, TEM analysis of a
Py–TT COF film removed from the substrate shows a highly
textured morphology with the ab-plane perpendicular to the
viewing direction, i.e., parallel to the substrate (Supplementary
Figure 3e, f). The pores consequently extend at an angle of 75° vs. the substrate surface (i.e., 15° vs. the viewing direction) and hence
are fully accessible to the surrounding atmosphere. The isostructural Py–1P COF is an equally well-crystallized
framework with a slightly smaller unit cell due to the shorter
terephthalaldehyde bridge (Supplementary Figure 1). The Py–Py
COF has a similar pseudo-quadratic overall geometry, but is
composed of alternating columns of the pyrene amine and aldehyde. The symmetry of the framework is thus reduced to P2/m with a
considerably smaller unit cell owing to the reduced length of the
bridge between the pyrene centres (Supplementary Figure 2). y
g
p
For the characterization of the Py–1P and Py–Py COF thin
films, see Supplementary Figure 11. Following
our
initial
considerations,
COFs
comprising
donor–acceptor motifs of alternating electron-rich and -deficient
building blocks are expected to show a solvatochromic response
towards molecules in their pores. Indeed, exposing the initially
orange-red Py–TT COF powder to an atmosphere of 98% relative
humidity causes a colour change to dark brown within a few
seconds (Fig. 2e). The corresponding diffuse reflectance spectra
reveal that this colour change stems from the appearance of new
optical transitions in the 550–850 nm range. This effect is fully
reversible as the colour reverts to the initial orange-red hue upon
drying. The Py–1P and Py–Py COFs also respond to water vapour
(Supplementary Figures 1e and 2e). The colour shifts, however,
are less pronounced, presumably owing to the much smaller
donor–acceptor contrast between their building blocks. The
origin of the solvatochromic colour shifts in our COFs will be
discussed in more detail below. Solvatochromism. As in the case of the COF powder, exposing
the Py–TT COF film to a humidified N2 stream results in a colour
change from orange to dark red. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w actual COF structure can cause the deviations in intensity that we
observe for some of the higher-index reflections. actual COF structure can cause the deviations in intensity that we
observe for some of the higher-index reflections. The
2D
grazing-incidence
wide-angle
X-ray
scattering
(GIWAXS) pattern of the Py–TT COF film exhibits a number
of well-defined reflections that can be indexed as shown in Fig. 3a. The distribution of the reflections indicates that the COF film is
highly textured with the imine-linked layers extending parallel to
the substrate surface (Supplementary Figure 3a, b). Individual
COF domains grow hereby at random rotation about the
substrate normal (planar disorder) (Supplementary Figure 3c,
d). We found that this texture is identical for different substrates
(c-cut sapphire, fused silica, polycrystalline ITO), suggesting that
the uniaxial preferred orientation is generated by the anisotropy
of the framework34,36. The
2D
grazing-incidence
wide-angle
X-ray
scattering
(GIWAXS) pattern of the Py–TT COF film exhibits a number
of well-defined reflections that can be indexed as shown in Fig. 3a. g
With pore diagonals of 2.4 and 2.0 nm (corner-to-corner and
bridge-to-bridge, respectively) in the refined structure model, the
Py–TT COF is expected to be a mesoporous material. Its nitrogen
sorption isotherm exhibits a type IVb isotherm shape with a
sharp step at p/p0 = 0.08, confirming the mesoporosity (Fig. 2c)40. Quenched solid density functional theory (QSDFT) analysis using
an equilibrium model for cylindrical pores yields a very narrow
pore size distribution with a maximum of 2.1 nm, in excellent
agreement
with
the
structure
model. The
Brunauer–Emmett–Teller (BET) surface of the Py–TT COF is
1960 ± 50 m2 g−1 with a total pore volume of 1.22 ± 0.05 cm3 g−1. These results are in very good agreement with the porosity values
derived from the structure model, confirming that the pores of
the framework are fully open and accessible. The electronic coupling throughout the film was analysed for
films grown on ITO substrates with electron- or hole-selective
contact layers (TiO2 or MoOx, respectively). Transport measure-
ments of these vertical-stack single-carrier devices yield charge-
carrier mobilities of (4.02 ± 0.04) × 10−6 and (1.02 ± 0.01) × 10−7
cm2 V−1 s−1 for holes and electrons, respectively (see Supple-
mentary Figures, section G). Transmission electron microscopy (TEM) reveals the forma-
tion of a periodic framework with domain sizes of 50–200 nm
(Fig. 2d). ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w Pyrene-based COFs have not only proven
to yield extremely well-ordered frameworks with large crystal
domains in our recent studies37, but are also geometrically
compatible with a wide range of aromatic and heteroaromatic
aldehyde counterparts33,34,38, enabling us to optimize the solva-
tochromic response within a single COF family. bridge and doubles the number of weakly accepting imines. A much
stronger charge-transfer character can be achieved in combinations
with electron-deficient heterocycles, such as the thieno-[3,2-b]
thiophene-2,5-dicarboxaldehyde, TT(CHO)2, in the Py–TT COF. Combinations with more electron-deficient aldehyde counter-
parts are expected to produce electronic transitions with a varying
degree of charge-transfer character across the conjugated imine
bond. For optimal solvatochromic response, however, these
charge-transfer transitions are not only required to possess
sufficient oscillator strength, but must also be sensitive to polarity
changes in the pores. COF bulk materials. The Py–Py, Py–1P and Py–TT COFs were
initially synthesized as bulk powders under solvothermal condi-
tions (see the Methods section and Supplementary Methods for
details, and Supporting Figures, section Q for infrared spectra and
thermogravimetric analysis). g
y
The powder X-ray diffraction (PXRD) pattern of the Py–TT
COF contains a number of sharp reflections, including several
well-defined higher-order reflections, and is devoid of any visible
amorphous background (Fig. 2a). Rietveld refinement employing
the density functional theory (DFT)-optimized C2/m-symmetric
structure model shown in Fig. 2b (see the Supplementary
Methods for details) provides a very good fit to the experimental
data. However, the large number of light atoms in the unit cell
and peak broadening due to the inherent flexibility of imine-
linked COFs impede the refinement of individual atom positions. Hence, slight differences between the structure model and the In view of these considerations, we chose three increasingly
electron-deficient aldehyde counterparts (Fig. 1). Pairing Py(NH2)4
with the tetradentate 1,3,6,8-tetrakis(4-formylphenyl)pyrene, Py
(CHO)4, in the Py–Py COF is anticipated to produce the smallest
donor–acceptor contrast in this context, derived mainly from the
slightly polarized, electron-accepting imine bond. Switching from
pairing two tetradentate building blocks to a combination of the
tetradentate amine with a linear acene dialdehyde, 1P(CHO)2, as
realized in the Py–1P COF, increases the polarity within the linear NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w 110
200, 020
220
330
440
001, hk1
a
Dry
Wet
b
c
Increasing
polarity
d
= 560 nm
rise = 0.21 s
fall = 0.15 s
90%
10%
e
Blue: electron
density loss
Orange: electron
density gain
S0 – S1
f
20
4
3
2
Absorbance
Absorbance
1
0
0.98
0.98
0.69
0.64
0.59
Dry
0.84
0.79
0.74
H2O partial pressure, (p/p0)
H2O partial pressure, (p/p0)
560 nm
0.69
0.64
0.59
Dry
0.84
0.79
0.74
–2
–1
0
ΔA (OD)
1
2
300
400
500
Wavelength (nm)
Wavelength (nm)
600
300
400
500
600
15
10
5
0
4
3
2
1
0
300
400
500
Wavelength (nm)
Time (s)
–1
0.0
0.5
1.0
Absorbance
1.5
0
1
2
3
600
–10
–5
0
qx (nm–1)
Solvent (ET
N polarity)
H2O
(1.00)
(0.76)
(0.65)
(0.46)
(0.10)
(0.01)
MeOH
EtOH
MeCN
Toluene
Hexane
Dry
qz (nm–1)
5
10
Fig. 3 Solvatochromism of Py–TT COF oriented thin films. a GIWAXS pattern of a 360 nm thick Py–TT COF film grown on a sapphire substrate. The
intensity of hk0 reflections is concentrated directly above the sample horizon, whereas the 001 and other low-index hk1 reflections appear close to the
substrate normal. b UV–Vis absorption spectra of the Py–TT COF film recorded at different relative pressures of H2O in N2. Increasing water content
causes a strong absorption increase in the 520–640 nm region, accompanied by a reduced absorption in the 440–500 nm and 280–380 nm regions. Insets, photographs of the COF film in the dry and water-saturated states. c Corresponding plot of the humidity-induced absorbance changes, Ahumid−Adry,
at different H2O relative pressures. The grey line indicates the wavelength used for the response time measurements (see below). d UV–Vis spectra of the
same COF film in saturated atmospheres of various solvents. The solvatochromic shift increases monotonically with increasing ETN polarity of the solvents. e Solvatochromic response of the Py–TT COF film towards step changes between dry and H2O-saturated N2 streams. Ten individual data sets (black dots)
recorded at λ = 560 nm have been averaged (red line). The response times τrise and τfall are determined between the 10% and 90% thresholds. f TD-DFT
calculated electron density difference upon the one-electron excitation from the ground state (S0) to the first singlet excited state (S1). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w This transition
involves a reduction of the electron density on the pyrene moieties and an electron density gain of the thienothiophene bridges, and hence possesses a
pronounced charge-transfer character. Electron density isosurfaces are displayed at an isovalue of 5e−5 Dry
Wet
b
4
3
2
Absorbance
1
0
0.98
H2O partial pressure, (p/p0)
0.69
0.64
0.59
Dry
0.84
0.79
0.74
300
400
500
Wavelength (nm)
600 110
200, 020
220
330
440
001, hk1
a
20
15
10
5
0
–10
–5
0
qx (nm–1)
qz (nm–1)
5
10 c
0.98
0.69
0.64
0.59
Dry
0.84
0.79
0.74
H2O partial pressure, (p/p0)
560 nm
–2
–1
0
ΔA (OD)
1
2
Wavelength (nm)
300
400
500
600 b a c Blue: electron
density loss
Orange: electron
density gain
S0 – S1
f Increasing
polarity
d
Absorbance
4
3
2
1
0
300
400
500
Wavelength (nm)
600
Solvent (ET
N polarity)
H2O
(1.00)
(0.76)
(0.65)
(0.46)
(0.10)
(0.01)
MeOH
EtOH
MeCN
Toluene
Hexane
Dry f = 560 nm
rise = 0.21 s
fall = 0.15 s
90%
10%
e
Time (s)
–1
0.0
0.5
1.0
Absorbance
1.5
0
1
2
3 d e Fig. 3 Solvatochromism of Py–TT COF oriented thin films. a GIWAXS pattern of a 360 nm thick Py–TT COF film grown on a sapphire substrate. The
intensity of hk0 reflections is concentrated directly above the sample horizon, whereas the 001 and other low-index hk1 reflections appear close to the
substrate normal. b UV–Vis absorption spectra of the Py–TT COF film recorded at different relative pressures of H2O in N2. Increasing water content
causes a strong absorption increase in the 520–640 nm region, accompanied by a reduced absorption in the 440–500 nm and 280–380 nm regions. Insets, photographs of the COF film in the dry and water-saturated states. c Corresponding plot of the humidity-induced absorbance changes, Ahumid−Adry,
at different H2O relative pressures. The grey line indicates the wavelength used for the response time measurements (see below). d UV–Vis spectra of the
same COF film in saturated atmospheres of various solvents. The solvatochromic shift increases monotonically with increasing ETN polarity of the solvents. e Solvatochromic response of the Py–TT COF film towards step changes between dry and H2O-saturated N2 streams. Ten individual data sets (black dots)
recorded at λ = 560 nm have been averaged (red line). NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w The response times τrise and τfall are determined between the 10% and 90% thresholds. f TD-DFT
calculated electron density difference upon the one-electron excitation from the ground state (S0) to the first singlet excited state (S1). This transition
involves a reduction of the electron density on the pyrene moieties and an electron density gain of the thienothiophene bridges, and hence possesses a
pronounced charge-transfer character. Electron density isosurfaces are displayed at an isovalue of 5e−5 system reported to date, and places it among the fastest
nanostructured humidity sensors40–43. structural changes that might alter the coupling between the
building blocks or COF layers. Solid-state nuclear magnetic
resonance measurements give no indication of a different
chemical environment in the water-saturated COF (Supplemen-
tary Figure 10). If the Py–TT COF is exposed to humidity, the
PXRD reflection intensities, especially of hk0 reflections, are
reduced considerably (Supplementary Figure 9a, b). This effect,
however, is fully reversible and can be attributed to modified
structure factors due to the water molecules in the pores
(Supplementary Figure 9c, d). All reflection positions, peak
shapes and widths, and hence the unit cell and framework
symmetry, remain unchanged during the humidity cycles. Given
the three-dimensional configuration and interlocked stacking of
the COF layers, even minor deformations or rotations of the
bridges would be reflected in modified unit cell parameters. The
absence of structural changes is further supported by the extreme
stability
of
the
material,
which
seems
hardly
possible
if
deformations or sliding of the COF layers were involved. Moreover, the as-synthesized COFs are, despite their acid- In addition to this extremely fast response, the Py–TT COF
films display excellent reversibility and reproducibility during
repeated switching (Supplementary Figure 7a). Furthermore, the
COF film is stable over at least 4000 humidity and solvent vapour
switching cycles and storage in ambient air for 250 days, without
showing any apparent changes in its absorption spectra and
GIWAXS patterns (Supplementary Figure 7b–e). For a possible application as a high-performance solvatochro-
mic sensor, easy read-out, fast response times, reversibility and
reproducibility are of key importance. Employing the COF thin
film as a vapour-sensitive light filter, a continuous read-out was
realized in combination with a green light-emitting diode (LED)
and a light-dependent resistor (Supplementary Figure 17). A
video demonstrating this proof of concept is included as Supple-
mentary Movie. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w As the solvent molecules need to
diffuse through the entire film in order to saturate the
solvatochromic colour change, we anticipated a strong correlation
between film thickness and response time. Indeed, both response
times get shorter for thinner films, whereby the fastest response of
0.11 s/0.09 s (rise/fall) was achieved using a 160 nm thick COF
film (Supplementary Figure 6). To the best of our knowledge, this
represents the fastest response time of a solvatochromic sensing The growth of oriented COF films on non-epitaxial substrates
has recently also been realized for imine-linked frameworks35. We adapted this method for the growth of the Py–TT, Py–1P and
Py–Py COFs. Solvothermal syntheses in slightly diluted solutions
yielded smooth and homogeneous films of the three frameworks
on fused silica, sapphire or indium-tin-oxide (ITO) substrates
with tuneable thickness between 160 and 360 nm, depending on
the reaction time (see the Methods section and Supplementary
Methods for details). NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications 4 4 ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w catalysed formation, not protonated (Supplementary Figure 12). Protonation of the imines, which is possible with strong acids,
produces a different and more red-shifted absorption profile than
the solvatochromism. between an LED and a light-dependent resistor. Experimental
data and TD-DFT calculations strongly suggest that the solva-
tochromism is of purely electronic origin and does not involve
structural or chemical changes in the framework – a fact that we
believe is not only key to the extremely fast response times and
outstanding stability of the material, but might also have impli-
cations for the use of COFs in the broader context of optoelec-
tronics. In particular, the observation that in these materials
electronic transitions can be manipulated reversibly and that
intramolecular charge-transfer can be facilitated via the inclusion
of chemically inert guest molecules could impact the development
of stimuli-responsive organic electronics. Future chemical mod-
ifications to the COF backbone or the pore walls could be used to
adapt the sensitivity and selectivity of the solvatochromic
response, broadening the range of possible applications for these
materials. The Py–TT COF displays a positive solvatochromism, i.e., the
absorption is red-shifted with increasing polarity. In well-studied
molecular dyes such as phenol blue45, this is observed for
combinations of a low-polarity ground state with a polar first
excited state29. In that case, the excited state is stabilized to a
greater extent than the ground state with increasing polarity of
the surrounding medium, hence lowering the energy required for
photoexcitation. To take a closer look at the electronic structure of the COF, we
performed time-dependent density functional theory (TD-DFT)
calculations (PBE0/6-31G(d)) for a single-layer Py–TT molecular
fragment (see the Supplementary Figures, section S for details). The electron density difference upon the one-electron excitation
from the ground state to the first singlet excited state reveals that
this transition involves a reduction of the electron density on the
pyrene moieties, accompanied by an electron density gain of the
thienothiophene bridges (Fig. 3f). Hence, this lowest-energy
optical transition has significant charge-transfer character, as we
anticipated from our initial considerations. The effect of a
surrounding solvent was probed by employing the conductor-like
polarizable continuum model46. In support of the experimental
data, the calculated absorption in water is red-shifted with respect
to the absorption in vacuum (705 nm vs. 685 nm). Methods
P
TT COF Py–TT COF synthesis. COF bulk powder syntheses were performed under argon
atmosphere in polytetrafluoroethylene (PTFE)-sealed glass reaction tubes (6 mL
volume). Solvents and acetic acid were obtained in high-purity grades from
commercial suppliers and were, unless shipped under argon, degassed and satu-
rated with argon prior to use. Py–TT COF synthesis. COF bulk powder syntheses were performed under argon
atmosphere in polytetrafluoroethylene (PTFE)-sealed glass reaction tubes (6 mL
volume). Solvents and acetic acid were obtained in high-purity grades from
commercial suppliers and were, unless shipped under argon, degassed and satu-
rated with argon prior to use. Py(NH2)4 (14.0 mg, 20 µmol) and thieno-[3,2-b]thiophene-2,5-dicarboxaldehyde
(7.8 mg, 40 µmol) were filled into a reaction tube, followed by the addition of
mesitylene (667 µL), benzyl alcohol (333 µL) and 6 M acetic acid (100 µL). The tube
was sealed and kept at 120 °C for 3 days. After cooling to room temperature, the
precipitate was collected by filtration, washed with MeCN and dried in air, yielding
a bright red powder. These findings are further supported by the photoluminescence
(PL) characteristics of the Py–TT COF (Supplementary Figure 8). If the COF is exposed to a humid atmosphere, the PL is red-
shifted, indicating a stabilization of the excited state by the pore
medium. This is accompanied by a reduction in PL intensity by
more than 95% compared to the dry material, suggesting that the
increased dielectric screening due to the water molecules helps to
overcome the Coulomb barrier and sustain a more charge-
separated state46,47. This sensitivity to the brought-in charge-
transfer character causes the solvatochromic colour shifts. Py–TT COF thin film synthesis. COF thin films were synthesized in 100 mL
autoclaves equipped with a 28 mm diameter glass liner. Fused silica (Spectrosil
2000), sapphire (UQG Optics, c-axis cut) and ITO-coated glass (VisionTec, 12–15
ohms per sq) substrates were cleaned in detergent solution, water, acetone and
isopropanol, and activated with an O2-plasma for 5 min directly before use. The
substrates were placed horizontally in PTFE sample holders with the activated
surface face-down. Py(NH2)4 (7.0 mg, 10 µmol) and thieno-[3,2-b]thiophene-2,5-dicarboxaldehyde
(4.0 mg, 20 µmol) were filled into an autoclave, followed by the addition of
mesitylene (1333 µL) and benzyl alcohol (666 µL). A substrate (fused silica,
sapphire or ITO) was inserted, followed by the addition of 6 M acetic acid (200 µL). The autoclave was sealed and heated to 120 °C for 4 days. ARTICLE The absolute
energies of the transitions are about 0.4 eV lower than the
experimental values due to limitations of the TD-DFT method. Methods
P
TT COF After cooling to room
temperature, the substrate was immersed in dry MeCN and dried with compressed
air. Thinner films were grown at shorter reaction times ranging from 4 h to 2 days. While the above findings provide strong evidence for a purely
electronic nature of the solvatochromism itself with no structural
or chemical changes involved, the morphology of our materials is
crucial for obtaining observable colour shifts and fast response
times. Thin films of a Py–1P molecular fragment and of an
amorphous
Py–1P
network,
despite
being
chemically
and
electronically almost identical to the crystalline Py–1P COF, do
not show any measurable solvatochromism (see the Supplemen-
tary Figures, section P for details). Only the COF with its regular
microporosity provides the required accessibility on a molecular
length scale, allowing the water or solvent molecules to rapidly
penetrate into the material and trigger the electronic changes. Structure characterization. PXRD measurements were performed using a Bruker
D8 Discover with Ni-filtered Cu Kα radiation and a LynxEye position-sensitive
detector. The 2D GIWAXS data were recorded with an Anton Paar SAXSpace system
equipped with a GeniX Cu Kα microsource and a Dectris Eiger R 1M detector. The
samples were positioned at a tilt angle of 2.3° and a sample-detector distance of
135 mm. TEM was performed with an FEI Titan Themis equipped with a field emission
gun operated at 300 kV. Optical absorption spectroscopy. UV–Vis spectra were recorded using a Perkin-
Elmer Lambda 1050 spectrometer equipped with a 150 mm InGaAs integrating
sphere. Time-resolved absorption measurements were performed at fixed detector
gain and slit settings. Diffuse reflectance spectra were collected with a Praying
Mantis (Harrick) accessory and were referenced to barium sulphate powder as
white standard. The specular reflection of the sample surface was removed from the
signal by spatial filtering. NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06161-w y
In order to clarify the origin of the solvent-induced colour
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vent or water vapours. Growing these COFs as highly crystalline
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device by using the COF film as a vapour-sensitive light filter Gas flow experiments. Gas flow experiments were performed using a gas flow
controller system (F-201-C-RBA-33-V, Bronkhorst Hi-Tec) and a liquid mass flow
controller with a controlled evaporation mixer (W-101A-110, Bronkhorst Hi-Tec),
where the solvents were evaporated at temperatures above their boiling points. Solvents were obtained from commercial suppliers in high-purity anhydrous grades
and were used as received. The flow cell was home-built from a 10 × 10 mm fused
silica cuvette (Hellma Analytics) equipped with a tightly fitting PTFE lid and 2 mm
diameter PP hoses connected to the gas flow system. NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications 6 Data availability 24. Ding, S.-Y. et al. Thioether-based fluorescent covalent organic framework for
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sonable request. 25. Zhang, Y. et al. Covalent organic frameworks as pH responsive signaling
scaffolds. Chem. Commun. 52, 11088–11091 (2016). 25. Zhang, Y. et al. Covalent organic frameworks as pH responsive signaling
ff ld
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52 11088 11091 (2016) scaffolds. Chem. Commun. 52, 11088–11091 (2016). 26. Das, G. et al. Chemical sensing in two dimensional porous covalent organic
nanosheets. Chem. Sci. 6, 3931–3939 (2015). Received: 1 February 2018 Accepted: 16 August 2018 Received: 1 February 2018 Accepted: 16 August 2018 ARTICLE constructed and analysed the single-carrier devices. L.A. and F.A. analysed the data with
contribution from R.H.F. and wrote the manuscript. F.A. and T.B. supervised the project. All authors discussed the results and contributed to the manuscript. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
article’s Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit http://creativecommons.org/
licenses/by/4.0/. Author contributions 23. Li, Z., Zhang, Y., Xia, H., Mua, Y. & Liu, X. A robust and luminescent covalent
organic framework as a highly sensitive and selective sensor for the detection
of Cu2+ions. Chem. Commun. 52, 6613–6616 (2016). L.A. and F.A. conceived and designed the project. L.A. synthesized and characterized the
materials. E.W.E., M.H. and T.C. performed the theoretical calculations. A.G.H. con-
ceived and built the sensor device. L.A. and M.B. contributed to the sensor device. D.D.N. 7 NATURE COMMUNICATIONS | (2018) 9:3802 | DOI: 10.1038/s41467-018-06161-w | www.nature.com/naturecommunications ARTICLE ARTICLE © The Author(s) 2018 Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467-
018-06161-w. Competing interests: The authors declare no competing interests. Reprints and permission information is available online at http://npg.nature.com/
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English
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Proper time to the black hole singularity from thermal one-point functions
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The Journal of high energy physics/The journal of high energy physics
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Published for SISSA by
Springer Received: November 11, 2020
Accepted: January 5, 2021
Published: March 11, 2021 Received: November 11, 2020
Accepted: January 5, 2021
Published: March 11, 2021 Received: November 11, 2020
Accepted: January 5, 2021
Published: March 11, 2021 Open Access, c⃝The Authors.
Article funded by SCOAP3. Keywords: AdS-CFT Correspondence, Spacetime Singularities, Black Holes in String
Theory, Conformal Field Theory Proper time to the black hole singularity from thermal
one-point functions https://doi.org/10.1007/JHEP03(2021)131 Contents
1
Introduction
1
2
One-point functions from higher-derivative corrections
3
3
One-point functions from the geodesic approximation
4
4
Thermal one-point functions for planar black branes
6
4.1
Analytic computation
6
4.2
Geodesic approximation
7
4.3
A more detailed saddle-point analysis
8
5
More general Schwarzschild black holes
11
5.1
Four-dimensional black holes
11
5.2
Five-dimensional black holes
14
6
Black holes with an inner horizon
15
7
Conclusions and discussion
17
7.1
Summary
17
7.2
Three-dimensional case
18
7.3
Two-point functions and thermal one-point functions of higher-spin operators 19
A Normalization of the correlators
20
A.1 Normalization in the geodesic approximation
21
B Prefactor
21
C Geodesic approximation for three-point functions
22
D Toy model for one-point functions in nearly-AdS2
23
E Thermal one point functions for three dimensional black holes
25
F Operator mixing
28 Contents
1
Introduction
1
2
One-point functions from higher-derivative corrections
3
3
One-point functions from the geodesic approximation
4
4
Thermal one-point functions for planar black branes
6
4.1
Analytic computation
6
4.2
Geodesic approximation
7
4.3
A more detailed saddle-point analysis
8
5
More general Schwarzschild black holes
11
5.1
Four-dimensional black holes
11
5.2
Five-dimensional black holes
14
6
Black holes with an inner horizon
15
7
Conclusions and discussion
17
7.1
Summary
17
7.2
Three-dimensional case
18
7.3
Two-point functions and thermal one-point functions of higher-spin operators 19
A Normalization of the correlators
20
A.1 Normalization in the geodesic approximation
21
B Prefactor
21
C Geodesic approximation for three-point functions
22
D Toy model for one-point functions in nearly-AdS2
23
E Thermal one point functions for three dimensional black holes
25
F Operator mixing
28 JHEP03(2021)131 Proper time to the black hole singularity from thermal
one-point functions JHEP03(2021)131 Matan Grinberga,b and Juan Maldacenac
aDepartment of Physics, Princeton University,
Princeton, New Jersey, U.S.A. bDepartment of Applied Mathematics and Theoretical Physics, University of Cambridge,
Cambridge, U.K. cInstitute for Advanced Study,
Princeton, New Jersey, U.S.A. E-mail: matang@princeton.edu, malda@ias.edu Matan Grinberga,b and Juan Maldacenac
aDepartment of Physics, Princeton University,
Princeton, New Jersey, U.S.A. bDepartment of Applied Mathematics and Theoretical Physics, University of Cambridge,
Cambridge, U.K. cInstitute for Advanced Study,
Princeton, New Jersey, U.S.A. E-mail: matang@princeton.edu, malda@ias.edu E-mail: matang@princeton.edu, malda@ias.edu Abstract: We argue that the proper time from the event horizon to the black hole sin-
gularity can be extracted from the thermal expectation values of certain operators outside
the horizon. This works for fields which couple to higher-curvature terms, so that they can
decay into two gravitons. To extract this proper time, it is necessary to vary the mass of
the field. Keywords: AdS-CFT Correspondence, Spacetime Singularities, Black Holes in String
Theory, Conformal Field Theory Keywords: AdS-CFT Correspondence, Spacetime Singularities, Black Holes in String
Theory, Conformal Field Theory ArXiv ePrint: 2011.01004 ArXiv ePrint: 2011.01004 ArXiv ePrint: 2011.01004 Open Access, c⃝The Authors. Article funded by SCOAP3. 1
Introduction If you are going to fall into a Schwarzschild black hole, it would be helpful to know how
long you can live inside. Your lifetime inside is shorter than (or equal to) the time between
the bifurcation surface and the singularity. See figure 1. Given that this is an interesting
property of a black hole, we would like to be able to extract it by computing properties of
correlation functions outside the black hole. – 1 – ℓhor
τs
Figure 1. We define τs to be the time between the bifurcation surface and the singularity. ℓhor is
the (renormalized) distance from the horizon and the boundary. The time from the horizon to the
singularity along any timelike curve, such as the orange curve is smaller than τs. ℓhor
τs JHEP03(2021)131 Figure 1. We define τs to be the time between the bifurcation surface and the singularity. ℓhor is
the (renormalized) distance from the horizon and the boundary. The time from the horizon to the
singularity along any timelike curve, such as the orange curve is smaller than τs. Under some reasonable assumptions, the simplest correlation function — the one-point
function of a massive field — contains this information. In particular, one needs to examine
the dependence of this expectation value on the mass of the field. We argue that the time
to the singularity, τs, is contained in its exponential large-mass behavior Under some reasonable assumptions, the simplest correlation function — the one-point
function of a massive field — contains this information. In particular, one needs to examine
the dependence of this expectation value on the mass of the field. We argue that the time
to the singularity, τs, is contained in its exponential large-mass behavior ⟨O⟩∼(powers of m) × exp [−imτs −mℓhor] ,
for
Im(m) < 0 ,
(1.1) (1.1) where we have assumed that m has a negative imaginary part. In other words, we can say
that the time to the singularity arises from a “phase” in the one-point function. Of course,
the one-point function is real for real m, but it develops this “phase” for complex m.1 This
expression requires some assumptions about the coupling of the massive field to gravity,
which we specify below. Let us first give a quick rationale for this formula and we will make more precise
statements later in the paper. 2In three bulk dimensions, thermal one-point functions arise due to particles wrapping the horizon [2 1The word “phase” is in quotation marks because, for complex m, the term involving imτs is not a pu
phase. It is just the term with an extra i in the exponential.
2 1
Introduction A minimally coupled field has a quadratic action which leads to a vanishing one-point
function. However, a non-zero value could result if higher-derivative corrections to the
action, such as a coupling between the field and the squared Weyl tensor, are included. Physically, this means that the particle in question can decay into two gravitons. This
assumption is true if the initial field is a generic massive string mode in string theory. On a
black hole background, this gravitational coupling leads to a source term for the field and
therefore, a one-point function [1].2 We are treating the field as a probe of the background,
ignoring its backreaction. In the large-mass approximation, correlation functions of the
field can be approximated in terms of geodesics. The geodesic starts at the insertion point
of the operator. The other end is integrated over spacetime, weighed by the background
value of the squared Weyl tensor. In a saddle-point approximation, we should balance the
“force” from the geodesic with that exerted by the spatial variation of the Weyl tensor. Because of the large mass, the geodesic contribution dominates everywhere except very – 2 – close to the singularity. For this reason, the saddle point is at a (complex) radial position
very near the singularity. Therefore, the saddle-point approximation gives us the time to
the singularity as in (1.1). See figure 1. The real part in the exponent involves the distance
from the operator insertion to the horizon. There are some further details and qualifications that we will spell out later in the
paper. In the context of a simple example of AdS/CFT, such as the case of N = 4 four-
dimensional supersymmetric Yang-Mills [3–5], we are considering one-point functions on a
black hole background to leading order in the large-N approximation. The massive field can
be a massive string state in the bulk with mass on the order of the string scale. The mass
can be varied by varying the t’ Hooft coupling of the gauge theory, since m ∝λ1/4 [4, 5]. We can also give it an imaginary part by taking λ complex, in which case (1.1) holds. JHEP03(2021)131 Our discussion is in the spirit of [6], though the analytic continuation we use looks a
bit simpler. The information we get is also simpler. We only claim that it gives us the
time to the singularity. 4Causality based bounds on α were discussed in [7, 8]. 3Couplings to the Ricci scalar or Ricci tensor can be removed by field redefinitions.
4 1
Introduction On the other hand, the procedure in [6] gives a more direct signal
from the singularity. The rest of the paper is organized as follows. In section two, we explain how higher-
derivative corrections give rise to thermal one-point functions. In section three, we discuss
how to compute the one-point functions for large mass by using a geodesic approximation. In section four, we discuss in detail the example of a black brane. In section five, we explore
various geodesics that can contribute for more general black holes. In section six, we discuss
some aspects of black holes with inner horizons. Finally, we present some conclusions. 4Causality based bounds on α were discussed in [7, 8]. 2
One-point functions from higher-derivative corrections However, if two
dimensions coincide, then we can have mixing at leading order in the large-N expansion. The fact that this mixing is larger than for generic dimensions leads to poles in correlators
as function of the dimension. More precisely, when we think about the regularized version
of the operator O, it can mix with lower-dimension operators. When we compute the
one-point function, we are interested in the one-point function of the operator with the
large dimension. In the large-N limit, this is well defined as long as ∆is not at one of the
resonant dimensions. For ∆> 2d, the integral (2.2) diverges. This divergence is a common feature of AdS
Witten diagrams involving fields that can decay into lighter fields. In this case, the field ϕ
can decay into two gravitons. By analytically continuing in the dimension, we can define
finite integrals, as is standard [9]. In this case, the resulting function has poles at certain
values of ∆. These values are the dimensions of multi-graviton operators that have non-
zero vacuum expectation values in the black hole background. One possible sequence of
operators corresponds to powers of the stress tensor and lead to poles at ∆= nd, for
n ≥2. These poles result from enhanced operator mixing when there is a “resonance”. For
generic operator dimensions, the mixing is suppressed by powers of 1/N. However, if two
dimensions coincide, then we can have mixing at leading order in the large-N expansion. The fact that this mixing is larger than for generic dimensions leads to poles in correlators
as function of the dimension. More precisely, when we think about the regularized version
of the operator O, it can mix with lower-dimension operators. When we compute the
one-point function, we are interested in the one-point function of the operator with the
large dimension. In the large-N limit, this is well defined as long as ∆is not at one of the
resonant dimensions. JHEP03(2021)131 2
One-point functions from higher-derivative corrections We consider the Lagrangian S =
1
16πGN
Z 1
2(∇ϕ)2 + 1
2m2ϕ2 + αϕW 2
,
(2.1) (2.1) containing a single massive field and the simplest higher-derivative coupling to the gravita-
tional field. Here W 2 = WµνδσW µνδσ is the square of the Weyl tensor.3 Here we consider
a scalar field, but one can write similar couplings for higher-spin fields. The coupling α
is expected to be small, α ∝α′ ∝
1
√
λ.4 Note that (2.1) is a coupling that appears in the
classical theory at leading order in the GN expansion. Let us consider this theory in AdSd+1. In AdS, W 2 = 0 and the one-point function is
also zero, as generically required by conformal symmetry. On the other hand, for a black
hole the Weyl tensor is nonzero, and this nonzero value sources the field ϕ. In Euclidean
space, we can write the one-point function as ⟨O(0)⟩∝α
Z
EBH
dd+1x √g G(0; x) W 2 ,
(2.2) (2.2) where G is the boundary-to-bulk propagator for the massive field. The integral is over the
Euclidean black hole. This integral is convergent for small enough masses, namely, ∆< 2d. where G is the boundary-to-bulk propagator for the massive field. The integral is over the
Euclidean black hole. This integral is convergent for small enough masses, namely, ∆< 2d. – 3 – Here, ∆is the scaling dimension, given by [4, 5] Here, ∆is the scaling dimension, given by [4, 5] ∆= d
2 +
s
d2
4 + m2R2 ,
∆∼mR ,
for mR ≫1 . (2.3) (2.3) For ∆> 2d, the integral (2.2) diverges. This divergence is a common feature of AdS
Witten diagrams involving fields that can decay into lighter fields. In this case, the field ϕ
can decay into two gravitons. By analytically continuing in the dimension, we can define
finite integrals, as is standard [9]. In this case, the resulting function has poles at certain
values of ∆. These values are the dimensions of multi-graviton operators that have non-
zero vacuum expectation values in the black hole background. One possible sequence of
operators corresponds to powers of the stress tensor and lead to poles at ∆= nd, for
n ≥2. These poles result from enhanced operator mixing when there is a “resonance”. For
generic operator dimensions, the mixing is suppressed by powers of 1/N. 3
One-point functions from the geodesic approximation For large mass, mRAdS ∼∆≫1, we can use the geodesic approximation for the propagator
in (2.2). This amounts to approximating G ∼e−mℓ,
(3.1) (3.1) where ℓis the (renormalized) proper length between the boundary and a bulk point. The
prefactor in (3.1) can also be written down, see appendix B. When we insert this into (2.2), we find that the propagator has a strong dependence
on position due to the large exponent in (3.1). Furthermore, (3.1) is strongly peaked near
the boundary, which leads to a divergence there — the same one we mentioned above when
∆is large. This divergent contribution can be interpreted as arising from the ϕ particle
decaying into gravitons near the boundary, which gives us the expectation value of the
corresponding multi-trace operator of the stress tensor. Notice that a conceptually similar
feature arises when we compute the vacuum AdS three-point functions between O and two
stress tensors, using the geodesic approximation. This integral near the boundary gives
rise to the poles in the three-point function. In appendix C we discuss a simple example. The interesting contribution to the one-point function comes from a solution where we
balance the pull from the propagator (3.1) and the W 2 term. This can happen only where
the W 2 term is varying rapidly. This does not happen anywhere in the Euclidean black
hole. However, we can analytically continue the integral to the region near the singularity – 4 – where W 2 is diverging and thus, we can find a balance between the two terms. In order
to continue the geodesic beyond the horizon, we need to pick a branch. We must decide
whether to continue it as ℓ= ℓhor + iτ or as ℓ= ℓhor −iτ. This is selected by giving an
imaginary part to m, say m = m−iϵ. Then one of these continuations results in a decreasing
exponential, the one with ℓhor + iτ. This decreasing exponential is what we expect from a
saddle-point evaluation and we will later justify it more explicitly in a special case. At the saddle point, we have the equation −im + ∂τ log W 2 = 0
→
im +
c
τ∗−τs
= 0
→
τ∗−τs = −ic
m ,
(3.2) (3.2) JHEP03(2021)131 where c is an order one positive constant. We see that for large mass, the saddle point
τ∗is near the singularity at τs. 3
One-point functions from the geodesic approximation The displacement away from the singularity is imaginary. This implies that we cannot view this as a point in the Lorentzian black hole. Still, it is
close to the singularity in the sense that the leading-order approximation for the integral
is given by evaluating ⟨O⟩∝
q
g(τ∗)W 2(τ∗)e−ml(τ∗) ∝exp [−m ℓhor −im τs] × (powers of m) . (3.3) (3.3) The first term comes from evaluating the propagator at the singularity. The deviation
away from the singularity in (3.2) gives a subleading correction. Similarly, the W 2 term
in (3.2) only gives powers of m. We see then, that the small displacement in the imaginary
direction in (3.2) is not important and the final answer involves the time to the singularity. Let us make some comments: Let us make some comments: Let us make some comments: • We are using the fact that we can vary m in order to focus on the m-dependence of
the correlator. This is appropriate in the case of black holes in string theory, where
we can keep the black hole metric fixed and vary the string length, which varies the
mass of the fields. • In the particular case of N = 4 supersymmetric Yang-Mills, the change in the mass
of the field, or the string length, can be achieved by varying the ’t Hooft coupling of
the theory. • When we claim that the exponential dependence on m only comes from the propaga-
tor, we are assuming that the coupling α in the Lagrangian (2.1) does not itself have
an exponential dependence on m. Indeed, in the N = 4 SYM example, it has only
a power law dependence on the coupling. Generically in string theory, it is expected
to have a power-law dependence on the string length. • The dependence of the one-point function on the temperature, or the mass of the
black hole, is contained within ℓhor. We will see examples below. • The dependence of the one-point function on the temperature, or the mass of the
black hole, is contained within ℓhor. We will see examples below. • We have not shown that the particular saddle point we picked is the dominant one,
or that it even contributes. We will return to this question later. Depending on the
size of the imaginary part of m, other saddles can contribute more. 5[1] did the d = 3 case, but, as we will see, the hard part is the same for all d. 3
One-point functions from the geodesic approximation • Until now, we have discussed the case of a Schwarzschild black hole with its spacelike
singularity. We will later discuss black holes with inner horizons. • Until now, we have discussed the case of a Schwarzschild black hole with its spacelike
singularity. We will later discuss black holes with inner horizons. – 5 – 4
Thermal one-point functions for planar black branes In this section we consider black branes in various dimensions. In this case, we can do the
analytic computation as well as the geodesic analysis. We find a match between these two
approaches. 4.1
Analytic computation Here, CN is a normalization coefficient that depends on how we
normalize the operator O, see (A.7). Note that CN does not have an exponential dependence
on h.6 In the final expression (4.8), the only important factor for us will be the 1/ sin πh. We see that (4.8) has poles at ∆= nd for n ≥2. The integral expression is convergent
only for ∆< 2d, since the hypergeometric function behaves like w−2h+1 for small w. Here
we defined the integral by analytically continuing h. The small-w region, which gives rises
to the divergences and the poles, corresponds to the region near the boundary of AdSd+1. As previously mentioned, we interpret the poles as arising from mixing with operators that
are powers of the stress tensor, T n, n ≥2. For black branes, operators involving derivatives
of the stress tensor are zero [10, 11], implying that expectation values of operators of the
schematic form T∂2mT vanish.7 JHEP03(2021)131 Giving a small negative imaginary part to ∆(or equivalently to h or m), we can avoid
these poles and obtain a large-|∆| exponential behavior of the form (for z0 = 1): ⟨O⟩∼e−iπ( ∆
d ) 4−( ∆
d ) ∼e−iπ( mR
d )4−( mR
d ) ,
for Im(∆) ∝Im(m) < 0 . (4.9) If the imaginary part of m had been positive, we would need to change i →−i in (4.9). The factor of 4−∆comes from the gamma functions in (4.8). Notice that (4.8) is real for real h. However, as we give h a small negative imaginary
part, one of the exponentials in the sine factor of (4.8) dominates and gives rise to the
“phase” in (4.9). 4.1
Analytic computation This computation was done in [1]5 and we now review it. The black brane metric in
AdSd+1 is JHEP03(2021)131 ds2 = R2
z2
"
−f(z)dt2 + dz2
f(z) + d⃗x2
#
,
f(z) ≡1 −zd
zd
0
,
z0 = d
4πβ . (4.1) (4.1) Since the temperature is the only scale, the temperature dependence is fixed as ⟨O⟩∝z−∆
0
∝T ∆. (4.2) (4.2) We are then left with the problem of fixing the overall coefficient. For this purpose, we can
adjust the temperature so that z0 = 1. We are then left with the problem of fixing the overall coefficient. For this purpose, we can
adjust the temperature so that z0 = 1. To construct the propagator, we solve the wave equation with only radial dependence. After defining
∆
d h ≡∆
d ,
w ≡zd
zd
0
,
(4.3) (4.3) we find equations that are independent of d. We pick two solutions, one regular at infinity
and one at the horizon: we find equations that are independent of d. We pick two solutions, one regular at infinity
and one at the horizon: ginf(w) = wh 2F1(h, h, 2h; w) ,
(4.4)
ghor(w) = wh 2F1(h, h, 1; 1 −w) . (4.5) (4.4)
(4.5) (4.4) (4.5) We then construct the Green’s function for the canonically normalized field as (w,w′) = −
1
Rd−1d
Γ(h)2
Γ(2h)
ginf(w)ghor(w′)θ(w′−w)+ginf(w′)ghor(w)θ(w−w′)
. (4.6) (4.6) Inserting this into (2.2) and using the fact that, for the metric (4.1), the squared Weyl
tensor is
(
)(
)2
2d W 2 = d(d −2)(d −1)2
R4
z
z0
2d
∝w2,
(4.7) (4.7) we get the final expression for the one-point function: we get the final expression for the one-point function: ⟨O⟩= −CN
s
16πGN
Rd−1
α
R2
(d −2)(d −1)2
d
Γ(h)2
Γ(2h)
Z 1
0
dw wh 2F1(h, h, 1; 1 −w)
= −CN
s
16πGN
Rd−1
πα
R2
4πT
d
∆(d −2)(d −1)2
d
Γ(h)2
Γ(2h)
h(1 −h)
sin πh
. (4.8) did the d = 3 case, but, as we will see, the hard part is the same for all d. 5[1] did the d = 3 case, but, as we will see, the hard part is the same for all d. – 6 – In the second line, we have restored temperature dependence by using (4.2) and inserting
an extra factor of z−∆
0
. 7In this expression, the derivatives are acting on both factors of T in such a way as to yield a conformal
primary. 6We can also do this computation more generally for a W 21+k coupling. It involves, R 1
0 dwwh+
2F1(h, h, 1; 1 −w) = Γ(1 + h + 2k)Γ(2 −h + 2k)/Γ(2 + 2k)2. This correctly reproduces (4.8) for k = 0. 6We can also do this computation more generally for a W 21+k coupling. It involves, R 1
0 dwwh+2k
2F1(h, h, 1; 1 −w) = Γ(1 + h + 2k)Γ(2 −h + 2k)/Γ(2 + 2k)2. This correctly reproduces (4.8) for k = 0.
7In this expression, the derivatives are acting on both factors of T in such a way as to yield a conformal
primary. 4.2
Geodesic approximation The candidate saddle-point approximation (3.3) involves the integral ℓ= R
Z ∞
0
dz
z
√
1 −zd = R
d
Z ∞
0
dw
w√1 −w ,
for z0 = 1. (4.10) (4.10) This integral diverges at small w, which is the region near the boundary. In addition, we
must decide how to go around the branch cut that starts at the horizon, w = 1. The
singularity is at w = ∞. The small-w divergence can be regularized in the same way as
the divergence of the two-point function in empty AdS, see appendix A.1. This gives the
renormalized length to the singularity as ℓ= ℓhor −iτs = R
d lim
wc→0
Z ∞
wc
dw
w√1 −w + log wc
= R
d [−iπ + log(4)] . (4.11) (4.11) To compute the imaginary part, we had to pick a path around the cut starting at w = 1 in
the first integral. We could have chosen either sign and we discuss this choice in the next
subsection. Setting the one-point function to ⟨O⟩∼e−mℓ, we reproduce (4.9). We see that the
factor of 4−∆/d comes from the renormalized length up to the horizon. When z0 ̸= 1, this
also reproduces the temperature dependence. – 7 – SHORT
LONG
ρ
HORIZON
BOUNDARY
×
Figure 2. Euclidean cigar with two geodesics: long (orange) and short (blue). SHORT
LONG
ρ
HORIZON
BOUNDARY
× HORIZON Figure 2. Euclidean cigar with two geodesics: long (orange) and short (blue). JHEP03(2021)131 8The actual proper distance is R
d ρ. 4.3
A more detailed saddle-point analysis Here we sketch a more systematic saddle-point analysis for the integral (4.8). It is conve-
nient to choose a variable ρ defined by zd = w =
1
(cosh ρ
2)2 ,
(4.12) (4.12) which is such that we can interpret ρ as proportional to the proper distance from the
horizon.8 The first step is to define the integral for large h. The problem here is the divergence
near w = 0, where the integrand behaves like Z
dw(Aw1−h+Bwh) ∼
Z
dρ
Ae(h−2)ρ+Be−(h−1)ρ
,
for w ≪1,
or ρ ≫1. (4.13) (4.13) For small w and large positive ρ, we have that w ∝e−ρ. This is the region near the AdS
boundary. If we make h complex, then it is possible to make the integral convergent by
tilting the integration contour into the imaginary direction. It is possible to tilt it in such
a way that both terms are convergent. This defines a convergent integral, which is the
integral that we would like to approximate using the saddle-point method. We fix a bulk point, and look for a geodesic that goes from this point to the point
on the boundary where the operator is inserted. We are supposed to integrate over the
angular direction of the bulk point. This integral is the same as integrating over the position
of the insertion of the boundary operator. So we fix an arbitrary position of the angular
coordinate for the bulk point, but we allow the geodesic to end at any value of the euclidean
time direction on the boundary. The point where its ends will be chosen by minimizing
the length of the geodesic. There are two geodesics that go from a given bulk point to the
boundary. One goes straight to the boundary, the other goes to the horizon (the tip of
the cigar) and then to the boundary. We call them the “short” and the “long” geodesics,
respectively, see figure 2. We need to sum over the contributions of these two geodesics. These long and short geodesics are responsible for the two terms we have in (4.13). The
second term is the long geodesic contribution, which is convergent at large ρ. The first
term is the short geodesic contribution, which diverges on the original contour. – 8 – ω - PLANE
Cn
ω = 1
ω = 0
Cconv ... 4.3
A more detailed saddle-point analysis ρ = −iπ
ρ = −3iπ
ρ = iπ
ρ = 0
ρ - PLANE
ω - PLANE
Cn
Clong
Cconv
SADDLE
POINTS
Cs−left
Cn
ω = 1
ω = 0
Cconv
(a)
(b)
Figure 3. In (a) we see the w-plane. The boundary is at w = 0 and the horizon at w = 1. In blue,
we see the naive contour Cn. In red, we have depicted the integration contour Cconv that leads to a
convergent answer when Im(h) < 0. The singularity is at w = ∞. In (b) we see the ρ-plane. ρ = 0
is the horizon and there are multiple images of the singularity at ρ = (1 + 2n)iπ. The convergent
contour is depicted in red. It can be deformed to the steepest-descent contours shown in green. These pass through the saddle points. We are also left with the contour Cs−left, from the short
geodesic contribution. Additionally, we have Clong computing the long geodesic contribution, shown
in orange. Along the short and long contributions, the propagator takes the form ehρ and e−hρ,
respectively. ω - PLANE ρ - PLANE ... ρ = −iπ
ρ = −3iπ
ρ = iπ
ρ = 0
Cn
Clong
Cconv
SADDLE
POINTS
Cs−left
(b) JHEP03(2021)131 (b) (a) Figure 3. In (a) we see the w-plane. The boundary is at w = 0 and the horizon at w = 1. In blue,
we see the naive contour Cn. In red, we have depicted the integration contour Cconv that leads to a
convergent answer when Im(h) < 0. The singularity is at w = ∞. In (b) we see the ρ-plane. ρ = 0
is the horizon and there are multiple images of the singularity at ρ = (1 + 2n)iπ. The convergent
contour is depicted in red. It can be deformed to the steepest-descent contours shown in green. These pass through the saddle points. We are also left with the contour Cs−left, from the short
geodesic contribution. Additionally, we have Clong computing the long geodesic contribution, shown
in orange. Along the short and long contributions, the propagator takes the form ehρ and e−hρ,
respectively. After we choose the tilted contour indicated above, we can rotate the contour differently
for the short and long geodesic contributions. For the long geodesic, we simply bring it
to the original position, at ρ ∈[0, ∞]. 4.3
A more detailed saddle-point analysis For the short geodesic, we must approach the
continuation more carefully. We set Im(h) < 0. (4.14) (4.14) Then the convergent contour is one that is tilted towards the negative imaginary direction
by an angle greater than the angle of the complex number −ih∗. In other words, we start
with a contour which begins at ρ = 0 and goes along ρ = −ih∗(1 −iϵ)σ, with σ ≫1 in the
complex plane for large |ρ|. See figure 3(b). Let us return to the full integral. The choice of variables (4.12) is such that the
exponent in the propagator is simple G ∝e−mℓshort(ρ) + e−mℓlong(ρ) ∝e−mℓhor
h
Fshort(ρ)ehρ + Flong(ρ)e−hρi
,
(4.15) (4.15) where where ℓshort(ρ) = ℓhor −R
d ρ ,
and
ℓlong(ρ) = ℓhor + R
d ρ . (4.16) (4.16) – 9 – The prefactors, F(ρ), in (4.15) do not have exponential dependence on m and are discussed
in appendix B. For now, we will ignore them. The square of the Weyl tensor (4.7) is W 2 ∝w2 ∝
1
(cosh ρ
2)4 = exp
−4 log
cosh ρ
2
. (4.17) (4.17) In order to make this term competitive with the propagator term, we can replace W 2 →W 2k. For h < 2k, there is a saddle point along the original integration contour, for real and
positive ρ. However, we are really interested in the case where h ≫k. Notice that (4.17)
diverges at JHEP03(2021)131 ρ = −(1 + 2n) iπ,
n ∈Z. (4.18) (4.18) In fact, we find saddle points at In fact, we find saddle points at ∂ρ
hρ −mℓhor −4 log
cosh ρ
2
= 0
−→
ρ = −(1 + 2n) πi + η ,
(4.19) (4.19) where η is a small quantity, with a positive real part, given explicitly by tanh η
2 = 2
h. (4.20) (4.20) The original tilted contour, Cconv in figure 3, can be rotated clockwise to the negative
real-axis direction. In doing so, this integral can be expressed as a sum of steepest-descent
contours passing through the saddle points (4.19).9 All saddle points contribute equally,
except for a factor of e−2iπh. The sum is then proportional to ⟨O⟩∝4−h
∞
X
n=0
e−(1+2n) iπh ∝4−h
e−iπh
1 −e−2iπh ∝
4−h
sin πh . 9A further derivative of the left most expression in (4.19) is close to positive at the saddle point (4.19)
when h is close to real. This means that the steepest descent contour indeed goes vertically through the
saddle points as in figure 3(b). 10The bottom line is that we have only rigorously derived the first saddle n = 0 in (4.21), but not the
rest, n > 0 in (4.21). 4.3
A more detailed saddle-point analysis (4.21) (4.21) In evaluating the exponent, we have only kept the leading term in the large-h expansion
and are ignoring powers of h in (4.21). If h has a negative imaginary part, higher-order
terms in this sum are more and more suppressed. However, it is interesting that they
sum up to the inverse sine that we had in the exact answer (4.8). The overall factor
of 4−h = 4−∆/d in (4.21) comes from the regularized distance from the boundary to the
horizon ℓhor in (4.11). The integral of the short geodesic along the negative real axis, labeled Cs−left in figure 3,
has a similar form to that of the long geodesics and thus, could cancel. To verify this, we
need to compute the prefactors in (4.15) and check that they indeed cancel, see appendix B. These prefactors have additional singularities and we have not fully understood their effects,
see appendix B for a longer discussion.10 In summary, we began by considering a black brane. We computed the exact answer by
doing the explicit integral in (4.8). We considered the geodesic approximation in section 4.2. – 10 – We further justified this approximation through a more detailed saddle-point analysis in
section 4.3, which explained why the contour passes through the saddle point. We also
came upon the added benefit of finding subleading saddles that sum to a 1/ sin(πh) factor. A similar procedure for the Veneziano amplitude was discussed in appendix A of [12]. We further justified this approximation through a more detailed saddle-point analysis in
section 4.3, which explained why the contour passes through the saddle point. We also
came upon the added benefit of finding subleading saddles that sum to a 1/ sin(πh) factor. A similar procedure for the Veneziano amplitude was discussed in appendix A of [12]. 5
More general Schwarzschild black holes Here we will explore the case of more general black holes. A simple generalization is
to consider an AdSd+1 Schwarzschild black holes with a spherical boundary. These have
metrics of the form JHEP03(2021)131 ds2 = R2
−f(r)dt2 + dr2
f(r) + r2dΩ2
d−1
! ,
(5.1)
f(r) = r2 + 1 −
µ
rd−2 . (5.2) ds2 = R2
−f(r)dt2 + dr2
f(r) + r2dΩ2
d−1
! ,
(5.1) (5.1) ! f(r) = r2 + 1 −
µ
rd−2 . (5.2) f(r) = r2 + 1 −
µ
rd−2 . (5.2) The black brane case is recovered in the µ →∞limit. We have not been able to solve the wave equation analytically in this case. In principle,
one could do a careful saddle-point analysis. Instead of doing this, we note that in our
previous example, a crucial point was to understand the proper distances to the singularity. We again define ˆρ in terms of the proper distance11 dˆρ =
dr
p
f(r) ,
(5.3) (5.3) and set ˆρ = 0 at the horizon. For large ˆρ, we have a discussion similar to the one before in
the sense that in order to make the integral convergent, we pick out a tilted contour, which
spirals to infinity in the r-plane. A new feature is that we now have poles at ∆= 2d + 2n
from operators like T∂2nT. These had vanishing vacuum expectation values for black
branes, but not for this more general case [11]. These operators, in combination with the
previous ones, T n, give poles at ∆= n for odd d and ∆= 2n for even d. Again, we expect to move the contour into the negative ˆρ direction and through this
process, we expect to pick out saddles near r = 0. The single point r = 0 corresponds to
many points in ˆρ. We will not figure out the precise structure of the covering space where
ˆρ lives, but we will compute some of the leading values of ˆρ. These can be obtained by
integrating (5.3) along various contours. One approach for this is to consider a contour in
the r-plane that starts at the horizon and gets to r = 0 in various ways. For example, see
figure 4. This is not a real substitute for a full steepest-descent analysis, but it provides us
with some information about what to expect. 5.1
Four-dimensional black holes As a first example, let us consider the case of d = 3, corresponding to a black hole in AdS4. We write f(r) in (5.1) as f(r) = (r −r+)(r −r1)(r −r∗
1)
r
,
r1 = −r+
2 + i
r
1 + 3
4r2
+ ,
µ = r+(1 + r2
+) . (5.4) (5.4) 11This is normalized slightly differently than the ρ variables we previously introduced, ˆρ = ρ/d. 11This is normalized slightly differently than the ρ variables we previously introduced, ˆρ = ρ/d. – 11 – r+
r2 = r∗
1
r1
HORIZON
SINGULARITY
eC1
C1
C2
C0
Figure 4. We see the r complex plane and some branch cuts involved in the definition of √f. Integrating (5.3) along the contours indicated, we get the proper distance to the singularity along
various contours. We expect that all of these contribute. Contours C1 and eC1 give the same answer,
but the second one is convenient to obtain (5.6). JHEP03(2021)131 Figure 4. We see the r complex plane and some branch cuts involved in the definition of √f. Integrating (5.3) along the contours indicated, we get the proper distance to the singularity along
various contours. We expect that all of these contribute. Contours C1 and eC1 give the same answer,
but the second one is convenient to obtain (5.6). When we consider 1/
p
f(r), we can run the branch cuts as indicated in figure 4. The
convergent contour for the short geodesic spirals clockwise around the complex r-plane
for large values of r. As we try to deform the contour into the region of a decreasing
propagator, we want to go under all these branch cuts. We will not analyze exactly how to
do so. However, we notice that as we move the contour, we will encounter the singularities
in W 2 at r = 0. It is interesting to find out where they first occur in the ˆρ-plane. Setting
the origin of the ˆρ-plane at the horizon, we find that the first singularity occurs at ˆρ0 = −iχ0 ,
χ0 ≡
Z r+
0
dr
p
−f(r) ,
(5.5) (5.5) and corresponds to the integral along the contour C0 in figure 4. 5.1
Four-dimensional black holes We can also reach r = 0
following the contour C1 in figure 4, giving ˆρ1 = −iπ + γ ,
γ = lim
rc→∞
"Z rc
r+
dr
p
f(r) −
Z 0
−rc
dr
p
f(r)
#
. (5.6) (5.6) This formula is derived by deforming the integral to eC1. We note that this γ is positive. We can also consider the contour C2 in figure 4: ˆρ2 = i (χ0 −2π) . (5.7) (5.7) Additionally, we can add −2iπ to all of the above by circling more times around infinity. We are not sure if these are all of them, or if all of these do indeed contribute. Additionally, we can add −2iπ to all of the above by circling more times around infinity. We are not sure if these are all of them, or if all of these do indeed contribute. – 12 – – 12 – However, if the imaginary part of ∆is large, then only ˆρ0 dominates. On the other
hand, if the imaginary part of ∆is very small, then the one involving ˆρ1 dominates, since
it has the largest real part. Thus, the question of whether or not the information about the
proper time to the singularity is contained in the leading term depends on the size of Im(h). The schematic form of the answer is ⟨O⟩∼e−mℓhor
ae−iχ0∆+ be−i(π+iγ)∆+ a∗e−i(2π−χ0)∆+ · · ·
1 −e−2iπ∆
! ,
(5.8) (5.8) where the dots indicate possible further exponentials. The denominator comes from the
circles around r = 0. The prefactors a, b have only power-law dependence on h. The
overall factor in (5.8) comes from the renormalized distance from infinity to the horizon
and is equal to JHEP03(2021)131 ℓhor = R lim
rc→∞
"Z rc
r+
dr
p
f(r) −log rc
#
. (5.9) (5.9) As a check, when we go to the black brane limit, r+ →∞, we find that χ0 ∼π/3
and γ ∼0,12 so that the exponents in (5.8) become e−iπ∆/3, e−iπ∆, e−5iπ∆/3. We further
expect that a becomes equal to b, so that As a check, when we go to the black brane limit, r+ →∞, we find that χ0 ∼π/3
and γ ∼0,12 so that the exponents in (5.8) become e−iπ∆/3, e−iπ∆, e−5iπ∆/3. We further
expect that a becomes equal to b, so that ⟨O⟩µ→∞∝e−mℓhor
e−iπ∆/3(1 + q + q2)
(1 −q3)
! 12Numerically, we found that we approach these values as χ = π/3 −0.25/r2
+ and γ = 0.43/r2
+ when
r+ →∞. 5.2
Five-dimensional black holes Here we insert d = 4 in (5.1). After redefining u ≡r2, we find that the proper length
involves dˆρ = 1
2
du
p
(u −u+)(u + u+ + 1)
−→
u −u+
1 + 2u+
= sinh2 ˆρ ,
(5.11) (5.11) where u+ parametrizes the position of the horizon. See figure 5. where u+ parametrizes the position of the horizon. See figure 5. where u+ parametrizes the position of the horizon. See figure 5. In this case, it is very easy to find the ˆρ positions of the r = u = 0 regions. They case, it is very easy to find the ˆρ positions of the r = u = 0 regions. They sit at ˆρ0 = −iχ0 ,
ˆρ1 = −i(π −χ0) ,
sin2 χ0 =
u+
1 + 2u+
,
(5.12) (5.12) and we can add any multiple of −iπ to these values. Here we see that for large u+, we get
χ0 = π/4. In this case then, we expect that these saddles combine as and we can add any multiple of −iπ to these values. Here we see that for large u+, we get
χ0 = π/4. In this case then, we expect that these saddles combine as ⟨O⟩∼e−mℓhore−iπ∆/4
1 + e−iπ∆/2
1 −e−iπ∆
! ∼
4−∆/4
sin
π∆
4
,
(5.13) (5.13) as we had in (4.21). Note that as we had in (4.21). Note that ℓhor
R = 1
2 lim
uc→∞
"Z uc
u+
du
p
(u−u+)(u+u++1) −loguc
#
= −1
2 log(1+2u+)+log(2). (5.14) (5.14) Assuming that the prefactors associated with all saddles are real, we find ⟨O⟩∝(1 + 2u+)∆/2 2−∆cos( π
2 −χ0)
sin
π∆
2
. (5.15) (5.15) Let us make some comments: • The poles at ∆= 2n are what we expect from the operators, T m and T∂2mT, when
the dimension of T is even (four in this case). This is slightly different than what we
had found for d = 3, where the poles were at ∆= n. • The poles at ∆= 2n are what we expect from the operators, T m and T∂2mT, when
the dimension of T is even (four in this case). This is slightly different than what we
had found for d = 3, where the poles were at ∆= n. 5.1
Four-dimensional black holes We see the integration contours in the u-plane that define ˆρ0 and ˆρ1. JHEP03(2021)131 JHEP03(2021)131 5.1
Four-dimensional black holes = z−∆
0
4−∆/3
e−iπ∆/3
(1 −e−2iπ∆/3) ,
(5.10) (5.10) where above we have used the large-r+ value of ℓhor (5.9) and defined q ≡e−2iπ∆/3. We
see that we reproduce the black brane result (4.21) for d = 3, with z0 ∼1/r+. Let us make some comments: where above we have used the large-r+ value of ℓhor (5.9) and defined q ≡e−2iπ∆/3. We
see that we reproduce the black brane result (4.21) for d = 3, with z0 ∼1/r+. Let us make some comments: • The poles in (5.8) are, in general, at integer values ∆= n. This comes from the
combination of operators T 3n (for d = 3) and T∂2mT. In the black-brane limit, the
latter have zero expectation value and only the poles at ∆= 3n are present. • The poles in (5.8) are, in general, at integer values ∆= n. This comes from the
combination of operators T 3n (for d = 3) and T∂2mT. In the black-brane limit, the
latter have zero expectation value and only the poles at ∆= 3n are present. • The positions of the poles are determined by the operator content of the theory. In
fact, the basic spacing is set by the integral of a full circle at large values of r, which
is fixed by the form of the theory near the boundary. • However, the numerator (i.e. the position of the zeros) depends on the details of the
black hole and its temperature. For large r+, we find that they precisely cancel some
of the poles. • In the small mass limit (r+ →0), we get χ0 ∼πr+/2, which sets the time to the
singularity for a black hole in flat space. In this regime we get γ ∼r+(−log r+). This logarithmic divergence is interpreted as coming from the region outside the
black hole, where AdS is approximated by flat space. We expect the effects of the
second root ˆρ1 (5.6) to be reflective of the contribution not from the interior of the
flat space black hole, but rather from the region near the center of AdS and outside
the black hole. We have not yet understood this in detail. – 13 – u = 0
C0
u+
−1 −u+
SINGULARITY
C1
Figure 5. We see the integration contours in the u-plane that define ˆρ0 and ˆρ1. SINGULARITY Figure 5. 5.2
Five-dimensional black holes • In the small-r+ limit, we see that χ0 →r+, which is what we expect for the time to
the singularity for the flat-space black hole in five dimensions. • In the small-r+ limit, we see that χ0 →r+, which is what we expect for the time to
the singularity for the flat-space black hole in five dimensions. – 14 – SINGULARITY
τin
ℓhor
ℓsing
−ℓsing
INNER HORIZON
OUTER HORIZON
Figure 6. Penrose diagram of a charged black hole in AdS. We have both an outer horizon and an
inner horizon. The one-point function would involve the length of geodesics roughly as indicated, as
well as an imaginary contribution that has the size of the order of the time between the inner and
outer horizons. The distance from the inner bifurcation surface to the singularity, ℓsing, also appears. SINGULARITY
τin
ℓhor
ℓsing
−ℓsing
INNER HORIZON
OUTER HORIZON JHEP03(2021)131 Figure 6. Penrose diagram of a charged black hole in AdS. We have both an outer horizon and an
inner horizon. The one-point function would involve the length of geodesics roughly as indicated, as
well as an imaginary contribution that has the size of the order of the time between the inner and
outer horizons. The distance from the inner bifurcation surface to the singularity, ℓsing, also appears. Figure 6. Penrose diagram of a charged black hole in AdS. We have both an outer horizon and an
inner horizon. The one-point function would involve the length of geodesics roughly as indicated, as
well as an imaginary contribution that has the size of the order of the time between the inner and
outer horizons. The distance from the inner bifurcation surface to the singularity, ℓsing, also appears. • We can also give a physical interpretation to the metric in the u < 0 region. This
corresponds to replacing the S3 with H3, and considering a hyperbolic black hole.13
Then, the point at u = −1 −u+ is the horizon and the time to the singularity for
this new black hole is R(π
2 −χ0). • We can also give a physical interpretation to the metric in the u < 0 region. This
corresponds to replacing the S3 with H3, and considering a hyperbolic black hole.13
Then, the point at u = −1 −u+ is the horizon and the time to the singularity for
this new black hole is R(π
2 −χ0). 13Recall that under θ →iρ, we have that ds2 = dθ2 +sin2 θ dΩ2
2 →ds2 = −[dρ2 +sinh2 ρ dΩ2
2]. For u < 0,
the u dΩ2
3 term in the metric is interpreted as (−u) ds2
H3, which now has a positive coefficient. 6
Black holes with an inner horizon Here we discuss some aspects of black holes with an inner horizon. We will consider
spherical charged black holes. Their Penrose diagram is shown in figure 6. We are ignoring
backreaction, so we do not expect any singularity in the inner horizon. We will present
some evidence that, in the large-mass expansion with Im(m) < 0, the “phase” of the one-
point function tells us about the time between the outer and inner horizons. The geodesics
can be interpreted as going to the left or right in the Penrose diagram, so that schematically
they look like they are going to the timelike singularities in figure 6. As a simple case, let us consider a five-dimensional charged AdS black hole with the
same metric as in (5.1) but with f(r) = r2 + 1 −µ
r2 + q2
r4 = (u −u+)(u −u−)(u + 1 + u+ + u−)
u2
,
u ≡r2 ,
(6.1) (6.1) where u+ > u−> 0. Here we have parametrized µ and q2 in terms of u+ and u−in
the regime where we have a smooth horizon. The structure of branch cuts when we write
dˆρ =
dr
√
f(r) = 1
2
du
√
uf(u) is depicted in figure 7. 13Recall that under θ →iρ, we have that ds2 = dθ2 +sin2 θ dΩ2
2 →ds2 = −[dρ2 +sinh2 ρ dΩ2
2]. For u < 0,
the u dΩ2
3 term in the metric is interpreted as (−u) ds2
H3, which now has a positive coefficient. 13Recall that under θ →iρ, we have that ds2 = dθ2 +sin2 θ dΩ2
2 →ds2 = −[dρ2 +sinh2 ρ dΩ2
2]. For u < 0,
the u dΩ2
3 term in the metric is interpreted as (−u) ds2
H3, which now has a positive coefficient. – 15 – C0
u+
−1 −u+ −u−
C1
u−
C2
C3
γ0
−iχ0
Figure 7. We depict the u = r2 plane and the various branch points in the computation of ρ. The
depicted contours give us various values for the “distance” to the singularity, which are expected to
contribute to the one-point function. The dotted lines indicate that we go to the second sheet by
crossing the cut. We have given two equivalent forms for the contour C0 — one of them emphasizes
the origin of the −iχ0 and γ0 contributions. Figure 7. 14We suggest this interpretation by noticing that, in the low-temperature limit, we expect an almost
decoupling of the physics of the AdS2 region from the rest. Furthermore, we do not expect a one-point
function in the strict AdS2 limit. The fact that we still receive a finite contribution in this limit suggests
to us that it is related to the connecting region. 6
Black holes with an inner horizon We interpret this as saying that this
corresponds to the contributions of the Weyl tensor in the neck region — the region that
connects the AdS5 geometry to the AdS2 region.14 Nevertheless, note that it still contains – 16 – the time τin between the inner and outer horizons! Notice that in (6.4) the distances ℓhor
and ℓsing are being subtracted. Therefore, when we look at the geodesics in figure 6, we
should not add the proper lengths of the spacelike sections, but subtract them. the time τin between the inner and outer horizons! Notice that in (6.4) the distances ℓhor
and ℓsing are being subtracted. Therefore, when we look at the geodesics in figure 6, we
should not add the proper lengths of the spacelike sections, but subtract them. Integrating along the contour C1 in figure 7, we get a possible saddle-point value ˆρ1. Compared to ˆρ0, it contains an additional imaginary part ˆρ1 = γ0 −i(π −χ0) ,
(6.5) (6.5) where we have used that the integral over a full large circle gives −iπ. The contours C2
and C3 in figure 7 give us where we have used that the integral over a full large circle gives −iπ. The contours C2
and C3 in figure 7 give us JHEP03(2021)131 ˆρ2 = −γ0 −i(π −χ0) ,
ˆρ3 = −γ0 −iχ0 . (6.6) (6.6) These look similar to the previous ones, except that the quantity γ0 appears with the
opposite sign. Such contributions would lead to very suppressed terms at low temperatures
(where γ0 →∞), since we do not have the cancellations mentioned in (6.4). More precisely,
ρ3 gives a term of the form ⟨O⟩⊃T 2∆′e−iχ0∆= T 2∆′e−iπ∆′ ,
(6.7) (6.7) where ∆′ is the dimension of the field in the AdS2 region, given by ∆′ = mRAdS2 = ∆
r+
2
q
1 + 2r2
+
,
for
r+ −r−→0 . (6.8) (6.8) We see that the time between the inner and outer horizons is πRAdS2 We see that the time between the inner and outer horizons is πRAdS2. Notice that, in contrast with the black brane case, the AdS2 limit yields a factor of
T 2∆′ (as opposed to T ∆′). This is connected to the fact that, in the limit of perfect SL(2)
symmetry of AdS2, the one-point functions are zero. 6
Black holes with an inner horizon We depict the u = r2 plane and the various branch points in the computation of ρ. The
depicted contours give us various values for the “distance” to the singularity, which are expected to
contribute to the one-point function. The dotted lines indicate that we go to the second sheet by
crossing the cut. We have given two equivalent forms for the contour C0 — one of them emphasizes
the origin of the −iχ0 and γ0 contributions. JHEP03(2021)131 The structure here is somewhat similar to that of the four-dimensional black hole. However, here all the branch cuts are on the real axis. It is interesting to consider the total
length ˆρ of the r = u = 0 singularity when we follow the contours indicated in figure 7. The first value, ˆρ0, has both real and an imaginary parts: (6.2) ˆρ0 = −iχ0 + γ0 ,
(6.2)
χ0 = 1
2
Z w+
w−
dw
√w
p
(w+ −w)(w −w−)(w + 1 + w+ + w−) ,
γ0 = 1
2
Z w−
0
dw
√w
p
(w+ −w)(w−−w)(w + 1 + w+ + w−) ,
(6.3) (6.3) where we have indicated more explicitly the integrals we are considering. The imaginary
part is given by the time χ0 between the inner and outer horizons, while the real part
contains the distance ℓsing = Rγ0 between the bifurcation surface of the inner horizon and
the singularity (see figure 6). The saddle (6.2) contributes as ⟨O⟩∝exp [m(−ℓhor + Rγ0 −iRχ0)] = exp [m(−ℓhor + ℓsing −iτin)] ,
(6.4) (6.4) where ℓhor is the regularized distance from the boundary to the horizon. In order to
understand the meaning of a positive value of γ0, it is convenient to consider the low-
temperature limit, where r−∼r+. In this case, both ℓhor and γ0 diverge because the
distance to the horizon goes to infinity. However, this divergence cancels out in (6.4). This
cancellation is obvious if we note that in this limit, 1/√f develops a pole at u = u+ = u−,
which the C0 contour goes around, see figure 7. In the extremal limit, this contribution is
not suppressed and becomes temperature independent. 6
Black holes with an inner horizon We present a simple toy model of
one-point functions in nearly-AdS2 in appendix D. 7.1
Summary In this paper, we have proposed that the time to the singularity is contained in the thermal
one-point functions. This information is extracted by analyzing the dependence on the
mass, with the assumption that the higher-derivative coupling depends only on a power of
the mass. This assumption is true in string theory. For large mass, we have argued that we can perform a saddle-point analysis in terms
of geodesics. Then we pointed out that there is a saddle point near the singularity once we
assume a natural coupling between the massive particle and two gravitons. This is not a
proof, as we did not show that this saddle point always contributes or that it is dominant. To present evidence for the contributions of this saddle we did the following. We analytically computed the thermal one-point function for a black brane and checked
that the proposal is correct in this particular case. Furthermore, we gave a more detailed – 17 – contour rotation argument that explains why the saddle point contributes, despite the fact
that it does not lie on the original integration contour. We suspect that a similar argument
can be made for other black holes, but we did not present a general rigorous argument. For more general black holes, we examined the form of various possible saddle-point
contributions, picking out the ones that we expect to contribute. For Schwarzschild-like
black holes containing a spacelike singularity, we found that the saddle point that gives (1.1)
is the dominant one when the imaginary part of the mass is sufficiently large. However,
there can be larger contributions when the imaginary part of the mass is small. For black holes with an inner horizon, the structure of the answer is a bit different,
see (6.4). The dominant contribution looks roughly like a geodesic that goes through the
outer horizon, to the inner horizon, and then to the singularity, see figure 6. The timelike
region produces a “phase” proportional to the time between the outer and inner horizons,
τin. The spacelike regions give contributions with opposite signs. This cancellation implies
that this contribution becomes temperature independent in the extremal limit. For this
reason, from the point of view of the original integral, we can interpret this as a contribution
from the region that connects the AdS2 space to the higher-dimensional background. 7.1
Summary There
are other subleading saddles which display a temperature dependence of the form T 2∆′ as
we approach the extremal limit. These are expected to be contributions from the nearly
AdS2 region. JHEP03(2021)131 It is interesting that these one-point functions can be computed in the bulk using
the Euclidean black hole through an integral involving only the exterior. It is only the
saddle-point approximation that brings in the interior. Note that the actual saddle is at
some complex value of the radial position. It is only because this value is very close to the
singularity that we can relate it to a property of the Lorentzian black hole. Of course, the interior of a collapsing black hole can be much more complicated and
we wonder if any of the considerations here can be extended to that case. The considerations of this paper give us some very indirect access to the interior. No-
tice that this time to the singularity is a property of the thermal state and is independent
of possible Lorentzian processes happening behind the horizon. For example, we can start
from the two-sided black hole and send a shock wave at very early time on the left-hand side
so that it sits just behind the future horizon of the right-hand side observer. The expec-
tation values of the right-hand side observer are unchanged. However, the real Lorentzian
time to the singularity, the one experienced by an observer falling through the shock wave,
will change. 7.3
Two-point functions and thermal one-point functions of higher-spin
operators Thermal one-point functions are relevant when we make an operator product expansion of
two-point functions in a thermal background [10, 13–17]. The reason that the thermal two-
point function is different than the vacuum two-point function is the fact that operators
that appear in the OPE acquire non-zero expectation values in the thermal state. These
expectation values can in principle be related to vacuum OPE data [10, 13]. JHEP03(2021)131 In the case of free theories, we have that an operator creates particles, and these par-
ticles propagate fairly independently from each other. This is related to the observation
that there is a significant contribution from higher-spin operators in the OPE, and that fur-
thermore, these operators acquire a vacuum expectation value in the thermal background. In contrast, for theories with an Einstein gravity dual, the OPE in the thermal state has
contributions only from multi-graviton states [10, 13–17]. This is associated with the fact
that the bulk particle feels it is moving in a gravitational background as a single particle,
sometimes at speeds less than the boundary light speed, see [18] for a recent discussion. One could then be curious about the fate of the higher-spin operators as we increase the
coupling of the boundary theory. We know that they acquire a large anomalous dimension,
which makes them look like massive particles in the bulk [4, 5]. Nevertheless, we still expect
them to develop expectation values in the thermal state. Our discussion explains the origin
of these expectation values. They are absent in the Einstein gravity approximation, but
they appear once we include the α′ corrections, even in the planar theory. These involve
couplings between the higher-spin fields and two or more gravitons. Such higher-derivative
corrections are present since these massive string states can decay into gravitons. These
then lead to one-point expectation values that can be estimated using the methods of this
paper. This gives a pleasing continuity to the description: the higher-spin operators are
always present, and with non-zero thermal expectation values, but their contributions are
suppressed when the boundary theory is strongly coupled. Notice that these expectation
values for higher-spin operators are already present in the classical theory. In other words,
in the normalizations of (2.1), the expectation value of ϕ is of order one in GN, or the 1/N 2
expansion, but they are suppressed by the gravity limit of small α′/R2. 7.2
Three-dimensional case When the bulk has three dimensions, there are no gravitons and no Weyl tensor. Fur-
thermore, in the case of an infinite black string, the one-point functions are zero due to
conformal symmetry. However, non-zero one-point functions do arise for a finite area BTZ
black hole [2]. These can be interpreted as arising from a three-point coupling between the
field in question and the square of another field, with this other particle forming a loop
around the black hole horizon. It would be interesting to see whether this mechanism also
leads to (1.1). Naively, the same logic that leads to (1.1) should lead to a similar result for – 18 – the three-dimensional case when we evaluate it perturbatively. We simply replace W 2 by
the part of the loop diagram in the bulk that wraps non-trivially along the horizon. Here,
we would also expect to obtain (1.1), since diagram will get very large near the singular-
ity. However, we could not see this formula from the analysis in [2]. It would be nice to
understand this better. Acknowledgments JM is grateful to Akash Goel for some initial collaboration on this subject. We are also
grateful to A. Almheiri, S. Giombi, A. Maloney, F. Popov, D. Stanford, E. Witten, Y. Zhao
for discussions. JM was supported in part by U.S. Department of Energy grant DE-SC0009988. MG
was supported in part by the Princeton University Department of Physics. – 19 – A
Normalization of the correlators Here we discuss the normalization of the one-point function. We will use the extrapolate
dictionary, defining the unnormalized correlators by taking limits of bulk correlators eO(x) = lim
z→0
h
z−∆φ(x, z)
i
,
(A.1) (A.1) where the metric is ds2 = (d⃗x 2
d + dz2)/z2, and φ is a canonically normalized scalar field where the metric is ds2 = (d⃗x 2
d + dz2)/z2, and φ is a canonically normalized scalar field S = 1
2
Z √g
(∇φ)2 + m2φ2
. (A.2) (A.2) JHEP03(2021)131 After going to Fourier space in the ⃗xd coordinates, the Green’s function obeys the equation ∂z
1
zd−1 ∂z ˜G(z, z′, k)
−
k2z2 + m2
zd+1
! ˜G(z, z′, k) = δ(z −z′) . (A.3) (A.3) The homogeneous solutions take the form of two Bessel functions: η1(z, k) = zd/2Iν(kz)
and η2(z, k) = zd/2Kν(kz), where the index is ν = ∆−d/2. The solution of (A.3) can then
be written as ˜G(z, z′, k) = −
η1(z, k)η2(z′, k)θ(z′ −z) + η1(z′, k)η2(z, k)θ(z −z′)
. (A.4) (A.4) For small z and z′, this behaves like For small z and z′, this behaves like ˜G(z, z′, k) ∼2d−1−2∆k2∆−d
Γ(d
2 −∆)
Γ(∆−d
2 + 1)
! (zz′)∆,
(A.5) (A.5) Fourier transforming back into position space or, Fourier transforming back into position space ⟨eO(x) eO(0)⟩= lim
z,z′→0(zz′)−∆G(z, z′, ⃗x) =
1
2πd/2
Γ(∆)
Γ(∆−d
2 + 1)
1
|x|2∆. (A.6) (A.6) We then conclude that the properly normalized operator is defined as O(x) = CN ˜O(x) = CN lim
z→0
h
z−∆φ(x, z)
i
,
CN ≡
v
u
u
t2πd/2 Γ(∆−d
2 + 1)
Γ(∆)
. (A.7) (A.7) This formula gives us the normalized one-point function. Starting from ⟨φ(z′, 0)⟩=
Z
dzdx √g G(z′, 0; z, x) ˆαW 2 ,
(A.8) (A.8) the boundary expectation value is obtained by extracting the z′∆piece of this expectation
value. We can take the z′ →0 limit first inside the integral, and then use the z′ < z form of
the propagator (4.6) to obtain (4.8). The factors of (16πGN) arise from the normalization
of (2.1). Similarly, the factors of R can be easily restored. – 20 – – 20 – A.1
Normalization in the geodesic approximation A simple way to determine the normalization in the geodesic approximation is the following. The unit normalization at short distances, ⟨O(x)O(0)⟩∼|x|−2∆, implies that operators
on the sphere behave like, ⟨O(θ)O(0)⟩∼[2 sin θ
2]−2∆. On opposite points, we then have
⟨O(π)O(0)⟩∼2−2∆. Writing the empty AdS metric as in (5.1) with f(r) = r2+1, we find that the two-point
function is given by e−∆ℓ. Here ℓis the total length, given explicitly by ℓ= 2
Z rc
0
dr
√
r2 + 1 = 2 log rc + 2 log(2) . (A.9) (A.9) JHEP03(2021)131 JHEP03(2021)131 This implies that we simply need to subtract a factor of log rc for each operator, with
no extra constant. Then the log(2) term correctly reproduces the expected answer. Of
course, we get the same prescription if we use the usual semicircular geodesics in Poincaré
coordinates. This implies that we simply need to subtract a factor of log rc for each operator, with
no extra constant. Then the log(2) term correctly reproduces the expected answer. Of
course, we get the same prescription if we use the usual semicircular geodesics in Poincaré
coordinates. B
Prefactor With translation symmetry, the propagator obeys the wave equation 1
rd−1 ∂r(f(r)rd−1∂rΨ) −m2Ψ = 0 ,
(B.1) (B.1) away from coincident points. The standard WKB method then gives solutions Ψ ∼F(r) exp
±m
Z r
dr′
p
f(r′)
! ,
F(r) ≡
1
r
d−1
2 p
f(r)
. (B.2) (B.2) The prefactor F gives rise to additional singularities at positions where f = 0. For the particular case of the singularity at the horizon, we can choose a new variable,
ρ (5.3). The equation near ρ = 0 is just that of the Bessel function, since the cigar looks
like two-dimensional Euclidean space. The regular solution is simply the I0(mρ) function,
which can be expanded for large mass as I0 ∝emρ
√mρ(1 + · · · ) −ie−mρ
√mρ(1 + · · · ) ,
for Im(mρ) < 0 ,
(B.3) (B.3) where each term is also multiplied by powers of (mρ)−1. This gives us the relative normal-
ization of the short and long geodesic contributions near ρ = 0. The fact that there is an
i for the long geodesic is reasonable because it is expected to have a negative mode. The
long geodesic integral involves the second factor in (B.3) where each term is also multiplied by powers of (mρ)−1. This gives us the relative normal-
ization of the short and long geodesic contributions near ρ = 0. The fact that there is an
i for the long geodesic is reasonable because it is expected to have a negative mode. The
long geodesic integral involves the second factor in (B.3) Ilong = −i
Z ∞
0
dρρe−mρ
√ρ ,
(B.4) (B.4) where we have indicated only the small-ρ behavior and neglected unimportant overall
factors. The factor of ρ comes from the volume of the circle. The short geodesic contribution
involves the first term in (B.3). Integrating along the contour Cs−left in figure 3, we find Ishort =
Z −∞
0
dρρ emρ
√ρ = i
Z ∞
0
dρ′ρ′ e−mρ′
√ρ′ ,
ρ = e−iπρ′ ,
(B.5) (B.5) – 21 – which cancels (B.4). There is a similar cancellation if we use the full prefactor for the black
brane
1 F(ρ) ∝
1
√sinh ρ ,
(B.6) (B.6) which replaces the 1/√ρ in (B.3). This cancellation is important for the result we are
obtaining. B
Prefactor If we had not had this cancellation, each integral would have only given powers
of m and would have been larger than the term going like e−iπ∆/d, which is very small for
Im(∆) < 0. The prefactor (B.6) also has singularities at ρ = −in, which seem to interfere with
our contour rotation argument. We have not understood how to treat these properly. Perhaps one should consider the saddle-point approximation in the two-dimensional space
of ρ and tE, where tE is the Euclidean time direction, after a suitable complexification. This
should be doable in terms of Lefschetz thimbles, see [19]. In the unlikely case that they
do not cancel, they would give contributions involving exponentials of ρ = −iπn. The first
coincides with the leading contribution we have kept. And the others would be subleading
if Im(m) < 0, so that they would not affect (1.1). JHEP03(2021)131 C
Geodesic approximation for three-point functions As shown in [20], the Witten diagram for the three-point function for large masses can be
approximated by a geodesic computation, as shown in figure 8(b). This reproduces the
large-∆i limit of the gamma functions appearing in the Witten diagram computed in [9]. The approximation involves writing each propagator in terms of geodesics and using a
saddle point for the integration over the interaction point. A real saddle point exists if the
masses obey m1 + m2 > m3 (up to permutations). However, if m1 + m2 < m3, then the interaction point gets driven to the boundary,
and more specifically, to the insertion point of the third operator. This is related to the
appearance of poles at ∆3 = ∆1 + ∆2 + 2n, which stem from the mixing of the O3 with
operators of the schematic form O1∂2nO2. Nevertheless, even in this case, it is possible to
show that we can reproduce the large-mass (large-∆) behavior from a complex solution. Since we just want to illustrate the phenomenon, we will choose a simple case with
m1 = m2 ̸= m3. We can consider all three points at the boundary of an H2 bulk subspace
with coordinates ds2 = dρ2 + cosh2 ρ dt2 . (C.1) (C.1) We put O1 and O2 at t = 0 and ρ = ±∞. We also place the third operator at ρ = 0
and t = +∞, see figure 8. By symmetry, the classical trajectory of the third particle is at
ρ = 0. The first particle follows a trajectory We put O1 and O2 at t = 0 and ρ = ±∞. We also place the third operator at ρ = 0
and t = +∞, see figure 8. By symmetry, the classical trajectory of the third particle is at
ρ = 0. The first particle follows a trajectory tanh ρ = cosh t −sinh t
tanh t0
,
0 ≤t ≤t0 ,
(C.2) (C.2) where t0 is the value of t at ρ = 0 (the intersection point with the third particle), see
figure 8. The second particle is at a symmetric configuration. – 22 – – 22 – (a)
(b)
t
ρ
t0
m3
m2 = m1
m1
ρ = +∞
ρ = 0
ρ = −∞
∼
1
3
2
1
2
t = 0
Figure 8. Geodesics for the three-point function. (a) The coordinates used in (C.1). C
Geodesic approximation for three-point functions Configuration
for the case m1 = m2 > m3/2. (b) Conventional picture in terms of the hyperbolic disk. (b)
∼
1
3
2 (a)
t
ρ
t0
m3
m2 = m1
m1
ρ = +∞
ρ = 0
ρ = −∞
1
2
t = 0 (b) (a) JHEP03(2021)131 Figure 8. Geodesics for the three-point function. (a) The coordinates used in (C.1). Configuration
for the case m1 = m2 > m3/2. (b) Conventional picture in terms of the hyperbolic disk. Evaluating its length, we get Evaluating its length, we get ℓ(t0) =
Z t0
ϵ
dt
q
cosh2 ρ + ρ′2 = 1
2 log
sinh 2t0
ϵ
= log cosh t0 + ρmax ,
(C.3) (C.3) where we have used the relation ρmax ∼1
2(−log ϵ+log tanh t0+log 2), and ρmax is a physical
cutoff, independent of t0. The final action is then where we have used the relation ρmax ∼1
2(−log ϵ+log tanh t0+log 2), and ρmax is a physical
cutoff, independent of t0. The final action is then S = R [2m1 log cosh t0 −m3t0] . (C.4) (C.4) Minimizing with respect to t0, we find Minimizing with respect to t0, we find tanh t0 = m3
2m1
,
or
tanh u0 = 2m1
m3
,
for t0 = u0 + iπ/2 . (C.5) (C.5) We see that t0 is complex for m3 > 2m1. The action (C.4) becomes We see that t0 is complex for m3 > 2m1. The action (C.4) becomes We see that t0 is complex for m3 > 2m1. The action (C.4) becomes S = iπ(2∆1 −∆3)/2 + (real) . (C.6) (C.6) This reproduces the “phase”, eiπ(2∆−1−∆3)/2 of the Γ ((∆1 + ∆2 −∆3)/2) factor in the
Witten diagram. By summing over saddles with t0 →t0+iπn, we reproduce a 1/ sin(π(2∆−
∆3)/2) factor present from the gamma function. The real part in (C.6) reproduces the
large-∆i limit of all other gamma factors. This reproduces the “phase”, eiπ(2∆−1−∆3)/2 of the Γ ((∆1 + ∆2 −∆3)/2) factor in the
Witten diagram. By summing over saddles with t0 →t0+iπn, we reproduce a 1/ sin(π(2∆−
∆3)/2) factor present from the gamma function. The real part in (C.6) reproduces the
large-∆i limit of all other gamma factors. The conclusion is that in this well-studied example, we also find that complex saddle
points reproduce the answer. D
Toy model for one-point functions in nearly-AdS2 D
Toy model for one-point functions in nearly-AdS2 We can consider the AdS2 metric We can consider the AdS2 metric ds2 = R2
"
−(r2 −r2
0)dt2 +
dr2
r2 −r2
0
#
. (D.1) (D.1) – 23 – −r0 < r < r0
r > r0
r < −r0
SINGULARITY
(r = rs ≪−r0)
INNER HORIZON
(r = −r0)
OUTER HORIZON
(r = r0)
CONNECTED HERE
TO ANOTHER SOLUTION
(r = rconnect)
Figure 9. Penrose diagram of nearly-AdS2 spacetime. The exterior region, r > r0, is then con-
nected to some of the spacetime at r = rconnect. The region beyond the inner horizon contains a
timelike singularity at r = rs ≪−r0. −r0 < r < r0
r > r0
r < −r0
SINGULARITY
(r = rs ≪−r0)
INNER HORIZON
(r = −r0)
OUTER HORIZON
(r = r0)
CONNECTED HERE
TO ANOTHER SOLUTION
(r = rconnect) INNER HORIZON
(r = −r0) JHEP03(2021)131 Figure 9. Penrose diagram of nearly-AdS2 spacetime. The exterior region, r > r0, is then con-
nected to some of the spacetime at r = rconnect. The region beyond the inner horizon contains a
timelike singularity at r = rs ≪−r0. In purely-AdS2, any expectation value has to be a constant, which we can subtract. Notice
that the region −r0 < r < r0 corresponds to the region between the outer and inner
horizons, see figure 9. If we now consider a space which is nearly-AdS2 (as it arises when we take the near-
extremal limit of a more general black hole) then the metric (D.1) will connect to some
other space at some large value, r = rconnect ≫r0. Similarly, we expect deviations in the
region behind the inner horizon (r = rs ≪−r0), see figure 9. We expect that these deviations will induce some effective coupling to the scalar field
of the form
Z Ssource =
Z
dtEdr √g f(r) φ(tE, r) ,
(D.2) (D.2) where f(r) is some function. The integral is over the Euclidean black hole, which contains
only the exterior region r ≥r0. As a toy model, we choose the function where f(r) is some function. The integral is over the Euclidean black hole, which contains
only the exterior region r ≥r0. As a toy model, we choose the function f(r) =
−rs
r −rs
2
,
(D.3) (D.3) which has some desirable features. First, it goes to zero at the physical boundary, (r→+∞). D
Toy model for one-point functions in nearly-AdS2 The additional integer powers of T or r0 present in (D.5), can be viewed as arising
from terms involving ηcn
R dtOHn, where H is the Hamiltonian.16 D
Toy model for one-point functions in nearly-AdS2 It also diverges at r = rs ≪0, which is in the region where we expect the singularity to lie. We keep rs fixed as we vary the temperature, or vary r0 ∝T. We note that the particular
function (D.3) was only chosen so that we can analytically compute the integrals below. We can solve for the propagator, as in the black brane case, after choosing the variable
w ≡
2r0
r+r0 . However here, in contrast with section 4.1, w continues beyond infinity, to
negative values, where it describes the region behind the inner horizon (r < −r0). see
figure 9. The expression for the one-point function then becomes, (assuming r0/|rs| ≪1) ⟨O⟩∝r∆
0
Z 1
0
dw
w2
w
w−ws
2
w∆2F1(∆,∆,1;1−w),
ws = 2r0
rs
< 0,
∆∼mR,
(D.4) (D.4) – 24 – where the integral is only over the black hole exterior. This gives where the integral is only over the black hole exterior. This gives ⟨O⟩∝r∆
0 Γ(∆+1)Γ(2−∆) 1
w2s
2F1
∆+1,2−∆,2; 1
ws
∝Γ(2−∆)Γ(2∆−1)
Γ(∆)
−rs
2
∆
+··· Γ(∆+1)Γ(1−2∆)
Γ(1−∆)
r2∆−1
0
−rs
2
1−∆
+ ···
(D.5) (D.5) where the dots represent extra integer powers of r0. Note that, for large ∆, both terms
lead to a “phase” factor of the form e−i∆π, which comes from the large-∆expansion of the
gamma functions. The πR here is indeed the time between the inner and outer horizons
for (D.1). The first term in (D.5) is like the temperature-independent term that was
discussed in (6.4). The second term gives the temperature-dependent term, as in (6.7),
since r0 ∝T.15 JHEP03(2021)131 Note that the temperature dependence of these two terms can also be obtained as
follows. Suppose we have a perturbation S = η
Z
dtO(t)
(D.6) (D.6) at the boundary. Assuming the conformally invariant two-point function ⟨O(t)O(0)⟩=
T 2∆
(sin(Tπt))2∆,
(D.7) (D.7) perturbing the theory with (D.6), gives ⟨O⟩∼η
Z
dt⟨O(t)O(0)⟩∝T 2∆−1 ,
(D.8) (D.8) where the last term comes from a rescaling of the integration variables or dimensional
analysis. This reproduces the temperature dependence of the second term in (D.5). The
first term, which is temperature independent, comes from the UV divergence of (D.8). This
simple integral (D.8) does not however, reproduce the “phase” factor that we encountered
above, so the story is not complete. 15In (6.7), the approximation does not distinguish between 2∆′ and 2∆′ −1. ∆′ in (6.7) is the same as
∆here — the scaling dimension in the AdS2 region.
16We thank the anonymous referee for this comment. 16We thank the anonymous referee for this comment. ∆here — the scaling dimension in the AdS2 region.
16 15In (6.7), the approximation does not distinguish between 2∆′ and 2∆′ −1. ∆′ in (6.7) is the same 15In (6.7), the approximation does not distinguish between 2∆′ and 2∆′ −1. ∆′ in (6.7) is the same
∆here — the scaling dimension in the AdS2 region.
16W th
k th
f
f
thi
t E
Thermal one point functions for three dimensional black holes Thermal one point functions are zero for an infinite black string due to conformal symmetry. However, as shown in [2] they are non-zero once we compactify the spatial direction and
obtain a BTZ black hole. As argued in [2], the one-point function can be viewed as arising
from a bulk diagram where the field in question, φ, interacts via a three-point coupling – 25 – χ
φ
Figure 10. Origin of the thermal one point function for a BTZ black hole [2]. Figure 10. Origin of the thermal one point function for a BTZ black hole [2]. JHEP03(2021)131 with another field, χ, that goes around the horizon of the BTZ black hole, see figure 10. As an example, consider an action of the form with another field, χ, that goes around the horizon of the BTZ black hole, see figure 10. As an example, consider an action of the form S ∝
Z
(∇φ)2 + m2φ2 + (∇χ)2 + µ2χ2 + g φχ2 ,
(E.1) (E.1) where φ and χ are bulk scalar fields, and φ is related to the operator whose one-point
function we are after. This one-point function comes from the interactions with the field
χ. More precisely, we get a contribution of the form ⟨O⟩∝
Z
BTZ
⟨χ2(x)⟩βGb(x, 0) ,
(E.2) (E.2) where β denotes temperature dependence. And Gb is a bulk to boundary propagator. In the
coincident point correlator for the field χ, ⟨χ2(x)⟩, we include only the contribution from
trajectories with non-zero winding around the spatial circle, disregarding the contribution
from the unwound trajectories which should be removed when we are in global AdS3. In
other words, when we go from global AdS3 to the BTZ black hole, we perform a quotient. In computing the χ correlator we use the method of images, keeping only the contributions
from non-trivial images. This means that ⟨χ2(x)⟩is some function of the radial coordinate. From now on the analysis is similar to what we did in higher dimensions. More explicitly,
we can write Euclidean metric as ds2 = 1
z2
(1 −z2)dt2 +
dz2
1 −z2 + z2dx2
! ,
t ∼t + 2π
(E.3) (E.3) where we have rescaled the time coordinate to set the temperature to 2π. E
Thermal one point functions for three dimensional black holes The length of
the x coordinate is then (E.4) x ∼x + ℓ,
ℓ= 2πL/β ,
(E.4) where β is the original temperature and L is the original length of the circle. If we momentarily ignore the compactification of x, we can view (E.3) as global Ad
where the angular direction is t. The propagator of the χ field is given by [21] G∆= C∆2∆
u∆
2F1
∆, ∆−1
2, 2∆−1; −2
u
(E.5) (E.5) – 26 – with u ≡2 sinh2 ˆd
2 ,
(E.6) u ≡2 sinh2 d
2 ,
(E.6) (E.6) where ˆd is the proper distance between the two points and ∆= 1 +
p
1 + µ2 (we have set
the AdS3 radius to one). Then we can write where ˆd is the proper distance between the two points and ∆= 1 +
p
1 + µ2 (we have set
the AdS3 radius to one). Then we can write ⟨χ2(x)⟩=
∞
X
n=1
G(un),
(E.7) (E.7) where we are summing over images, but have subtracted the completely coincident point
singularity. JHEP03(2021)131 We have that un is given, in terms of the distance ˆdn from a general point to its image,
by (E.6),
ˆ
ˆ
ℓ un = 2 sinh2 ˆdn
2 ,
sinh
ˆdn
2 = sinh( nℓ
2 )
z
. (E.8) (E.8) We see from these expressions that when z →∞, ˆdn or un →0 and the terms in the
sum (E.7) diverge. In fact, since this expression appears in (E.2) we therefore expect that
there will be saddle point solutions as we had previously. We will now check this explicitly. Consider a particular dimension for the χ field ∆= 1 where the propagator (E 6) We see from these expressions that when z →∞, ˆdn or un →0 and the terms in the
sum (E.7) diverge. In fact, since this expression appears in (E.2) we therefore expect that
there will be saddle point solutions as we had previously. We will now check this explicitly. sum (E.7) diverge. In fact, since this expression appears in (E.2) we therefore expect that
there will be saddle point solutions as we had previously. We will now check this explicitly. E
Thermal one point functions for three dimensional black holes Consider a particular dimension for the χ field, ∆= 1, where the propagator (E.6)
becomes relatively simple Consider a particular dimension for the χ field, ∆= 1, where the propagator (E.6)
becomes relatively simple G ∝−2
u F
1, 1
2, 1; −2
u
∝
1
√u
p
1 + u/2 ∝
1
sinh ˆd
. (E.9) (E.9) Let us first insert the n = 1 from (E.7) in the expression (E.2). Defining w = z2, we fin Let us first insert the n = 1 from (E.7) in the expression (E.2). Defining w = z2, we find
⟨O⟩∝
Z 1
0
dz
z3 ⟨χ2(z)⟩n=1Gb(z) ∝
1
sinh ℓ
2
Z 1
0
dw
wh−1
q
w + sinh2 ℓ
2
2F1(h, h, 1; 1 −w) ,
(E.10) ⟨O⟩∝
Z 1
0
dz
z3 ⟨χ2(z)⟩n=1Gb(z) ∝
1
sinh ℓ
2
Z 1
0
dw
wh−1
q
w + sinh2 ℓ
2
2F1(h, h, 1; 1 −w) ,
(E.1 with h ≡∆φ
2 . Note that Gb is the bulk to boundary propagator for an operator of dimension
∆φ, not to be confused with the χ field propagator in (E.9). The integral gives17 ⟨O⟩∝Γ(h)Γ(1 −h)
sinh2 ℓ
2
3F2
h, 1
2, 1 −h; 1, 1; −
1
sinh2 ℓ
2
! (E.11) (E.11) If we now expand for large ℓ, we can evaluate the hypergeometric function at zero and we get
⟨O⟩
Γ(h)Γ(1
h) × (i d
f h) ×
−ℓ≈
1
× (i d
f h) ×
−ℓ
(E 12) If we now expand for large ℓ, we can evaluate the hypergeometric function at zero and we g ⟨O⟩∝Γ(h)Γ(1 −h) × (indep. of h) × e−ℓ≈
1
sin(πh) × (indep. of h) × e−ℓ,
(E.12) (E.12) This agrees with our previous e−iπh phase factor. The other terms in the sum over n can
be obtained by replacing ℓ→nℓin (E.11) and give a similar h dependence. The expected
factor of T ∆φ is obtained by rescaling the temperature back from 1/(2π) to the actual
temperature. Therefore, we have checked that the BTZ result is reproducing the expectations we
had in general, giving us a “phase” which is related to the time between the horizon and the
17See Gradshteyn and Ryzhik formula (7.512.9). 17See Gradshteyn and Ryzhik formula (7.512.9). 17See Gradshteyn and Ryzhik formula (7.512.9). E
Thermal one point functions for three dimensional black holes Therefore, we have checked that the BTZ result is reproducing the expectations we
had in general, giving us a “phase” which is related to the time between the horizon and the – 27 – BTZ singularity. We have reproduced this only for the case of ∆χ = 1. We also expect it
to be true for more general values, but we have not explicitly performed the computation. g
y
p
y
χ
p
to be true for more general values, but we have not explicitly performed the computation. Note that the answer has an exponential suppression e−ℓ= e−∆χℓ, as discussed in [22],
since the one-point function must vanish for the black string case. Note that the answer has an exponential suppression e−ℓ= e−∆χℓ, as discussed in [22],
since the one-point function must vanish for the black string case. References [1] R.C. Myers, T. Sierens and W. Witczak-Krempa, A Holographic Model for Quantum Critical
Responses, JHEP 05 (2016) 073 [Addendum ibid. 09 (2016) 066] [arXiv:1602.05599]
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Feynman Integrals, JHEP 02 (2020) 159 [arXiv:1910.11852] [INSPIRE]. [13] S. El-Showk and K. Papadodimas, Emergent Spacetime and Holographic CFTs, JHEP 10
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arXiv:2010.09734 [INSPIRE]. [19] E. F
Operator mixing In this appendix we discuss in more detail the operator mixing problem. Let us use the “extrapolate” dictionary to compute the one-point function [23]. In
this case, we are supposed to compute the expectation value of the bulk field, ⟨φ(z′)⟩, as
a function of the bulk coordinate z′. We can then expand for small z′ and pick out the
expectation value from the behavior JHEP03(2021)131 ⟨φ(z′)⟩∼⟨O⟩(z′)∆,
(F.1) (F.1) where ⟨O⟩is, by definition, the coefficient of the (z′)∆term. For the black brane case, the expectation value of the bulk field is ⟨φ⟩∼
Z
dz
zd+1 z2dG(z|z′) ,
(F.2) (F.2) where G(z|z′) is the propagator. In writing (4.8) we have assumed that z′ is very small and
approximated the propagator in terms of the solution that is smooth at the horizon times
a factor of (z′)∆(which is correct for z > z′). In particular, we neglected the contribution
from the region z < z′. This is valid if ∆< 2d, when the integral (4.8) is convergent. If
∆> 2d, the integral (F.2) is still finite, however its leading behavior as z′ →0 is obtained as (z′)⟩∝(z′)d−∆
Z z′
0
dz
zd+1 z2dz∆+ (z′)∆
Z zh
z′
dz
zd+1 z2dzd−∆∝c1(z′)2d + c2(z′)∆,
(F.3) (F.3) where we indicated only the small z, z′ behavior and c1, c2 are constants. The first term
does not behave at all like an operator of dimension ∆. Instead, it behaves like an operator
of dimension 2d. The idea is that this term is related to the expectation value of the square
of the stress tensor and that the field φ contains both a contribution from the stress tensor,
as well as a contribution from the primary field of dimension ∆that we are after. The
latter is now contained in a subleading term. In writing (4.8) we have neglected the first
term in (F.3) and approximated z′ = 0 in the lower integration limit of the second term. The analytic continuation of the integral that we discussed is designed to pick out the piece
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English
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Multi-rater developmental trajectories of hyperactivity–impulsivity and inattention symptoms from 1.5 to 17 years: a population-based birth cohort study
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European child & adolescent psychiatry
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* Sylvana M. Côté
sylvana.cote.1@umontreal.ca European Child & Adolescent Psychiatry (2019) 28:973–983
https://doi.org/10.1007/s00787-018-1258-1 European Child & Adolescent Psychiatry (2019) 28:973–983
https://doi.org/10.1007/s00787-018-1258-1 ORIGINAL CONTRIBUTION Abstract The developmental course of hyperactivity–impulsivity and inattention symptoms from infancy to adolescence has not
been documented in a population sample. The aim of this study was to describe the developmental course of hyperactiv-
ity–impulsivity and inattention symptoms from 1.5 to 17 years using multiple informants, and to identify perinatal risk fac-
tors associated with following elevated (high-risk) trajectories. Using a population-based birth cohort (n = 1374), symptom
ratings from mothers (1.5–8 years), teachers (6–13 years) and participant self-reports (10–17 years) were combined using
group-based multi-trajectory modeling to identify informants’ convergence in identifying high-symptom trajectories of
hyperactivity–impulsivity and inattention over time. Perinatal risk factors associated with high-symptom trajectories were
identified using stepwise logistic regression. The study found that symptoms of hyperactivity–impulsivity broadly declined
from 1.5 to 17 years while symptoms of inattention remained constant. 21.4% of participants followed elevated trajectories of
hyperactivity–impulsivity and 20.2% followed elevated trajectories of inattention; 11.6% followed elevated trajectories of both
types of symptoms concurrently. Risk factors for high-risk trajectories of hyperactivity–impulsivity were low maternal educa-
tion, prenatal alcohol exposure, non-intact family, maternal depression, and low child IQ; for high-risk inattention they were
prenatal street drug exposure, early motherhood, low maternal education, maternal depression and low child IQ. Risk factors
for trajectories of high-risk hyperactivity–impulsivity and inattention concurrently were low maternal education, maternal
depression, and low child IQ. The combination of longitudinal assessments from multiple informants (i.e., mother, teacher,
participant-reports) provides a new way to characterize hyperactivity–impulsivity and inattention phenotypes over time. Keywords Attention-deficit/hyperactivity disorder · ADHD · Risk factors · Longitudinal Multi‑rater developmental trajectories of hyperactivity–impulsivity
and inattention symptoms from 1.5 to 17 years: a population‑based
birth cohort study Francis Vergunst1 · Richard E. Tremblay1,2,3 · Cédric Galera4 · Daniel Nagin5 · Frank Vitaro1,6 · Michel Boivin1,7,8 ·
Sylvana M. Côté1,4 t1 · Richard E. Tremblay1,2,3 · Cédric Galera4 · Daniel Nagin5 · Frank Vitaro1,6 · Michel Boivin1,7,8 ·
1 4 Received: 28 May 2018 / Accepted: 19 November 2018 / Published online: 1 December 2018
© The Author(s) 2018 4
INSERM U1219, Université de Bordeaux, Bordeaux, France
5
Carnegie Mellon University, Pittsburgh, PA, USA
6
School of Psycho‑Education, University of Montreal,
Montreal, Canada
7
School of Psychology, Université Laval, Quebec, Canada
8
Institute of Genetic, Neurobiological, and Social Foundations
of Child Development, Tomsk State University, Tomsk,
Russian Federation 4
INSERM U1219, Université de Bordeaux, Bordeaux, France Participants and procedure Data were obtained from the Quebec Longitudinal Study of
Child Development (QLSCD) approved by the Quebec Insti-
tute of Statistics and the St-Justine Hospital Research Center
ethics committees. A population sample of 2120 children
born in 1997/1998 in the province of Quebec, were identi-
fied through birth registries. Families were included if the
pregnancy lasted between 24 and 42 weeks and the mother
could speak either French or English. Data were collected
through structured interviews conducted by trained research-
ers. Relevant health and sociodemographic characteristics of
the children, family and parents were obtained at 5 months. Behavioral ratings of hyperactivity–impulsivity and inatten-
tion were obtained from mother reports (1.5, 2.5, 3.5, 4.5, 5,
6 and 8 years), teacher reports (6, 7, 8, 10, 12 and 13 years),
and participant-reports (10, 12, 13, 15 and 17 years). Written
informed consent was provided by parents at each interview. Symptom ratings were derived from items in the early
childhood behavior scale from the Canadian National Lon-
gitudinal Study of Children and Youth [15]. The instru-
ment incorporates items from the Child Behavior Checklist
[16], the Ontario Child Health Study Scales [17], and the
Preschool Behavior Questionnaire [18]. Assessments at
ages 15 and 17 years were made using the Mental Health
and Social Inadaptation Assessment for Adolescents [19]. Hyperactivity–impulsivity items were: Can’t sit still, is
restless or hyperactive; Impulsive, acts without thinking;
and Difficulty waiting his/her/your turn in games/activi-
ties. Inattention items were: Cannot concentrate, cannot
pay attention for long; Is inattentive; and Easily distracted, Data were obtained from the Quebec Longitudinal Study of
Child Development (QLSCD) approved by the Quebec Insti-
tute of Statistics and the St-Justine Hospital Research Center
ethics committees. A population sample of 2120 children
born in 1997/1998 in the province of Quebec, were identi-
fied through birth registries. Families were included if the
pregnancy lasted between 24 and 42 weeks and the mother
could speak either French or English. Data were collected
through structured interviews conducted by trained research-
ers. Relevant health and sociodemographic characteristics of
the children, family and parents were obtained at 5 months. Behavioral ratings of hyperactivity–impulsivity and inatten-
tion were obtained from mother reports (1.5, 2.5, 3.5, 4.5, 5,
6 and 8 years), teacher reports (6, 7, 8, 10, 12 and 13 years),
and participant-reports (10, 12, 13, 15 and 17 years). Written
informed consent was provided by parents at each interview. Aims The aims of this study were twofold. First, to map the
development of hyperactivity–impulsivity and inattention
symptoms from infancy to adolescence, and identify groups
of children following elevated trajectories, using annual
symptom ratings obtained from mothers (early childhood,
1.5–8 years), teachers (middle childhood, 6–13 years) and
the participants (adolescence, 10–17 years). Second, to
identify early risk factors associated with high-symptom
trajectories of hyperactivity–impulsivity, inattention, and
both symptom categories concurrently, using multivariable
modeling. The present study uses data from a population sample
followed from infancy (age 5 months) to adolescence (age
17 years) to identify groups of children following distinct
developmental trajectories of hyperactivity–impulsivity
and inattention symptoms. Symptom ratings were obtained
from three sources—mothers, teachers, and participant-
reports—determined by the child’s social and educational
contexts (pre-school, elementary school, high school). This approach has several advantages. First, while par-
ents are well placed to describe the children’s behavior
in infancy, teachers become the most reliable source of
behavioral information once they reach school age [4]. Second, teacher assessments benefit from normative com-
parisons, something that parental assessments may lack. Third, as children become adolescents, self-rated assess-
ments become a reliable source of behavioral information
[5, 6]. Fourth, symptom ratings from multiple informants
are important for clinical practice and research as they
can help to accurately identify the clinical phenotype of
the child [7, 8]. Introduction Attention-deficit/hyperactivity disorder (ADHD) is an
etiologically complex neurodevelopmental condition asso-
ciated with significant negative long-term outcomes. Sev-
eral studies have described the developmental course of Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00787-018-1258-1) contains
supplementary material, which is available to authorized users. Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00787-018-1258-1) contains
supplementary material, which is available to authorized users. * Sylvana M. Côté
sylvana.cote.1@umontreal.ca
1
Sainte‑Justine University Hospital, Université de Montréal,
Montreal, Canada
2
Department of Pediatrics and Psychology, University
of Montreal, Montreal, Canada
3
School of Public Health, Physiotherapy and Population
Science, University College Dublin, Dublin, Ireland * Sylvana M. Côté
sylvana.cote.1@umontreal.ca 5
Carnegie Mellon University, Pittsburgh, PA, USA 1
Sainte‑Justine University Hospital, Université de Montréal,
Montreal, Canada 6
School of Psycho‑Education, University of Montreal,
Montreal, Canada 2
Department of Pediatrics and Psychology, University
of Montreal, Montreal, Canada 7
School of Psychology, Université Laval, Quebec, Canada 8
Institute of Genetic, Neurobiological, and Social Foundations
of Child Development, Tomsk State University, Tomsk,
Russian Federation 3
School of Public Health, Physiotherapy and Population
Science, University College Dublin, Dublin, Ireland (0123
1 3456789)
3 974 European Child & Adolescent Psychiatry (2019) 28:973–983 recall bias. Finally, many studies focus on clinical samples
or on at-risk males (e.g., pre-term or low birthweight),
which limits their generalizability to females and to the
general population. hyperactivity–impulsivity and inattention symptoms, but
findings remain mixed with respect to symptom continuity
and change across childhood and adolescence [1–3]. An
important obstacle for studies of this kind is the absence
of an accepted methodology that allows a single rater to
assess symptoms from infancy through adolescence. How-
ever, recent innovations in biostatistics allow us to account
for issues of temporality and multi-informant assessments,
providing a new way to characterize complex behavioral
phenotypes such as ADHD over time. Perinatal social factors difficulty pursuing any activity. These items correspond with
DSM-V criteria for ADHD and correlate highly with those
used in other standardized measures of childhood behavioral
problems such as the Strengths and Difficulties Question-
naire [20]. These measures have been extensively used in
ADHD research as proxies of ADHD diagnosis particularly
in epidemiological samples from the general population and
consider ADHD as a quantitative trait [9, 21]. Scores were
summed and divided by the number of items then standard-
ized on a 0–10 scale. Symptoms were rated on a frequency
scale (never/not true = 0, sometimes/somewhat true = 1,
often/very true = 2). Alpha scores for hyperactivity–impul-
sivity and inattention were, respectively, 0.85 and 0.89 for
mother ratings (1.5–8 years), 0.91 and 0.93 for teacher rat-
ings (6–13 years), and 0.74 and 0.82 for participant-reports
(10–17 years). Correlations for overlapping mother–teacher
ratings were, for hyperactivity–impulsivity and inattention,
respectively, 0.36 and 0.36 at 6 years and 0.37 and 0.44 at
8 years; correlations for overlapping teacher–participant-
reports were 0.24 and 0.38 at 10 years, 0.30 and 0.37 at
12 years, and 0.29 and 0.29 at 13 years. Family socioeconomic status (SES) was calculated from the
family’s overall income, and the mother’s and father’s num-
ber of years of education and occupational prestige [24]. SES scores were standardized with a mean of 0 and standard
deviation of 1. Family structure was coded as 1 if the fam-
ily was not intact (i.e., child not living with both biological
parents; 21.0% of the sample) and 0 if the family was intact
(child living with both their biological parents irrespective
of conjugal relationship). Insufficient household income
(24.5% of the sample) was calculated based on Statistics
Canada’s guidelines which account for family area of resi-
dence, number of occupants in the household, and family
income over the past year. Early motherhood was coded as
1 if she was 21 years or younger at the birth of her first child
(22.5% of the sample). Low parental education was coded
as 1 if the mother/father had never obtained a high school
diploma (16.0% of mothers, 17.6% of fathers). Baseline characteristics and early risk factors Family dysfunction at age 5 months was assessed using the
McMaster Family Assessment Device [15]. The 12-item
instrument measures communication, showing and receiving
affection, control of disruptive behavior, and problem resolu-
tion. Scores are z-standardized. Mother–child interactions
were assessed using the responsiveness scale of the home
observation for measurement of the environment–infant ver-
sion [25]. Hostile–reactive parenting, overprotection, paren-
tal self-efficacy, and perceived parental impact were assessed
using The Parental Cognition and Conduct Toward the Infant
Scale [26]. Scores for each dimension were z-standardized. Information on family and child characteristics was obtained
from parents at 5 months. For categorical variables, the pres-
ence of risk was coded as 1 and its absence as 0. Child characteristics The sex of the child was coded as 1 for boys and 0 for
girls. Methylphenidate hydrochloride (Ritalin) use was
coded as 1 for any methylphenidate taken between 6 and
15 years (14.4% of the sample). Child IQ was assessed at
41 months using the Wechsler Intelligence Scale for Chil-
dren Block Design [22]. Child temperament was assessed
at age 5 months using the difficult temperament scale from
the well-validated Infant Characteristics Questionnaire [23]. Parental psychopathology Parents were asked whether before completing high school
they had displayed any of five different conduct problems
matching DSM-IV criteria for conduct disorder and antiso-
cial personality disorder. Parental depression, also obtained
at 5 months, was assessed using the abbreviated version
of the Center for Epidemiologic Studies Depression Scale
(12-item) [27]. Parents reported the frequency of depressive
symptoms in the past week. Items were coded on a 4-point
scale and are z-standardized. Prenatal and perinatal factors Information about the child’s birth was obtained from medi-
cal records, defined as: premature birth if < 37th week of
gestation (4.9% of children), low birthweight if < 2500 g
(3.3% of children). Parental tobacco, alcohol and street drug
use during pregnancy were collected when the child was
5 months old. Tobacco, alcohol and drug exposure were,
respectively, coded as 1 if the mother smoked at least one
cigarette per day (25.3% of mothers), drank at least once per
week (3.3% of mothers) or used any drugs (1.4% of mothers)
during pregnancy. Early risk factors Identifying early risk factors for hyperactivity–impulsiv-
ity and inattention symptoms is crucial because they con-
stitute the principal pathway through which preventative
interventions can be used to reduce risk of onset of ADHD
and associated behavioral disorders. Existing studies of
early risk factors for ADHD have important limitations
[9]. First, most are relatively short and focus on a single
developmental period (e.g., early childhood) and conse-
quently fail to account for the developmental nature of the
disorder from infancy to adulthood [10]. Second, clinical
studies typically rely on discrete diagnostic categories at
a single time point, which does not provide information
on the developmental nature of symptom change across
development [11, 12]. Third, few risk factor studies of
hyperactivity–impulsivity and inattention distinguish
between the two symptom types, despite evidence that they
are best understood as two related but distinct phenotypic
dimensions [13, 14]. Fourth, information on risk factors is
frequently collected retrospectively, raising the problem of Symptom ratings were derived from items in the early
childhood behavior scale from the Canadian National Lon-
gitudinal Study of Children and Youth [15]. The instru-
ment incorporates items from the Child Behavior Checklist
[16], the Ontario Child Health Study Scales [17], and the
Preschool Behavior Questionnaire [18]. Assessments at
ages 15 and 17 years were made using the Mental Health
and Social Inadaptation Assessment for Adolescents [19]. Hyperactivity–impulsivity items were: Can’t sit still, is
restless or hyperactive; Impulsive, acts without thinking;
and Difficulty waiting his/her/your turn in games/activi-
ties. Inattention items were: Cannot concentrate, cannot
pay attention for long; Is inattentive; and Easily distracted, 3 3 European Child & Adolescent Psychiatry (2019) 28:973–983 975 Trajectory modeling Ratings of hyperactivity–impulsivity and inattention
between 1.5 and 17 years were modeled using group-based
multi-trajectory modeling [28, 29]. The method, based on 1 3 European Child & Adolescent Psychiatry (2019) 28:973–983 976 membership [29], a step that was not required for the analy-
sis of risk factors distinguishing the six trajectory groups. finite mixture modeling, identifies groups of distinctive
developmental trajectories over age or time. The approach
uses a generalization of the basic trajectory model in which
trajectory groups are defined by multiple trajectories. In the
present application, each group is defined by trajectories
obtained from annual symptoms from three raters: mothers
(1.5–8 years), teachers (6–13 years) and participant-reports
(10–17 years). The approach generates a set of trajectory
groups that represent the continuous symptom course from
1.5 to 17 years. The trajectory groups are displayed sepa-
rately for each rater (see figures). Model selection was based
on methodological as well as substantive considerations. At
the methodological level, it was based on the Bayesian Infor-
mation Criterion (BIC) and Akaike Information Criterion
(AIC) numbers and model adequacy tests, while at the sub-
stantive level, the model was selected based on parsimony
and maximum explanatory power given what is already
known about symptom change across development [29]. Further details about model selection, including model fit
statistics for the two next best fitting models, are presented
in the supplementary material (eTable 1). Separate models
were used to estimate hyperactivity–impulsivity and inat-
tention symptoms. Three logistic regression models were used to examine
early risk factors for high-symptom trajectories: one for inat-
tention, a second for hyperactivity, and a third for partici-
pants who followed high-symptom trajectories in both symp-
tom categories simultaneously. In each model, risk factors
were identified using two steps. First, we selected variables
by running bivariate logistic regressions between each pre-
dictor and the outcome (high vs. low trajectory). Variables
with p values < 0.25 were included in an initial multivariable
model (model 1). In the second step, backward selection
(variables are deleted if p ≥ 0.05) was used together with
step-by-step confounding control (model 2) [30]. Results
are presented as adjusted odds ratios. Participants with at least two data points for hyper-
activity–impulsivity and inattention for each rater were
included in the trajectory modeling (missing data patterns
are reported in eTable 2). Sample characteristics The initial sample comprised 2120 children. Children
with at least two symptom ratings for each rater (mother,
teacher, self-reports) were retained for the trajectory analysis
(n = 1374). Compared with the overall sample, these chil-
dren were more likely to be male, to come from higher SES
households, to have parents who completed high school, and
to live in intact families (Table 1). Multivariable analyses To test whether individual risk factors significantly distin-
guished among the six trajectory groups, we ran a series of
Wald-based Chi-square tests. Risk factors that were signifi-
cant at the 0.05 level were then included in a multivariable
model to identify risk factors that remained significant in the
context of multivariable analysis. Significant predictors were
again identified by Wald tests. An important limitation of
these tests is that they do not identify which trajectories were
distinguished by statistically significant risk factors. From
the perspective of developing population-based preventive
interventions, we were specifically interested in identify-
ing risk factors for following high-symptom trajectories. In
this context, groups of children with atypical (i.e., elevated)
symptom levels will be larger than groups of children with
the most extreme (i.e., clinical) symptom levels. Thus, to
identify children following persistently high symptom trajec-
tories, we combined groups 5 and 6 to create a single high-
symptom group and collapsed the remaining four trajectories
into a low-symptom group. We then repeated the risk factor
analysis within a logistic stepwise regression framework,
performed separately for the hyperactivity–impulsivity and
inattention symptom categories, then again for those chil-
dren who were following both high-symptom trajectories
simultaneously. To perform this second-stage analysis, par-
ticipants were assigned to the trajectory group they most
likely followed based on the posterior probability of group Trajectory modeling To examine the effects of missing
data on the risk factor analysis, inverse probability weight-
ings were generated (predictors of missingness were sex,
insufficient income and maternal depression) and added to
the multivariable logistic regression models as a sensitiv-
ity analysis. Variables used in the risk factor analysis had
between 1.9 and 11.6% missing data. Data were considered
missing at random, i.e., missingness is explained by other
observed variables [31]. All analyses were conducted using
Stata 14. Statistical significance was set at 0.05. Developmental trajectories of hyperactivity–
impulsivity and inattention The developmental trajectories of hyperactivity–impulsivity
and inattention symptoms are shown in Figs. 1 and 2. For
each symptom category, the overall trajectory of each group
is defined by the trajectories from three different raters. For
both symptom categories, six-group models provided the
best fit of the data based on the Bayesian information cri-
terion [28, 29] and performed well on all tests of model 1 3 3 3 European Child & Adolescent Psychiatry (2019) 28:973–983 European Child & Adolescent Psychiatry (2019) 28:973–983 977 Table 1 Comparison of
the study sample with the
representative sample
a Between 1.9 and 11.6% missing data
b P < 0.01
Characteristica
Popula-
tion sample
(n = 2120)
Study sample (n = 1374)
Sex of the child (male), no (%)
1080 (50.9%)
647b (47.1%)
Lifetime methylphenidate use, no (%)
288 (13.6%)
238b (17.3%)
IQ of the child, mean (SD)
6.20 (3.82)
6.44b (3.88)
Family socioeconomic status, mean (SD)
0.0 (1.0)
0.68b (1.0)
Maternal age at birth of first child (years), mean (SD)
25.8 (4.9)
25.9 (5.0)
Mother high school diploma or higher, mean (SD)
1778 (84.0%)
1175b (85.5%)
Mother no high school diploma, no (%)
339 (16.0%)
198 (14.4%)
Intact family, no (%)
1669 (78.7%)
1099 (80.0%)
Non-intact family, no (%)
443 (20.9%)
272 (19.8%)
Hyperactivity–impulsivity (teacher rated, 8 years), mean (SD)
2.24 (2.63)
2.14 (2.61)
Inattention (teacher rated, 8 years), mean (SD)
3.85 (3.32)
3.72 (3.34)
Fig. 1 Trajectories of hyperactivity–impulsivity symptoms from 1.5 to 17 years Table 1 Comparison of
the study sample with the
representative sample the study sample with the
representative sample
a Between 1.9 and 11.6% missing data
b P < 0.01
tion sample
(n = 2120)
Sex of the child (male), no (%)
1080 (50.9%)
647b (47.1%)
Lifetime methylphenidate use, no (%)
288 (13.6%)
238b (17.3%)
IQ of the child, mean (SD)
6.20 (3.82)
6.44b (3.88)
Family socioeconomic status, mean (SD)
0.0 (1.0)
0.68b (1.0)
Maternal age at birth of first child (years), mean (SD)
25.8 (4.9)
25.9 (5.0)
Mother high school diploma or higher, mean (SD)
1778 (84.0%)
1175b (85.5%)
Mother no high school diploma, no (%)
339 (16.0%)
198 (14.4%)
Intact family, no (%)
1669 (78.7%)
1099 (80.0%)
Non-intact family, no (%)
443 (20.9%)
272 (19.8%)
Hyperactivity–impulsivity (teacher rated, 8 years), mean (SD)
2.24 (2.63)
2.14 (2.61)
Inattention (teacher rated, 8 years), mean (SD)
3.85 (3.32)
3.72 (3.34)
Fig. Developmental trajectories of hyperactivity–
impulsivity and inattention 1 Trajectories of hyperactivity–impulsivity symptoms from 1.5 to 17 years
Fig. 2 Trajectories of inattention symptoms from 1.5 to 17 years Fig. 1 Trajectories of hyperactivity–impulsivity symptoms from 1.5 to 17 years Fig. 2 Trajectories of inattention symptoms from 1.5 to 17 years Fig. 2 Trajectories of inattention symptoms from 1.5 to 17 years 1 3 European Child & Adolescent Psychiatry (2019) 28:973–983 978 adequacy [29, 32]. Model adequacy tests for the three best
fitting models are presented in eTable 1. childhood, then remained elevated through adolescence. Overall, all six hyperactivity–impulsivity symptom trajec-
tories show a general decline from infancy to adolescence,
irrespective of the trajectory group and rater. i
A small group of children (5.2%) exhibited persistently
elevated hyperactivity–impulsivity symptoms that gradually
declined from 1.5 to 17 years. This group, hereafter referred
to as “chronic declining”, exhibited high symptom levels
from infancy into early childhood, declining moderately
in middle childhood, then remaining persistently elevated
through adolescence. A second group (16.2% of children)
also followed a high-symptom trajectory (hereafter referred
to as “chronic”) that remained relatively constant from
1.5 to 17 years. In contrast to the chronic declining group,
these children began with average symptom levels in early
infancy that rose and surpassed the chronic group in middle The inattention symptom trajectories also revealed a
chronic declining and chronic group (3.8% and 16.4% of the
sample, respectively) following a similar course to children
in the hyperactivity–impulsivity symptom trajectories. In
contrast to hyperactivity–impulsivity trajectories which gen-
erally declined across development, inattention symptoms
remained more constant from infancy through adolescence. Table 2 shows the baseline child, parent and family char-
acteristics associated with trajectory group membership. a Between 1.9 and 11.6% missing data Trajectories of hyperactivity–impulsivity
and inattention symptoms i
The multivariable logistic regression models in which all
risk factors associated with p values of < 0.25 in the initial
screening (model 1) were significant for both hyperactiv-
ity–impulsivity (Wald Χ2 = 127.78, p = 0.0001) and inatten-
tion (Wald Χ2 = 110.04, p = 0.0001) and for both models the
fit was good: p = 0.63 and p = 0.82, respectively. For the mul-
tivariable analysis with backward selection and step-by-step
confounding control (model 2) the models were significant
for hyperactivity–impulsivity (Wald Χ2 = 35.58, p = 0.0001)
and inattention (Wald Χ2 = 46.70, p = 0.0001), and the fit was
good: p = 0.75 and p = 0.77, respectively. For the analysis
of risk factors associated with the high-symptom trajec-
tories for both symptom categories, models 1 and 2 were
significant (Wald Χ2 = 93.80, p = 0.0001; Wald Χ2 = 30.29,
p = 0.0001) and the fit for both was good (p = 0.51 and
p = 0.34, respectively). Two groups of children followed high-symptom trajectories
of hyperactivity–impulsivity. A chronic declining group,
comprising 5.2% of the sample, followed a persistently high
trajectory, while a second, comprising 16.2% of the sample,
followed a chronic trajectory. There was a general decline in
symptoms across development and across raters. This is con-
sistent with reports in the literature showing that hyperactiv-
ity–impulsivity symptoms generally decrease from infancy
into adulthood [1–3, 33–36]. For inattention, two groups of children followed high-
symptom trajectories: 3.8% on a chronic declining trajec-
tory and 16.4% on a chronic trajectory. The overall trend of
symptom levels across development was more constant than
for hyperactivity–impulsivity. Previous studies of inattention
symptoms are mixed with respect to the course of inatten-
tion symptoms across development: some show symptoms
decline from infancy to early childhood [2, 3, 33, 36] and
from middle childhood to adulthood [1], others indicate
symptoms remain constant in middle childhood (7–12 years)
[34], and yet others suggest they may increase from middle
childhood to adolescence [37]. Our data show that following
an increase between 1.5 and 3.5 years, inattention symptom Male sex, prenatal alcohol exposure, low maternal edu-
cation, non-intact family, maternal depression, difficult
temperament, and low child IQ were associated with high
hyperactivity–impulsivity symptom trajectories from 1.5
to 17 years. Developmental trajectories of hyperactivity–
impulsivity and inattention 1–4), children in the high-symptom groups (groups 5 and 6)
were more likely to be male, to come from lower SES house-
holds, to have parents who did not complete high school, and
to live in non-intact families. high trajectories in both symptom categories, male sex, low
maternal education, maternal depression, and low child IQ
were significant in the final model. To examine effects of
missing data, the multivariable regression models were re-
run after including the inverse probability weightings. This
did not alter the significance of the results. Discussion This is the first study to describe the course of hyperac-
tivity–impulsivity and inattention symptoms from 1.5 to
17 years in a representative population sample [10], to
generate symptom trajectories using an innovative multi-
informant approach, and to identify a range of risk factors
independently associated with high-symptom trajectories
from infancy to adolescence. By combining symptom ratings from three informants
over three distinct developmental periods—mother ratings
for early childhood, teacher rating for middle childhood and
the participants themselves during adolescence—we provide
one of the longest follow-ups of hyperactivity–impulsivity
and inattention symptoms published to date. Across the three
rater groups, high-symptom trajectories were identified for
both symptom types indicating concordance between raters
on the children presenting with the highest symptom levels. To create a single high-symptom group in each symp-
tom category, we combined groups 5 and 6 for hyperac-
tivity–impulsivity (n = 294, 21.4% of the sample) and did
the same for inattention (n = 277, 20.2% of the sample),
which were then compared with the remaining groups (1
to 4) in the respective symptom categories. Table 3 shows
the regression models testing the association between early
risk factors and high-symptom trajectories of hyperactiv-
ity–impulsivity and inattention. Risk factors associated with
the high-symptom trajectories in both symptom categories
concurrently (n = 160, 11.6%) are shown in the final column. Early risk factors for high hyperactivity–impulsivity
and inattention symptom trajectories The Wald test comparison of risk factors that signifi-
cantly distinguished among the six trajectories of hyper-
activity–impulsivity in the multivariable model were: sex
(Χ2 = 83.8, p = 0.001), child IQ Χ2 = 14.13, p = 0.015), dif-
ficult temperament (Χ2 = 58.28, p = 0.001), and maternal
depression (Χ2 = 17.30, p = 0.004); for inattention they were:
sex (Χ2 = 71.72, p = 0.001), child IQ (Χ2 = 58.17, p = 0.001),
difficult temperament (Χ2 = 15.72, p = 0.007), paternal anti-
social behavior (Χ2 = 14.03, p = 0.015), and maternal depres-
sion (Χ2 = 12.13, p = 0.033). Developmental trajectories of hyperactivity–
impulsivity and inattention Compared to children in the low-symptom groups (groups Table 2 Child, parent and family characteristics associated with trajectories of hyperactivity–impulsivity and inattention symptoms (n = 1374)
aBetween 1 9 and 11 6% missing data
Characteristica
Hyperactivity–impulsivity
Inattention
Low (n = 1080)
Chronic (n = 222)
Chronic
declining
(n = 72)
Low (n = 1097)
Chronic
(n = 225)
Chronic
declining
(n = 52)
Sex of the child (male), no (%)
431 (39.9%)
170 (76.6%)
46 (63.9%)
459 (41.8%)
152 (59.6%)
36 (69.2%)
Lifetime methylphenidate use, no (%)
115 (10.6%)
90 (40.5%)
33 (45.8%)
85 (7.7%)
124 (48.6%)
29 (55.7%)
IQ of the child, mean (SD)
6.64 (3.93)
6.70 (3.66)
5.67 (3.60)
6.88 (3.95)
4.64 (3.06)
5.0 (3.12)
Difficult temperament
2.36 (1.53)
2.77 (1.54)
3.59 (1.97)
2.44 (1.54)
2.50 (1.65)
2.90 (2.0)
Prenatal and perinatal factors
Premature birth, no (%)
43 (4.0%)
10 (4.5%)
7 (9.7%)
43 (3.9%)
13 (5.8%)
4 (7.7%)
Low birthweight, no (%)
31 (2.9%)
4 (0.5%)
5 (6.9%)
30 (2.7%)
9 (4.0%)
1 (1.9%)
Prenatal tobacco exposure, no (%)
247 (22.9%)
71 (32.0%)
25 (34.7%)
259 (23.6%)
68 (30.2%)
16 (30.8%)
Prenatal alcohol exposure, no (%)
33 (3.1%)
9 (4.1%)
5 (6.9%)
35 (3.2%)
10 (4.4%)
2 (3.9%)
Prenatal street drug exposure, no (%)
10 (0.9%)
9 (4.1%)
2 (2.8%)
12 (1.1%)
5 (2.2%)
4 (7.7%)
Perinatal social factors
Non-intact family, no (%)
193 (17.9%)
59 (26.6%)
20 (27.7%)
200 (18.2%)
59 (26.2%)
13 (25.0%)
Insufficient income, no (%)
210 (19.4%)
61 (27.5%)
10 (13.9%)
206 (18.8%)
68 (30.5%)
17 (32.7%)
Early motherhood (≤ 21-years), no (%)
196 (18.1%)
73 (32.9%)
23 (31.9%)
198 (18.0%)
73 (32.4%)
21 (40.38%)
Low maternal education, no (%)
130 (12.0%)
52 (23.4%)
16 (22.2%)
137 (12.5%)
52 (23.1%)
9 (17.3%)
Low paternal education, no (%)
153 (14.2%)
45 (20.3%)
15 (20.8%)
160 (14.6%)
42 (18.7%)
11 (21.2%)
Postnatal family factors
Family dysfunction, mean (SD)
1.65 (1.40)
1.80 (1.53)
1.82 (1.42)
1.65 (1.44)
1.82 (1.31)
1.76 (1.39)
Hostile–reactive parenting, mean (SD)
1.0 (1.17)
1.20 (1.22)
1.32 (1.36)
1.05 (1.18)
1.06 (1.17)
1.39 (1.40)
Over protection, mean (SD)
4.52 (2.21)
4.50 (2.19)
5.02 (2.34)
4.49 (2.22)
4.81 (2.16)
4.61 (2.41)
Consequential parenting, mean (SD)
6.96 (1.38)
6.85 (1.37)
6.67 (1.39)
6.96 (1.37)
6.82 (1.37)
6.64 (1.64)
Parental self-efficacy, mean (SD)
8.99 (0.96)
8.97 (0.89)
8.86 (1.0)
8.97 (0.95)
9.06 (0.93)
8.83 (1.07)
Perceived parental impact, mean (SD)
8.52 (1.71)
8.41 (1.76)
7.76 (2.23)
8.51 (1.70)
8.29 (1.95)
8.26 (1.97)
Mother–child interaction, mean (SD)
9.67 (0.73)
9.71 (0.58)
9.73 (0.69)
9.67 (0.72)
9.78 (0.51)
9.56 (0.97)
Parental psychopathology
Maternal adolescent antisocial behav-
ior, mean (SD)
0.75 (0.88)
1.01 (1.0)
1.06 (1.07)
0.78 (0.91)
0.93 (0.94)
0.83 (0.95)
Paternal adolescent antisocial behav-
ior, mean (SD)
0.65 (0.92)
0.82 (1.02)
0.86 (0.82)
0.65 (0.91)
0.82 (1.05)
0.91 (0.96)
Maternal depression, mean (SD)
1.27 (1.21)
1.68 (1.49)
1.88 (1.56)
1.30 (1.28)
1.48 (1.23)
2.25 (1.60)
Paternal depression, mean (SD)
0.99 (0.95)
1.04 (1.91)
1.20 (1.29)
0.98 (0.94)
1.06 (0.93)
1.39 (1.37) y characteristics associated with trajectories of hyperactivity–impulsivity and inattention symptoms (n = 1374) 1 3 979 European Child & Adolescent Psychiatry (2019) 28:973–983 1–4), children in the high-symptom groups (groups 5 and 6)
were more likely to be male, to come from lower SES house-
holds, to have parents who did not complete high school, and
to live in non-intact families. Trajectories of hyperactivity–impulsivity
and inattention symptoms Male sex, prenatal street drug exposure, early
motherhood, low maternal education, maternal depression,
and low child IQ were significantly associated with high
inattention symptom trajectories. For children following 1 3 European Child & Adolescent Psychiatry (2019) 28:973–983 980 Table 3 Multiple logistic regression models predicting high-symptom trajectories of hyperactivity–impulsivity and inattention
P di t
i
ifi
t t th 0 05 l
l
i di
t d i b ld
Predictors
Hyperactivity–impulsivity
Inattention
Hyperactivity–impulsivity and
inattention
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Adjusted OR (95% CI)
Model 1
Model 2
Model 1
Model 2
Model 1
Model 2
Sex of the child (male), no (%)
4.32 (3.04–6.15) 4.32 (3.05–6.10) 2.56 (1.82–3.60)
2.66 (1.90–3.72)
4.63 (2.85–7.52) 4.78 (2.97–7.69)
IQ of the child, mean (SD)
0.93 (0.89–0.98) 0.93 (0.89–0.98)
1.0 (0.90–1.11)
0.84 (0.80–0.86)
1.06 (0.93–1.21) 0.86 (0.80–0.92)
Difficult temperament, mean
(SD)
1.19 (1.08–1.32) 1.21 (1.09–1.33) 0.83 (0.79–0.88)
0.85 (0.79–0.91)
Prenatal and perinatal factors
Premature birth, no (%)
1.64 (0.78–3.48)
1.34 (0.61–2.93)
Low birthweight, no (%)
Prenatal tobacco exposure,
no (%)
1.21 (0.80–1.81)
0.71 (0.46–1.09)
0.78 (0.45–1.33)
Prenatal alcohol exposure,
no (%)
2.60 (1.18–5.72) 2.49 (1.67–5.33) 1.42 (0.59–3.39)
2.38 (0.88–6.39)
Prenatal street drug exposure,
no (%)
1.36 (0.35–5.38)
3.87 (1.04–14.45) 3.80 (1.08–13.35) 2.06 (0.43–9.87)
Perinatal social factors
Non-intact family, no (%)
1.42 (0.89–2.23) 1.55 (1.01–2.39)
1.0 (0.63–1.60)
1.45 (0.83–2.54)
Insufficient income, no (%)
0.64 (0.40–1.05)
1.0 (0.63–1.57)
0.53 (0.29–0.98)
Early motherhood
(≤ 21-years), no (%)
1.28 (0.82–1.99)
1.73 (1.12–3.66)
1.69 (1.14–2.52)
1.49 (0.88–5.39)
Low maternal education, no
(%)
2.05 (1.22–3.42) 2.34 (1.49–3.68) 1.89 (1.14–3.15)
1.69 (1.05–2.72)
2.97 (1.63–5.39) 3.04 (1.18–5.06)
Low paternal education, no
(%)
1.09 (0.67–1.76)
0.87 (0.54–1.41)
1.21 (0.68–2.18)
Postnatal family factors
Family dysfunction, mean
(SD)
0.90 (0.79–1.04)
1.0 (0.87–1.35)
Hostile–reactive parenting,
mean (SD)
1.04 (0.90–1.21)
0.98 (0.82–1.18)
Over protection, mean (SD)
1.02 (0.94–1.19)
0.99 (0.89–1.10)
Consequential parenting,
mean (SD)
0.97 (0.86–1.10)
0.99 (0.87–1.11)
0.98 (0.84–1.44)
Parental self-efficacy, mean
(SD)
Perceived parental impact,
mean (SD)
0.93 (0.81–1.05)
0.99 (0.89–1.10)
0.97 (0.85–1.10)
Mother–child interaction,
mean (SD)
1.19 (0.91–1.54)
Parental psychopathology
Maternal adolescent antiso-
cial behavior, mean (SD)
1.10 (0.91–1.32)
1.01 (0.84–1.22)
Paternal adolescent antisocial
behavior, mean (SD)
1.09 (0.91–1.29)
1.18 (0.99–1.40)
1.08 (0.87–1.34)
Maternal depression, mean
(SD)
1.25 (1.09–1.44) 1.24 (1.10–1.41) 1.19 (1.03–1.38)
1.19 (1.05–1.35)
1.31 (1.11–1.56) 1.31 (1.14–1.53)
Paternal depression, mean
(SD)
1.11 (0.92–1.33)
1.04 (0.82–1.32) 1 3 981 European Child & Adolescent Psychiatry (2019) 28:973–983 from lower SES backgrounds could reduce the generaliz-
ability of the study findings. Trajectories of hyperactivity–impulsivity
and inattention symptoms This study does not consider
late onset of hyperactivity and inattention symptoms, so the
effect of these un-measured putative late-onset cases on the
symptom trajectories is unknown. This should be examined in
future studies. Finally, this study did not account for genetic
factors and genetic confounding (e.g., gene–environment
correlation) cannot be ruled out. Recent evidence suggests
that genetic factors may interact with exposure to pre- and
perinatal risk factors for ADHD with effects decreasing over
time [44]; this suggests that age may also need to be taken
into account when examining risk factors. Future work on
early risk factors should employ genetically informed designs,
where possible, and consider the effects of age. remain steady from early childhood to late adolescence irre-
spective of rater type. The trajectory models, when compared across the three
raters, reveal several distinct features. First, there is more
variation in the spread of trajectory groups derived from
mother than teacher or self-report ratings. This heterogeneity
could reduce overlap in scores between the raters. However,
the purpose of the present study was to identify groups of
individuals whose trajectories for one or more of the raters
but not necessarily for all differed from the others. In this
regard, it is still substantively interesting if a group were to
differ for only one of the raters. Second, there is a conspicu-
ous rise in children’s self-reported symptoms ratings in early
adolescence. This symptom pick-up could reflect an actual
increase in symptoms associated with entering middle ado-
lescence, or, though speculative, changes in the child’s sub-
jective perception of their own behavior, possibly in relation
to changing peer norms or increasing academic expectations
and school pressure. f
There is an ongoing debate within the ADHD literature
concerning the use of symptom ratings obtained from multi-
ple informants. Much of the discussion focuses on the issue
of low agreement between informants (e.g., parents and ado-
lescents) which is highly relevant in the context of clinical
diagnosis. While this issue warrants further methodological
work, the present study was not concerned with the symptom
course of ADHD per se, or the risk factors associated with
a formal diagnosis of the disorder; rather, the aim was to
describe the developmental course of hyperactivity–impul-
sivity and inattention symptoms from infancy to adolescence
in a population sample, based on the perspective of the avail-
able raters at each of these developmental periods. Trajectories of hyperactivity–impulsivity
and inattention symptoms We sug-
gest that the use of repeated ‘snapshots’ of symptoms over
time, with overlapping assessments in middle and late child-
hood, may help to more accurately capture and characterize
the developmental course of the hyperactivity–impulsivity
and inattention phenotypes [7, 8]. Early risk factors for hyperactivity–impulsivity
and inattention symptoms There is now ample evidence sup-
porting a link between pre and postnatal depression in moth-
ers and ADHD symptoms in offspring [41], but more work is
needed to unpick the psychosocial and biological—particularly
genetic—contributions which are known to interact in com-
plex ways to increase risk [42]. Low maternal education has
also been linked to increased risk of ADHD, but mechanisms
underlying this association remain uncertain [43]. Early risk factors for hyperactivity–impulsivity
and inattention symptoms Among children in this study, 21.4% followed high-symptom
trajectories of hyperactivity–impulsivity across raters, 20.2%
high-symptom trajectories of inattention across raters, and
11.6% followed both concurrently. Risk factors that distin-
guished among the six trajectory groups based on the initial
Wald tests were similar to those identified in the final logistic
stepwise regression models. These risk factors are consistent
with those reported in several previous studies [9, 38–40]. The
risk factors most consistently linked to following high-symp-
tom trajectories of hyperactivity–impulsivity and inattention,
as well as both simultaneously, were maternal depression and
low maternal education. There is now ample evidence sup-
porting a link between pre and postnatal depression in moth-
ers and ADHD symptoms in offspring [41], but more work is
needed to unpick the psychosocial and biological—particularly
genetic—contributions which are known to interact in com-
plex ways to increase risk [42]. Low maternal education has
also been linked to increased risk of ADHD, but mechanisms
underlying this association remain uncertain [43]. Among children in this study, 21.4% followed high-symptom
trajectories of hyperactivity–impulsivity across raters, 20.2%
high-symptom trajectories of inattention across raters, and
11.6% followed both concurrently. Risk factors that distin-
guished among the six trajectory groups based on the initial
Wald tests were similar to those identified in the final logistic
stepwise regression models. These risk factors are consistent
with those reported in several previous studies [9, 38–40]. The
risk factors most consistently linked to following high-symp-
tom trajectories of hyperactivity–impulsivity and inattention, A further challenge raised by the multi-rater approach
concerns whether symptom ratings made by different raters
are equally valid. Evidence from several recent studies sug-
gests that the predictive validity of adolescent self-reports is
lower than that of parent reports—at least in certain contexts
such as predicting longer-term life outcomes. This has led
to the suggestion that parent reports should be favored over
self-reports where possible [45, 46]. One explanation for
this observation is that children underestimate the severity
of their own symptoms [47]. If correct, this finding implies
that the self-rated trajectories presented in this study may
be a conservative estimate of symptoms compared to what
would be observed in the real world. as well as both simultaneously, were maternal depression and
low maternal education. Conclusions 3. Musser ED, Karalunas SL, Dieckmann N et al (2016) Attention-
deficit/hyperactivity disorder developmental trajectories related
to parental expressed emotion. J Abnorm Psychol 125:182–195. https://doi.org/10.1037/abn0000097 This study found that approximately one-fifth of children
follow relatively high symptom trajectories of hyperactiv-
ity–impulsivity and inattention, with roughly 11% follow-
ing elevated trajectories in both symptom categories simul-
taneously. Hyperactivity–impulsivity symptoms broadly
declined from 1.5 to 17 years while inattention symptoms
remained constant. A range of perinatal risk factors were
associated with following high-symptom trajectories from
infancy to adolescence. The study presents a new approach
for combining ratings from multiple sources to describe
symptom continuity and change over time. 4. DuPaul GJ (1991) Parent and teacher ratings of ADHD symp-
toms: psychometric properties in a community-based sample. J
Clin Child Psychol 20:245–253. https://doi.org/10.1207/s1537
4424jccp2003 3 j
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5. Schaughency E, McGee R, Raja SN et al (1994) Self-reported
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199402000-00004 6. Ustun B, Adler LA, Rudin C et al (2017) The World Health Organ-
ization adult attention-deficit/Hyperactivity Disorder Self-Report
Screening Scale for DSM-5. JAMA Psychiatry 74:520–526. https
://doi.org/10.1001/jamapsychiatry.2017.0298 Acknowledgements We are grateful to the parents of the children in
the Québec Longitudinal Study of Child Development (QLSCD). We
thank the Quebec Institute of Statistics (Nancy Illick, Bertrand Perron)
for data collection and management. 7. Martel MM, Markon K, Smith GT (2017) Research review: multi-
informant integration in child and adolescent psychopathology
diagnosis. J Child Psychol Psychiatry 58:116–128. https://doi. org/10.1111/jcpp.12611 Author contributions Drs Vergunst, Côté, Tremblay, Galera, and Nagin
designed the study. Drs Vergunst, Côté, and Nagin had full access to
the data and take responsibility for the integrity of the data and the
accuracy of the data analysis. Dr Vergunst conducted the statistical
analyses and drafted the manuscript. All authors contributed to critical
revision of the manuscript for important intellectual content. 8. APA (2013) Diagnostic and statistical manual of mental disorders,
5th edn. American Psychiatric Association, Washington, DC 9. Galéra C, Côté SM, Bouvard MP et al (2011) Early risk factors
for hyperactivity–impulsivity and inattention trajectories from age
17 months to 8 years. Arch Gen Psychiatry 68:1267–1275. https
://doi.org/10.1001/archgenpsychiatry.2011.138 10. Cherkasova M, Sulla EM, Dalena KL et al (2013) Developmental
course of attention deficit hyperactivity disorder and its predictors. Conclusions J Can Acad Child Adolesc Psychiatry 22:47–54 Funding This study was conducted with support from the Quebec
Health Research Fund, the Canadian Institutes of Health Research and
Canada Research Chair. The larger QLSCD study was also supported
by funding from the Québec Government’s Ministry of Health and
Ministry of Family Affaires, The Lucie and André Chagnon Founda-
tion, the Québec Health Research Fund (FQRS), The Québec Research
Fund for Society and Culture (FQRSC), Canada’s Social Science and
Humanities Research Council, the Canadian Institutes for Health
Research, the St-Justine Research Centre and the Québec Statistics
Institute (ISQ). The sponsors had no role in the design and conduct of
the study; collection, management, analysis, and interpretation of the
data; preparation, review, or approval of the manuscript; and decision
to submit the manuscript for publication. 11. Snowling M (2009) Editorial: multiple perspectives on ADHD:
implications for future research. J Child Psychol Psychiatry
50:1039–1041. https://doi.org/10.1111/j.1469-7610.2009.02145
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pid!” on epigenetics, programming and plasticity in child mental
health. J Child Psychol Psychiatry 51:113–115. https://doi.org/1
0.1111/j.1469-7610.2009.02213.x Strengths and limitations This study provides a novel approach for combining symptom
ratings from multiple sources, is one of the longest follow-ups
of hyperactivity-impulsivity and inattention symptoms con-
ducted to date, and the first to examine such a wide range of
perinatal risk factors associated with following high-symptom
trajectories over this extended period. However, the study has
several limitations. Attrition is common limitation of long-
term follow-up studies including this one. The loss of children Resolving differences in symptom ratings across multiple
informants is a longstanding problem in developmental psy-
chology and psychopathology and we do not purport to settle
the matter with the use of multi-trajectory modeling. We do,
however, view the methodology as a valuable methodologi-
cal device for combining the ratings across raters in a way
that transparently highlights commonalities and differences
in symptom ratings over time. 1 3 982 European Child & Adolescent Psychiatry (2019) 28:973–983 teachers’ ratings. J Clin Child Adolesc Psychol 53(42):197–207. https://doi.org/10.1080/15374416.2012.738456 Compliance with ethical standards 14. Frazier TW, Youngstrom EA, Naugle RI (2007) The latent
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archpsyc.62.8.896 1 3 1 3 1 3 3
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Interpregnancy intervals and perinatal and child health in Sweden: a comparison within families and across social groups
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Population Studies
A Journal of Demography Population Studies
A Journal of Demography Population Studies
A Journal of Demography ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rpst20 ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rpst20 Kieron Barclay1,2, Anna Baranowska-Rataj3, Martin Kolk2,4 and
Anneli Ivarsson3
1Max Planck Institute for Demographic Research, 2Stockholm University, 3Umeå University, 4Institute fo
Futures Studies Anneli Ivarsson3
1Max Planck Institute for Demographic Research, 2Stockholm University, 3Umeå University, 4Institute for
Futures Studies k Institute for Demographic Research, 2Stockholm University, 3Umeå University, 4Institute for
Futures Studies A large body of research has shown that children born after especially short or long birth intervals experience
an elevated risk of poor perinatal outcomes, but recent work suggests this may be explained by confounding by
unobserved family characteristics. We use Swedish population data on cohorts born 1981–2010 and sibling
fixed effects to examine whether the length of the birth interval preceding the index child influences the risk
of preterm birth, low birth weight, and hospitalization during childhood. We also present analyses stratified
by salient social characteristics, such as maternal educational level and maternal country of birth. We find
few effects of birth intervals on our outcomes, except for very short intervals (less than seven months) and
very long intervals (>60 months). We find few differences in the patterns by maternal educational level or
maternal country of origin after stratifying by the mother’s highest educational attainment. Supplementary material is available for this article at: http://dx.doi.org/10.1080/00324728.2020.1714701 Keywords: interpregnancy intervals; perinatal health; child health; low birth weight; preterm birth;
childhood hospitalization; population register data; sibling fixed effects; Sweden Keywords: interpregnancy intervals; perinatal health; child health; low birth weight; preterm birth;
childhood hospitalization; population register data; sibling fixed effects; Sweden [Submitted November 2018; Final version accepted October 2019] [Submitted November 2018; Final version accepted October 2019] have attempted to control for the shared family
environment by comparing siblings born to the
same
mother. Several
studies
in
high-income
countries
have
found
that
after
adopting
this
approach,
the
association
between
particularly
short or long birth intervals and poor perinatal out-
comes is completely removed (Ball et al. 2014;
Class et al. 2017; Hanley et al. 2017). In this study
we
use
Swedish
population
data
to
examine
whether IPI length—the time between the birth of
the next oldest sibling and the conception of the
index child—is associated with preterm birth and
low birth weight (LBW), as well as hospitalization
during various age windows of childhood. We also
examine whether different patterns are observed
among more vulnerable sections of the population,
such as children born to mothers with low levels of
education and children born to immigrant mothers. Potential differences across social groups have been
ignored in the most recent body of literature that © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://cre
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original wo Interpregnancy intervals and perinatal and child health
in Sweden: A comparison within families and across
social groups Kieron Barclay1,2, Anna Baranowska-Rataj3, Martin Kolk2,4 and
Anneli Ivarsson3
1Max Planck Institute for Demographic Research, 2Stockholm University, 3Umeå University, 4Institute for
Futures Studies Kieron Barclay1,2, Anna Baranowska-Rataj3, Martin Kolk2,4 and
Anneli Ivarsson3 pen Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.o
0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit Kieron Barclay , Anna Baranowska-Rataj , Martin Kolk & Anneli Ivarsson To cite this article: Kieron Barclay , Anna Baranowska-Rataj , Martin Kolk & Anneli
Ivarsson (2020) Interpregnancy intervals and perinatal and child health in Sweden: A
comparison within families and across social groups, Population Studies, 74:3, 363-378, DOI:
10.1080/00324728.2020.1714701 To link to this article: https://doi.org/10.1080/00324728.2020.1714701
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
View supplementary material
Published online: 13 Feb 2020. Submit your article to this journal
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View Crossmark data Population Studies, 2020
Vol. 74, No. 3, 363–378, https://doi.org/10.1080/00324728.2020.1714701 Population Studies, 2020 Vol. 74, No. 3, 363–378, https://doi.org/10.1080/00324728.2020.1714701 Introduction (2017) also found that IPIs of less than
18 months increased the risk of preterm birth,
though not the risk of LBW or SGA. Research on
infant mortality in less developed contexts using
sibling comparison models has found that short birth
intervals matter at lower levels of development but
that the negative effects are substantially weaker at
higher levels of development (Molitoris 2017; Moli-
toris et al. 2019). The focus of our study—health outcomes of chil-
dren beyond the first year of life, which we
examine by studying child hospitalization—has not
been examined in previous research. We would
expect the risk of hospitalization during childhood
to be related to birth interval length by a different
set of mechanisms from those affecting the risk of
preterm birth, LBW, and being small for gestational
age (SGA) and, in addition to adverse effects very
early in life, to also be related to the degree of par-
ental investment and attention available to each
child during childhood (Blake 1981). Having closely
spaced children, and particularly a larger number of
closely spaced children, would make it more difficult
for the parents to monitor the wellbeing of each
child, which might be related to the risk of hospitaliz-
ation from accidents as well as other diseases and ill-
nesses. Similarly, the focus on hospitalization allows
us to examine if the eventual poor perinatal out-
comes of closely spaced children have repercussions
on health later in childhood, as well as to identify
at what ages such effects are felt. Introduction Although several studies have found
that the very shortest intervals, 0–5 months, increase
the risk of preterm birth, Shachar et al. (2016) and
Class et al. (2017) also found that IPIs of less than
18 months increased the risk of preterm birth,
though not the risk of LBW or SGA. Research on
infant mortality in less developed contexts using
sibling comparison models has found that short birth
intervals matter at lower levels of development but
that the negative effects are substantially weaker at
higher levels of development (Molitoris 2017; Moli-
toris et al. 2019). has attempted to control for unobserved shared
frailty in the sibling group. (Klebanoff 2017). The logic behind this sibling com-
parison approach is that by controlling for otherwise
unobserved shared factors within the family, it is poss-
ible to isolate the effects of interval length itself, net of
risk factors shared among siblings that are potentially
correlated with the length of birth intervals. The first
known study to apply a sibling fixed effects analysis
to this research question—Ball et al. (2014), using
data from Australia—found that the association
between short IPIs (defined as 0–5 months) and the
risk of preterm birth, LBW, and SGA was almost
entirely removed after applying sibling fixed effects. This result was replicated using data from Canada
(Hanley et al. 2017). A study using data from
Sweden also found that short IPIs, again defined as
0–5 months, were no longer associated with the risk
of LBW or SGA when using a fixed effects analysis,
though the shortest IPIs did increase the odds of
preterm birth (Class et al. 2017). Similar analyses con-
ducted using data from the United States (US)
(Shachar et al. 2016; Mayo et al. 2017) have shown
that IPIs of 0–5 months are associated with the risk
of preterm birth, even after adjusting for shared
maternal frailty. A study using data from the Nether-
lands (Koullali et al. 2017) also found that an IPI of
0–5 months was associated with an increased risk of
LBW and preterm birth, though this study con-
ditioned on the mother having had a preterm birth
at parity one. Although several studies have found
that the very shortest intervals, 0–5 months, increase
the risk of preterm birth, Shachar et al. (2016) and
Class et al. Introduction A large body of work has examined how the length of
intervals between births is related to birth outcomes
and the health of the child. For the most part, this lit-
erature has consistently shown that interpregnancy
intervals (IPIs) that are particularly short (e.g. less
than 18 months) or particularly long (e.g. 60+
months) increase the risk of a range of poor out-
comes (Conde-Agudelo et al. 2006). Past studies
have also suggested that short birth intervals are
associated with poor long-term outcomes, such as
lower cognitive ability, achieving lower grades in
school, and being less likely to make subsequent edu-
cational transitions (Powell and Steelman 1990,
1993), suggesting either that there are consequent
effects of the poor perinatal outcomes or that the
short spacing between siblings also has a negative
effect on the development environment within the
household. Recently, however, a series of studies Kieron Barclay et al. 364 (Klebanoff 2017). The logic behind this sibling com-
parison approach is that by controlling for otherwise
unobserved shared factors within the family, it is poss-
ible to isolate the effects of interval length itself, net of
risk factors shared among siblings that are potentially
correlated with the length of birth intervals. The first
known study to apply a sibling fixed effects analysis
to this research question—Ball et al. (2014), using
data from Australia—found that the association
between short IPIs (defined as 0–5 months) and the
risk of preterm birth, LBW, and SGA was almost
entirely removed after applying sibling fixed effects. This result was replicated using data from Canada
(Hanley et al. 2017). A study using data from
Sweden also found that short IPIs, again defined as
0–5 months, were no longer associated with the risk
of LBW or SGA when using a fixed effects analysis,
though the shortest IPIs did increase the odds of
preterm birth (Class et al. 2017). Similar analyses con-
ducted using data from the United States (US)
(Shachar et al. 2016; Mayo et al. 2017) have shown
that IPIs of 0–5 months are associated with the risk
of preterm birth, even after adjusting for shared
maternal frailty. A study using data from the Nether-
lands (Koullali et al. 2017) also found that an IPI of
0–5 months was associated with an increased risk of
LBW and preterm birth, though this study con-
ditioned on the mother having had a preterm birth
at parity one. Previous empirical research Until very recently, the overwhelming body of evi-
dence has demonstrated convincingly that short
IPIs are bad for the health of the child as well as
the mother. For example, a meta-analysis of 67
studies by Conde-Agudelo et al. (2006) found that
short and long intervals were associated with poor
outcomes in high-income countries as well as low-
income countries. In both high- and low-income set-
tings, both short and long intervals were found to
be associated with the risk of poor outcomes such
as preterm birth, LBW, and SGA, while short birth
intervals were also associated with even more
severe outcomes, such as perinatal mortality, in
low-income contexts (Conde-Agudelo et al. 2006). A further meta-analysis by Conde-Agudelo et al. (2007) showed that particularly short and long birth
intervals were also associated with risks to maternal
health. On the strength of this evidence, the World
Health Organization (WHO) has issued universal
recommendations that potential mothers should
wait at least 24 months after the previous birth
before conceiving again (WHO 2006). These studies applying a within-mother comparison
approach have also found varying patterns in regard
to the very longest IPIs, usually defined as 60+
months. The Swedish study (Class et al. 2017) found
that these longest IPIs increased the odds of LBW,
preterm, and SGA births, while the Canadian study
found that the longest IPIs increased the odds of LBW
but not other outcomes, and the Australian study
found that they increased the odds of SGA (Ball et al. 2014; Hanley et al. 2017). The Centers for Disease
Control and Prevention in the US have suggested that
more research is needed to fully understand the
relationship between IPI length and health risks for
both the mother and the child (Copen et al. 2015). In the past five years, however, a series of studies on
the effects of birth spacing, comparing siblings who
are discordant on birth interval length, have called
these
long-standing
conclusions
into
question Interpregnancy intervals and perinatal and child health in Sweden 365 related to the physical adaptations that women
undergo when they first become pregnant (Zhu
et al. 1999). A long interval may lead to a physiologi-
cal transformation for the mother back to the physical
state of a woman who has not yet experienced a preg-
nancy, meaning that the mother is less physically
primed for childbearing. Previous empirical research This theory may explain
why both firstborn children and children born after
long intervals may be more likely to be born
preterm or LBW, because in neither case is the
mother physically primed for childbearing (Kramer
1987; Conde-Agudelo et al. 2006). Second, social
and environmental mechanisms that are relevant to
the risk of hospitalization essentially revolve around
sibling competition for finite parental resources,
where short birth intervals are expected to lead to
less parental attention and supervision for each child. A related body of research focusing on adult
health and mortality (Barclay and Kolk 2018) and
the educational and socio-economic consequences
of short birth intervals for outcomes later in life
(Powell and Steelman 1990, 1993; Buckles and
Munnich 2012; Barclay and Kolk 2017) has examined
birth intervals with varying results. Typically, adverse
effects, such as lower grades or lower educational
attainment, are found in studies not adequately con-
trolling for family background (Powell and Steelman
1990, 1993), but these negative effects disappear in
studies applying sibling comparisons (Barclay and
Kolk 2017, 2018). However, a study using sibling
comparisons and data from Ethiopia, India, Peru,
and Vietnam found that short spacing was associated
with lower height at age one (Miller and Karra 2017). Our examination of childhood health and hospitaliz-
ation bridges the divide between previous research
on perinatal outcomes and previous research focus-
ing on adult outcomes in high-income countries, by
examining whether birth interval lengths lead to
negative
consequences
in
the
sensitive
years
between birth and age ten, which themselves have
been shown to be a critical period for later life
health and socio-economic outcomes (Blackwell
et al. 2001; Palloni 2006; Haas 2008). Finally, selection and confounding mechanisms refer
to the fact that IPI length is not randomly distributed
in the population. For example, in the US, short birth
intervals are particularly likely to be unintended and
to be found among socio-economically and socio-demo-
graphically disadvantaged groups, such as teenage
mothers
and
racial
or
ethnic
minority
groups
(Gemmill and Lindberg 2013). However, short intervals
are also common among socio-economically advan-
taged mothers who delay first childbearing to older
ages and need to reduce birth interval length in order
to achieve desired fertility (Gemmill and Lindberg
2013). Long birth intervals may also be a consequence
of difficulty conceiving and therefore linked to lower
underlying fecundity and maternal health. Previous empirical research As a result,
it is important to adjust for all factors that are shared
among siblings in the sibling group, in order to try to
isolate the effects of birth intervals net of confounding
factors. As already discussed, when this approach is
applied, the long-standing conclusions regarding the
negative effects of short and long birth intervals are no
longer so clear (Ball et al. 2014; Shachar et al. 2016;
Barclay and Kolk 2017, 2018; Class et al. 2017; Hanley
et al. 2017; Koullali et al. 2017; Molitoris et al. 2019). Potential mechanisms linking interval length to
poor outcomes Although the focus of our study is not to identify or
evaluate the mechanisms that may link IPI length
to perinatal outcomes and child health, a brief
review of these potential mechanisms is valuable, in
order to contextualize the debate over whether the
length of birth intervals should matter or not for
child outcomes. Broadly speaking there are three
groups of explanations that may account for an
association between IPI length and child outcomes:
(1) physiological mechanisms; (2) social and environ-
mental mechanisms; and (3) selection and confound-
ing (Conde-Agudelo et al. 2012; Barclay and Kolk
2018). First, physiological mechanisms that may be
particularly
important
in
the
Swedish
context
include maternal nutrient depletion, folate depletion,
and
physiological
regression. Maternal
nutrient
depletion and folate depletion essentially refer to a
lack of recovery time between pregnancies, which
may mean that the foetus does not have access to
all of the resources needed to develop adequately
(Smits and Essed 2001; Conde-Agudelo et al. 2012). The physiological regression theory is related
to the risks associated with very long IPIs, and is Key contributions of this study In this study, we aim to extend the literature on the
association between IPI length and child outcomes
in two key ways. First, the most recent studies on
this topic applying a sibling comparison design have
focused on identifying the main effects of birth inter-
vals on perinatal outcomes, and have ignored the
potential for differences across social groups, such as
by maternal educational level or among children
born to immigrant mothers. Our first key contribution Kieron Barclay et al. 366 research has indicated that East African immigrants
in Sweden are more likely to experience longer
delays
in
establishing
contact
with
healthcare
centres during pregnancy, as well as to face verbal
miscommunication due to lack of interpreters at
healthcare centres, among other suboptimal factors
(Essén et al. 2002). Previous research has also docu-
mented differences in the risk of vitamin deficiencies,
which can critically affect the development of the
foetus (Sääf et al. 2011). Furthermore, potential
incompatibility between the diet in the country of
origin and the availability of food items in Sweden,
as well as ethnocultural dietary norms and practices
related to pregnancy, could potentially lead to food
choices that have detrimental health effects (Ahlqvist
and Wirfält 2000; Higginbottom et al. 2014). Given
that short IPIs can lead to maternal nutrient
depletion (Smits and Essed 2001), disparities of this
kind may magnify potential differences in negative
effects of birth spacing between the children of
mothers originating from different countries. is to examine whether the association between IPI
length and perinatal and child health outcomes
varies between these salient social groups. Specifically,
we examine whether the patterns differ between
mothers with tertiary education and mothers with
less than tertiary education, and also between children
born to: (1) native-born Swedish mothers; (2) immi-
grant mothers who were born in the other EU-15
nations, Norway, Switzerland, and non-European
OECD countries; (3) immigrant mothers born in
Central and Eastern Europe; and (4) immigrant
mothers born anywhere else in the world. Given that immigrant groups make up only a small
proportion of the population, negative effects of short
birth intervals among this more vulnerable section of
the population could be subsumed by the lack of an
association in the native-born population in a pooled
analysis of the full population. Key contributions of this study Furthermore, from pre-
vious research we know that immigrant mothers and
those with low levels of education, even net of the
overlap between the two groups, experience worse
birth outcomes, with an increased risk of preterm
birth and SGA (Rasmussen et al. 1995; Gissler et al. 2003; Luo et al. 2006); however, it should be noted
that the differences observed between native-born
Swedish mothers and immigrant mothers are smaller
than the differences observed between native-borns
and immigrants in many other countries (Bollini
et al. 2009). For example, some earlier studies in
Sweden
have
reported
negligible
differences
between immigrants and Swedes for severe birth out-
comes, such as perinatal death, though this might be
explained by the relative rarity of such cases
(Smedby and Ericson 1979; Oldenburg et al. 1997). We also know from previous research that edu-
cational attainment and country of origin are associ-
ated with health behaviours such as smoking and
alcohol
consumption
(Cnattingius
et
al. 1992;
Moussa et al. 2010; Urquia et al. 2013), which
greatly increase the risks of poor perinatal outcomes
(Cnattingius 2004) and health outcomes for children
(Wisborg et al. 1999; Davidson et al. 2010). These
differences in health behaviours also vary according
to the region of origin of immigrants, which is part
of the reason for stratifying our analyses. Our second key contribution is that we examine a
series of outcomes that have not been examined in
the previous literature: whether the risks of hospital-
ization during several age windows of childhood are
affected by the length of the birth interval between
siblings. We expect the risk of hospitalization from
different causes to vary by the age of the child, and
therefore we examine the risk of hospitalization in
relation to birth interval length in the first year of
life and at ages 1–3, 4–6, and 7–10. We argue that
this broader focus on health beyond the first year
of life makes an important contribution to under-
standing whether and how birth intervals have
long-term negative effects on individuals. (
y
g
)
Better educated mothers and those born in
Sweden may have more resources to monitor their
own health as well as that of their child, both
during pregnancy and afterwards, and to adopt com-
pensatory behaviours that reduce any potential nega-
tive effects of short IPIs. Key contributions of this study Part of the explanation for
these differences in birth outcomes is that mothers
from immigrant groups and mothers with lower
levels of education are more likely to suffer from
general socio-economic disadvantage and the conco-
mitant negative health effects (Westerling and Rosén
2002; Wiking et al. 2004; Torssander and Erikson
2009). Research has also suggested that mothers
from immigrant groups or with lower levels of edu-
cation face more barriers in taking full advantage
of prenatal care opportunities (Essén et al. 2002;
Heaman et al. 2013), and for some immigrant
groups there are also socio-cultural differences in
what are considered to be acceptable practices
during pregnancy (Essén et al. 2002). For example, Independent variable: Interpregnancy
intervals (IPIs) We calculate the number of months between the date
of birth of the earlier-born sibling and the date of
conception of the next sibling. Date of conception
is based on information on gestational age at birth
available in the Medical Birth Register. It is assessed
according to maternal reports on last menstrual
period and clinical judgment by the attending paedia-
trician (Socialstyrelsen 2003). IPIs are categorized as
0–6 months, 7–12 months, 13–18 months, 19–24
months (the reference category), 25–30 months, 31–
36 months, 37–42 months, 43–48 months, 49–54
months, 55–60 months, and >60 months. Data In this study, we use data available at the Umeå
SIMSAM Lab combining information from several
administrative registers in Sweden (Lindgren et al. 2016), specifically, the Multigenerational Register, Interpregnancy intervals and perinatal and child health in Sweden 367 the Medical Birth Register, and the National Patient
Register. The Multigenerational Register and the
Medical Birth Register include information on demo-
graphic events, most importantly the births of siblings
and the social background of children and their
parents. The National Patient Register provides
measures on all in-hospital care with respect to the
dates of admission and discharge. We select cohorts
of children born in Sweden between 1981 and 2010. For these cohorts, we can access all the relevant
maternal and child characteristics during pregnancy
and birth. Our primary estimation strategy is based
on implementing a sibling fixed effects approach,
which requires variance within the sibling group:
one-child families do not have an IPI, and there is
only one IPI in a two-child family. Therefore, we
exclude families with only one or two children. The
distribution of sibling group size in our data can be
seen in Figure S1. We also exclude firstborn children
because the preceding IPI is undefined for firstborns. Finally, we exclude families with multiple births and
children in blended families who have any half-sib-
lings. We exclude blended families because we want
to ensure that parents’ attention and investment is
focused on their own biological children rather than
any other children they might have, as this could otherwise confound our results. Overall, taking into
account these various exclusion criteria, we estimate
sibling fixed effects models based on 499,339 children
from 243,906 families. The flow chart in Figure 1 illus-
trates our analytical sample selection process. Independent variable: Interpregnancy
intervals (IPIs) Independent variable: Interpregnancy
intervals (IPIs) Low birth weight. Infants with birth weight less than
2,500 g are classified as children with LBW. Hospitalization. Based on data on the dates of
admission and discharge from the National Patient
Register, which includes all inpatient care in Sweden
(Ludvigsson et al. 2011), we created four binary indi-
cators of hospitalization, for the first year of life and
ages 1–3, 4–6, and 7–10. These indicators take a
value of zero if a child was not hospitalized for at
least one day at a specific age or a value of one if a
child was hospitalized at least once at that age. For our analysis of the pooled population, we con-
trast the results from the fixed effects models with the
results from ordinary least squares (OLS) models on
binary outcomes (i.e. linear probability models),
with the standard errors adjusted for clustering at
the sibling group level. For each of our six outcome
variables, we estimate the following two models: yi = a + b1 IPIi + b2 Sexi + b3 Birth Orderi
+ b4 MatAgei + b5 Size + 1
(1)
yij = aj + b1 IPIij + b2 Sexij
+ b3 Birth Orderij + b4 Mat Ageij + 1
(2) Dependent variables We consider a wide range of outcome variables
measuring health at birth and during the first ten Figure 1
Flow chart illustrating the analytical sample selection process. Note: Our analytical sample varies slightly between different analyses because of variation in the availability of complete data
on each of the six outcome variables, but the total number of unique individuals studied across all six outcomes that we
examine is 499,341. Figure 1
Flow chart illustrating the analytical sample selection process. Figure 1
Flow chart illustrating the analytical sample selection process. Note: Our analytical sample varies slightly between different analyses because of variation in the availability of complete data
on each of the six outcome variables, but the total number of unique individuals studied across all six outcomes that we
examine is 499,341. Kieron Barclay et al. 368 years of a child’s life: preterm birth, LBW, and hospi-
talization during the first ten years of life. sharing the same mother and father are treated as
repeated observations from the same family. The
choice of methodological approach was motivated
by the fact that biological siblings experience a
similar childhood environment in a way that a ran-
domly selected pair of individuals does not, and
introducing the sibling fixed effect allows us to
adjust for that shared environment. In addition, the
same family-specific factors that determine IPI
length may affect the risk of both adverse birth out-
comes and children’s health problems leading to hos-
pitalization. By using fixed effects sibling models, we
control for all shared family-specific factors, includ-
ing unobserved factors, which might otherwise bias
our estimates. This allows us to estimate the net
effect of IPI length on the various outcome variables
that we examine. Preterm births. Based on gestational age, we dis-
tinguish the following categories of preterm birth:
extremely
preterm
(less
than
28
weeks),
very
preterm (28–31 weeks), and moderate preterm (32–
36 weeks). Births after 37 completed weeks of preg-
nancy are considered as births at term. Stratified analyses (1) In this study we also examine how patterns of perina-
tal outcomes and childhood hospitalization by inter-
pregnancy intervals vary across children born to
mothers with different levels of education and differ-
ent countries of origin. Specifically, we first examine
whether the patterns differ between mothers with ter-
tiary education and mothers with less than tertiary
education, as defined by the highest level of education
achieved by 2010. Second, we examine whether the
patterns differ between children born to: (1) native-
born Swedish mothers (84 per cent of the analytical
sample); (2) immigrant mothers from the other EU-
15 nations (Austria, Belgium, Denmark, Finland,
France, Germany, Greece, Ireland, Italy, Luxem-
bourg, the Netherlands, Portugal, Spain, and the
United Kingdom), Norway, Switzerland, and non-
European OECD countries (4 per cent of the analyti-
cal population); (3) immigrant mothers from Central
and Eastern Europe (4 per cent of the analytical popu-
lation); and (4) immigrant mothers from the rest of the
world (8 per cent of the analytical population). (2) where yij is the outcome for individual i in sibling
group j for each of our six outcome variables. In
Model (1) we use a linear regression model to
examine how the IPI length experienced by individ-
ual i is associated with each outcome variable, adjust-
ing for sex, birth order, maternal age at time of birth,
and sibling group size, all of which covary with IPI
length and the various outcomes that we study. IPI
is entered into the model as a series of eleven
dummy variables based on six-month categories for
IPI length, from 0–6 months up to >60 months. In
Model (1), our analysis population is second- and
later-born children in sibling groups with at least
three
children;
that
is,
we
exclude
firstborns
because there is no value for the length of the preced-
ing interval. In Model (2), we introduce the sibling
fixed effect αj and remove the control for sibling
group size because that is adjusted for in the fixed
effects approach. We use the same analysis sample
for Model (2) as for Model (1). Preterm birth The estimates for the relationship between IPIs and
the probability of LBW are shown in Figure 2. Please take care to note that the y-axis scale varies
between panels (a), (b), and (c) across Figures 2–7. Full results tables with detailed output for the
results underlying Figure 2 can be found in Tables
S7–S9 in the supplementary material; these tables
also include information from F-tests, where we test
the joint significance of including the length of IPIs
in our regression models. The results from our models examining the relation-
ship between IPIs and the probability of preterm
birth are shown in Figure 3. Full results tables with
detailed output for the results underlying Figure 3
can be found in Tables S10–S12 in the supplementary
material. Panel (a) shows that estimates from both
the OLS and fixed effects models indicate an
increased risk of preterm birth for children born
after IPIs of less than 13 months and >60 months
relative to the reference category of 19–24 months. Relative to the baseline probability (0.035), the rela-
tive probability after an IPI of 0–6 months is 46.6 per
cent higher, and the relative probability after an IPI
of >60 months is 17.0 per cent higher. Panel (b)
shows that the increased probability of preterm
birth after short IPIs is similar regardless of the
mother’s educational level, but for long intervals is
only observed among mothers with less than tertiary
education. Panel (c) of Figure 3 shows point esti-
mates
indicating
an
increased
probability
of
preterm birth after an IPI of >60 months among chil-
dren born to all mothers, with the exception of those
originating from Central and Eastern Europe. Panel (a) in Figure 2 contrasts the results from the
within-family sibling comparison (i.e. Model 2, the
fixed effects model) with those from the regular
OLS models (Model 1) that do not adjust for unob-
served factors that are correlated with both birth
interval length and the risk of LBW. Panel (a)
shows that both the sibling comparison and OLS
models
indicate
that
IPIs
shorter
than
seven
months are associated with an increase in the prob-
ability of LBW. Indeed, the fixed effects models
show that the probability is 0.005 higher relative to
the reference category. Taking the baseline prob-
ability (0.019) into account, this is a relative increase
in the probability of LBW of 25.2 per cent. Descriptive statistics Descriptive statistics Table 1 shows summary statistics for the six main out-
comes that we focus on in this paper: LBW, preterm
birth, SGA, and hospitalization before the first birth-
day and at ages 1–3, 4–6, and 7–10. Detailed descrip-
tive tables can be seen in the supplementary material,
in Tables S1–S6. As can be seen in Table 1, the inci-
dence
of
LBW,
preterm
birth,
hospitalization
before the first birthday, and hospitalization at ages
4–6 and 7–10, is highest among children with IPIs
of less than 13 months (particularly less than seven
months). The incidence of LBW, preterm birth, and
hospitalization before the first birthday is also elev-
ated among children born after IPIs of >60 months. For hospitalizations at ages 1–3, the incidence is
highest among children born after the shortest IPIs,
but is not elevated for children born after the
longest IPIs of more than five years. Panel (c) shows the results stratified by maternal
country of origin. These show some notable within-
immigrant-group differences in the effects of short
birth intervals. For example, children born after
especially short IPIs (i.e. less than seven months)
to mothers from Central and Eastern Europe are
significantly more likely to be born with LBW
than other children born to mothers from Central
and Eastern Europe (after longer IPIs). However,
given the overlapping confidence intervals, we
cannot say that there are statistically significant
between-immigrant-group differences in the nega-
tive effects of especially short or especially long
birth intervals. Methods For our analyses of children born to mothers by
country of origin and educational level, we present
the results from only our fixed effects models Our primary estimation strategy is based on sibling
fixed
effects
models,
where
biological
children Interpregnancy intervals and perinatal and child health in Sweden 369 long IPIs are associated with any significantly
increased risk of LBW. long IPIs are associated with any significantly
increased risk of LBW. (Model (2)). In these analyses we run separate models
by mother country of origin and educational level. (Model (2)). In these analyses we run separate models
by mother country of origin and educational level. Panel (b), which is based on fixed effects sibling
comparison models, shows the results stratified by
maternal educational level. It shows that among
mothers with less than tertiary education, IPIs both
shorter than seven months and longer than 60
months are associated with an increased risk of
LBW. Hence, our results indicate that the negative
effects of very short and very long intervals shown
in the fixed effects estimates in panel (a) appear
somewhat more common for children with a more
disadvantaged parental background. Preterm birth However,
the sibling fixed effects models do not indicate that 370
Kieron Barclay et al. Table 1
Summary statistics for low birth weight (LBW), preterm birth, and hospitalization before first birthday and at ages
1–3, 4–6, and 7–10, by length of preceding interpregnancy interval (IPI): children born in Sweden 1981–2010 Table 1
Summary statistics for low birth weight (LBW), preterm birth, and hospitalization before first birthday and at ages
1–3, 4–6, and 7–10, by length of preceding interpregnancy interval (IPI): children born in Sweden 1981–2010
IPI length
(months)
LBW
(percentage)
Preterm
(percentage)
Hospitalized
aged 0
(percentage)
Hospitalized
aged 1–3
(percentage)
Hospitalized
aged 4–6
(percentage)
Hospitalized
aged 7–10
(percentage)
0–6
2.9
5.8
12.1
19.2
10.1
9.0
7–12
2.9
3.8
10.3
18.0
9.8
8.6
13–18
1.9
3.1
10.2
17.5
9.8
8.3
19–24
1.5
3.1
10.1
17.6
9.7
8.4
25–30
1.7
3.1
10.0
17.3
9.5
8.2
31–36
1.7
3.4
10.0
18.1
9.3
7.9
37–42
2.0
3.3
10.6
18.0
8.8
7.7
43–48
1.9
3.4
10.6
18.1
8.6
7.5
49–54
1.8
3.4
10.7
18.4
8.2
7.4
55–60
2.0
3.8
10.9
18.4
8.6
7.1
>60
2.2
4.5
12.1
17.9
8.3
6.6
All children
1.9
3.5
10.5
17.8
9.3
8.0
Note: Further details available in the supplementary material. Source: Swedish register data; authors’ own calculations. Note: Further details available in the supplementary material. Source: Swedish register data; authors’ own calculations. found in Tables S13–S15 in the supplementary
material. Panel (a) contrasts the results from our
fixed effects models with the regular OLS models
on the same sample population. The between-
family comparison shows an elevated probability of
hospitalization before age one for those born after
IPIs of less than seven months relative to the Hospitalization before the first birthday Interpregnancy intervals and perinatal and child health in Sweden
371 371 −0.010
0.000
0.010
0.020
0.030
Sibling comparison
Between−family comparison
a
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.040
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of preterm birth (<37 weeks gestation)
Preceding interpregnancy interval (months) −0.010
0.000
0.010
0.020
0.030
Sibling comparison
Between−family comparison
a
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.040
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of preterm birth (<37 weeks gestation)
Preceding interpregnancy interval (months)
Figure 3
Relationship between IPI length and the probability of preterm birth: (a) in the pooled sample (OLS
vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by maternal immigrant
status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Probability of preterm birth (<37 weeks gestation) Tertiary Preceding interpregnancy interval (months) Figure 3
Relationship between IPI length and the probability of preterm birth: (a) in the pooled sample (OLS
vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by maternal immigrant
status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. reference category, but no other meaningful relative
differences. The
fixed
effects
model,
however,
shows that short IPIs are barely related to the prob-
ability of hospitalization, but IPIs of 43 months or
longer decrease the risk of hospitalization. For
example, relative to the baseline probability (0.105),
the relative probability of hospitalization after an
IPI of >60 months is 31.1 per cent lower. The results
shown in panel (b) support the conclusion that this
is consistent regardless of maternal educational
level. Panel (c), however, suggests that this pattern
is visible only for Swedish mothers and those from
non-OECD and non-European countries. are associated with an increased probability of hospi-
talization at these ages. Hospitalization before the first birthday For example, relative to the
baseline probability (0.178), the relative probability
of hospitalization after an IPI of >60 months is 9.0
per cent higher. Panel (b) shows that the pattern
observed in the pooled sibling comparison analysis
is consistent regardless of maternal educational
level, while panel (c) suggests that this pattern is
driven by Swedish mothers rather than mothers
born outside Sweden. Hospitalization at ages 4–6 The results from our analyses of the relationship
between the IPI length and hospitalization at ages
4–6 are shown in Figure 6. Full results tables with
detailed output for the results underlying Figure 6
can be found in Tables S19–S21 in the supplemen-
tary material. Panel (a) in Figure 6 shows that the
association between IPI length and the probability
of hospitalization is weaker at ages 4–6 than at
ages 1–3, though there is some evidence that
shorter intervals, as well as IPIs of >60 months,
increase
the
probability
of
hospitalization. For
example,
relative
to
the
baseline
probability
(0.093), the relative probability of hospitalization Hospitalization before the first birthday Figure 4 shows the results for our first analyses of
health outcomes beyond those measured directly
after birth, focusing on hospitalization during the
first year of life. Full results tables with detailed
output for the results underlying Figure 4 can be −0.005
0.000
0.005
0.010
0.015
Sibling comparison
Between−family comparison
a
−0.010
−0.005
0.000
0.005
0.010
<Tertiary
Tertiary
b
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of low birth weight (<2,500 g)
Preceding interpregnancy interval (months) −0.005
0.000
0.005
0.010
0.015
Sibling comparison
Between−family comparison
a
−0.010
−0.005
0.000
0.005
0.010
<Tertiary
Tertiary
b
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of low birth weight (<2,500 g)
Preceding interpregnancy interval (months)
Figure 2
Relationship between IPI length and the probability of low birth weight: (a) in the pooled sample
(OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by maternal
immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. Reference category is an IPI of 19–24 months. Source: Swedish register data; authors’ own calculations. Preceding interpregnancy interval (months) Figure 2
Relationship between IPI length and the probability of low birth weight: (a) in the pooled sample
(OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by maternal
immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. Reference category is an IPI of 19–24 months. Source: Swedish register data; authors’ own calculations. Figure 2
Relationship between IPI length and the probability of low birth weight: (a) in the pooled sample
(OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by maternal
immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. Reference category is an IPI of 19–24 months. Source: Swedish register data; authors’ own calculations. Hospitalization at ages 1–3 Figure 5 shows the results from models examining
the relationship between the length of the preceding
IPI and hospitalization at ages 1–3. Full results tables
with detailed output for the results underlying Figure
5 can be found in Tables S16–S18 in the supplemen-
tary material. The between-family comparisons in
panel (a) show that both very short IPIs and longer
IPIs are associated with an increased probability of
hospitalization at ages 1–3, while the fixed effects
results show that only IPIs of 31 months or longer 372
Kieron Barclay et al. −0.040
−0.020
0.000
0.020
Sibling comparison
Between−family comparison
a
−0.060
−0.040
−0.020
0.000
0.020
<Tertiary
Tertiary
b
−0.100
−0.050
0.000
0.050
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at age 0
Preceding interpregnancy interval (months)
Figure 4
Relationship between IPI length and the probability of hospitalization before first birthday: (a) in the
pooled sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c)
by maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. 372
Kieron Barclay et al. −0.040
−0.020
0.000
0.020
Sibling comparison
Between−family comparison
a
−0.060
−0.040
−0.020
0.000
0.020
<Tertiary
Tertiary
b
−0.100
−0.050
0.000
0.050
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at age 0
Preceding interpregnancy interval (months) Preceding interpregnancy interval (months) Figure 4
Relationship between IPI length and the probability of hospitalization before first birthday: (a) in the
pooled sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c)
by maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Hospitalization at ages 1–3 −0.010
0.000
0.010
0.020
Sibling comparison
Between−family comparison
a
−0.020
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.100
−0.050
0.000
0.050
0.100
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 1−3
Preceding interpregnancy interval (months) −0.010
0.000
0.010
0.020
Sibling comparison
Between−family comparison
a
−0.020
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.100
−0.050
0.000
0.050
0.100
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 1−3
Preceding interpregnancy interval (months)
Figure 5
Relationship between IPI length and the probability of hospitalization at ages 1–3: (a) in the pooled
sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Preceding interpregnancy interval (months) Figure 5
Relationship between IPI length and the probability of hospitalization at ages 1–3: (a) in the pooled
sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Interpregnancy intervals and perinatal and child health in Sweden 373 −0.005
0.000
0.005
0.010
0.015
Sibling comparison
Between−family comparison
a
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.040
−0.020
0.000
0.020
0.040
0.060
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 4−6
Preceding interpregnancy interval (months) −0.005
0.000
0.005
0.010
0.015
Sibling comparison
Between−family comparison
a
−0.010
0.000
0.010
0.020
0.030
<Tertiary
Tertiary
b
−0.040
−0.020
0.000
0.020
0.040
0.060
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 4−6
Preceding interpregnancy interval (months)
Figure 6
Relationship between IPI length and the probability of hospitalization at ages 4–6: (a) in the pooled
sample (OLS vs. Hospitalization at ages 7–10 The results for our analyses of hospitalization at later
childhood ages are consistent with the weakening
relationship between the length of IPIs and prob-
ability of hospitalization at ages 4–6. Figure 7 shows
that there are no clear patterns of hospitalization
by the IPI length in either the pooled sample, by
maternal educational level, or by the country of
origin of the mother. Full results tables with detailed
output for the results underlying Figure 7 can be
found in Tables S22–S24 in the supplementary
material. Hospitalization at ages 1–3 fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Preceding interpregnancy interval (months) Preceding interpregnancy interval (months) Figure 6
Relationship between IPI length and the probability of hospitalization at ages 4–6: (a) in the pooled
sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. Supplementary analyses after an IPI of >60 months is 7.8 per cent higher. The
results from models stratified by maternal edu-
cational level, in panel (b), show that among chil-
dren born to mothers with a tertiary education, the
probability of hospitalization is higher for those
born after IPIs of >60 months. We do not observe
those
within-group
differences
among
children
born to mothers with less than a tertiary education. The results shown in panel (c) do not allow us to
infer that there are significant differences across
immigrant groups in the effects of very long IPIs
on the probability of hospitalization at ages 4–6. We also conducted a number of supplementary ana-
lyses. Previous research has shown that children born
‘early-term’ (37–38 weeks of gestation) experience
relatively worse long-term health outcomes than chil-
dren born ‘late-term’ (39+ weeks). We found that
children born after intervals shorter than 19 months
were particularly less likely to be born late-term,
and this pattern persisted regardless of the edu-
cational level or country of origin of the mother. These results can be seen in Tables S25–S27 in the
supplementary material. We also conducted several
additional analyses to check the robustness of our
results to restricting the analytical sample to families
with exactly three children (Tables S28–S33), and
with additional controls for birth month and the sex
composition of the sibling group at the time of birth
(Tables S34–S39). Those results were fully consistent
with the results presented earlier. Discussion In this study we have examined the effects of IPI
length on the probability of poor perinatal outcomes
and the risk of hospitalization during childhood, as 374
Kieron Barclay et al. −0.010
−0.005
0.000
0.005
0.010
Sibling comparison
Between−family comparison
a
−0.020
−0.010
0.000
0.010
0.020
<Tertiary
Tertiary
b
−0.060
−0.040
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 7−10
Preceding interpregnancy interval (months)
Figure 7
Relationship between IPI length and the probability of hospitalization at ages 7–10: (a) in the pooled
sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. −0.010
−0.005
0.000
0.005
0.010
Sibling comparison
Between−family comparison
a
−0.020
−0.010
0.000
0.010
0.020
<Tertiary
Tertiary
b
−0.060
−0.040
−0.020
0.000
0.020
0.040
0−6
7−12
13−18
19−24
25−30
31−36
37−42
43−48
49−54
55−60
>60
Sweden
EU−15 + Nordic + OECD
Central and Eastern Europe
Other
c
Probability of hospitalization at ages 7−10
Preceding interpregnancy interval (months) Preceding interpregnancy interval (months) Figure 7
Relationship between IPI length and the probability of hospitalization at ages 7–10: (a) in the pooled
sample (OLS vs. fixed effects models); (b) by maternal educational level (fixed effects models); and (c) by
maternal immigrant status (fixed effects models): children born in Sweden 1981–2010. Notes: Error bars are 95 per cent confidence intervals. The reference category is an IPI of 19–24 months. Source: As for Figure 2. well as how these patterns vary according to the
mother’s level of education and country of origin. adjusting for shared risk factors within the sibling
group. However, our results support those of other
recent studies using data from the US (Shachar
et al. 2016; Mayo et al. 2017) and the Netherlands
(Koullali et al. 2017), which found that very short
intervals (0–5 months) were associated with the risk
of LBW, preterm birth, or both, even after adjusting
for shared maternal frailty. Some of these studies,
such as the work using Canadian data, have also
found that very long IPIs (60+ months) increase the
risk of LBW, as our study did. Discussion Based on the results
of this study, we would like to echo the recent calls
for more research on this topic (e.g. Copen et al. 2015; Klebanoff 2017), and particularly for further
work to explain why birth intervals seem to matter
for perinatal health in some high-income contexts
but not others. Overall, we found that after controlling for shared
factors within the sibling group, IPI length does not
generally influence the probability of the child
experiencing poor perinatal outcomes. The excep-
tions to this are that very short and very long IPIs
do increase the probabilities of LBW and preterm
birth. For example, the probabilities of LBW and
preterm birth for children conceived after IPIs of
less than seven months are 25.2 and 46.6 per cent
higher, respectively, than the probabilities of LBW
and preterm birth for children conceived after IPIs
of 19–24 months. However, it should be said that
these very short IPIs are relatively uncommon,
accounting for only 2.9 per cent of intervals in our
analytical population. As a consequence of the low
prevalence of such short IPIs, the overall population
health impact of these short intervals is likely to be
small. In this study we also extended the literature by
examining health outcomes during childhood in
relation to IPI length; this had not been done
before with sibling comparison models. We examined
hospitalization during several different age windows
in the first ten years of childhood. The results from
these analyses suggest that IPI length is more impor-
tant for the probability of hospitalization before age
four. Intriguingly, our estimates suggest that longer
birth intervals are protective against hospitalization These results address the recent series of studies
that have raised questions about whether IPI length
matters for perinatal health in high-income countries. These studies have shown that very short IPIs do not
matter for the risk of low birth weight, preterm birth,
and being small for gestational age in Australia (Ball
et al. 2014) and Canada (Hanley et al. 2017) after Interpregnancy intervals and perinatal and child health in Sweden 375 in the first year of life, but that they increase the risk
of hospitalization at later ages, up to age seven. This
pattern is difficult to explain, but may be related to
medical practice norms regarding how sick infants
are treated. Discussion In Sweden doctors typically prefer a
child to be at home with the parents if at all possible,
rather than being hospitalized (Socialstyrelsen 1993;
Braveman et al. 1995; Johansson et al. 2010). Further-
more, those infants who are identified with health
problems at birth are more likely to be kept at the
hospital until the problems are solved, meaning
that this hospitalization would not be recorded as a
separate
event
from
the
hospital
birth
itself. Although we can only speculate, this might explain
why children born after very short or very long inter-
vals experience worse perinatal outcomes, but also a
lower risk of hospitalization in the first year of life. especially long birth intervals are not associated
with poor long-term educational, socio-economic,
and health outcomes in Sweden (Barclay and Kolk
2017, 2018), but it is not clear whether this previous
finding was because birth intervals did not matter
even for perinatal health outcomes in contemporary
Sweden, or whether the null finding for the long-term
effects might be due to some kind of moderating
effect of the Swedish welfare state in negating disad-
vantage early in life. Our results largely confirm pre-
vious results on the small impact of birth intervals on
children’s outcomes, though we found a substantial
negative effect of extremely short IPIs on perinatal
outcomes. Given that previous literature has shown
that LBW and preterm birth can have serious long-
term consequences for health and for educational
and
socio-economic
attainment
(Conley
and
Bennett 2000; Black et al. 2007; Swamy et al. 2008),
our study suggests that there may be a moderating,
ameliorating effect of medical, social, or environ-
mental conditions in Sweden that breaks the link
between the negative effects of extremely short
IPIs on perinatal outcomes and on poor long-term
socio-economic, educational, and health outcomes. We also extended previous research on this topic
by examining whether there are differences in the
effects of IPIs on perinatal and child health by
maternal educational level and maternal country of
origin. Overall, we did not find significant differences
in the effects of maternal educational level or country
of origin on the probability of poor perinatal out-
comes or hospitalization during childhood. Discussion Given
known differences in factors such as health beha-
viours and opportunities for navigating the health-
care system by maternal educational level and
country of origin, it is interesting that we did not
find many differences in the effects of IPI length on
perinatal outcomes across these different social cat-
egories. This suggests either that these differences
in behaviour across social groups are smaller than
believed, or that they have relatively little impact
on the risk of poor perinatal and child health out-
comes after especially short or long IPIs in a high-
income setting such as Sweden. It might also be the
case that the medical and social system in Sweden
is able to moderate such differences in maternal
health and maternal health behaviours adequately
through both prenatal and postnatal care. However,
we also note that our analyses by maternal country
of origin were underpowered, despite using the
population registers, and this may be the main
reason for our not being able to detect statistically
significant differences in child health outcomes by
maternal country of origin. Although this study has many strengths, it is also
important to acknowledge the limitations. Chief
among these is that, in order to estimate our fixed
effects models, we needed to focus on families with
at least three children because of the requirement
to observe variance in IPI length within the sibling
group. Although excluding one-child families was
unavoidable as we could not observe any birth inter-
val in this group, we also excluded two-child families,
the most common sibling group size in Sweden. There is an inevitable trade-off between the general-
izability of our findings to the full population and the
great benefit of being able to control for all unob-
served factors shared among siblings that might be
driving the relationship between IPI length and peri-
natal and child health. Given our chosen approach,
we need to be careful about generalizing our findings
to two-child sibling groups, as it is possible that the
effects of IPIs on perinatal and child health are
quite different in two-child families in comparison
to families with three or more children. Nevertheless,
we feel that this is a relatively small problem. Discussion First,
we would expect the mechanisms that could link
IPI length to perinatal and child health, such as
maternal nutrient depletion or sibling competition
for resources, to be more severe in larger sibling
groups than smaller ones. Second, by studying
families with at least three children we did still
study the majority of empirically observed IPIs in
the population, as larger sibling groups contribute Examining the effects of IPI length on childhood
hospitalization in this study also allowed us to
bridge the gap between recent research using a
sibling comparison approach on perinatal health out-
comes and research on long-term educational, socio-
economic, and health outcomes in Sweden. Previous
research has shown that even especially short and Kieron Barclay et al. 376 with support from the Swedish Research Council and
by strategic support from Umeå University. Kieron
Barclay was partly supported by a European Research
Council
grant
awarded
to
Mikko
Myrskylä
(COSTPOST:336475). far more intervals than two-child sibling groups. For
example, a four-child sibling group provides three
times as many intervals as a two-child sibling group. Another limitation of our analysis is that the fixed
effects analysis did not control for factors that vary
within the family. Although we did control for some
factors that vary within the family, such as maternal
age at birth and birth order, there may be time-
varying factors not captured by those variables. If,
for example, there are negative spillover effects of
having another sibling with LBW, this would not
necessarily have been adjusted for in our analysis. References Ahlqvist, Margary and Elisabet Wirfält. 2000. Beliefs con-
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Design of Polymer Extrusion Dies Using Finite Element Analysis
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7,200 Chapter 10 Abstract A computational fluid dynamics (CFD) model has been developed to compute the pres-
sure, temperature, velocity, viscosity and viscous dissipation in the high-density poly-
ethylene (HDPE) extrusion process. The numerical approach agrees fairly well with the
experimental data recorded during the extrusion process of the material. The extrusion
spider die was designed to produce high-density polyethylene pipes of 32 mm inner
nominal diameter and 2.4 mm thickness. In order to investigate if the spider legs are
able to perform under the pressure occurred using the maximum flow rate provided by
the single screw extruder of this study, a stress analysis was conducted on a single spi-
der leg. This fluid-structure interaction (FSI) problem was solved using the COMSOL
Multiphysics software. Finally, the results obtained from the FE analysis were applied
in the design and fabrication of the spider die, selecting IMPAX (tool steel) as fabrica-
tion material. Keywords: finite element analysis, pressure flow, HDPE, extrusion die, spider, arbitrary
Lagrangian-Eulerian (ALE) Design of Polymer Extrusion Dies Using Finite Element
Analysis G.N. Kouzilos, G.V. Seretis, C.G. Provatidis and
D.E. Manolakos
G.N. Kouzilos, G.V. Seretis, C.G. Provatidis and
D.E. Manolakos 1. Introduction The production of extruded polyethylene film, rods, tubes and pipes is a common industrial
process that has been the subject of major investigations over many years [1–3]. The designing
of extrusion dies for the production of such geometries requires a deep knowledge. It is usu-
ally based on experimental trial-and-error approaches, involving, therefore, the use of huge © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
ttribution License (http://creativecommons org/licenses/by/3 0) which permits unrestricted use distribution
© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. d use, distr se, distri Extrusion of Metals, Polymers, and Food Products 182 amounts of time and material resources [4–6]. According to manufacturers, 10–15 iterations
are required to optimize complex profile geometries [1]. The extrusion die is one of the most
important parts in extrusion processing. The extrusion die design process can become too
difficult to execute, or its cost can increase up to prohibitive levels, when complex geometry
thermoplastic profiles are concerned. Optimizing process parameter problems is routinely performed in the manufacturing indus-
try, particularly in setting final optimal process parameters. Final optimal process parameter
setting is recognized as one of the most important steps in plastics extrusion for improving
the quality of extruded products. Yilmaz et al. [7] optimized the geometric parameters of a profile extrusion die, using several
objective function definitions by Simulated Annealing-Kriging Meta-Algorithm. Objective
functions are defined based on the uniformity of velocity distribution at the die exit. For this,
computational fluid dynamics (CFD) simulations are performed for N = 70 die geometries. Appropriate geometric parameters (t and L) of the die are variables for the optimization
problem. The optimization of an extrusion die designed for the production of a wood-plastic composite
(WPC) decking profile is investigated by Gonçalves et al. [8]. The optimization was performed
with the help of numerical tools, more precisely, by solving the continuity and momentum
conservation equations that govern such a flow, and aiming to balance properly the flow dis-
tribution at the extrusion die flow channel outlet. A nonlinear optimization technique was applied by Mamalis et al. [9] to the numerical model
to pinpoint the processing conditions, namely inlet pressure, inlet temperature of the melt,
temperature of the die walls and temperature of the spider legs. The work described, hereinafter, is aiming to the development of an optimum design for
a pipe die with spider used for the extrusion of high-density polyethylene (HDPE) tubes. For this purpose, a computational fluid dynamics (CFD)-based model using the generalized
Newtonian approach was employed to investigate pressure drop, flow and temperature uni-
formity in the die. 2. Extrusion die design zones In order to determine the die pressure, that is, the pressure developed in the inner surfaces of
the die, the analytical approach, which is presented below, was used. The extrusion die was
considered to consist of five different zones. In each zone, a different stage of the extrusion
process was taking place. In zone 1, the fluid enters the die (input or inlet). In zone 2, the fluid
diverts from the extrusion axis. In this stage of the extrusion process, the distribution of the
fluid begins on the top of the mandrel cone and, subsequently, the fluid is driven to zone 3
through a ring-shaped leak. In zone 3, the fluid is leaking in the spider legs, which are fitting
in the male end of the die. A relaxation zone (zone 4) follows zone 3. The last stage is zone 5, where the pipe is being formed at its expected morphological characteristics. The extrusion
die zones are presented schematically in Figure 1. Figure 1. Zones of the extrusion die. Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
1 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
1 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 183 Figure 1. Zones of the extrusion die. where the pipe is being formed at its expected morphological characteristics. The extrusion
die zones are presented schematically in Figure 1. where the pipe is being formed at its expected morphological characteristics. The extrusion
die zones are presented schematically in Figure 1. 3. Mathematical model Extrusion of Metals, Polymers, and Food Products
184 Δ P tube = L [ 2 m (m + 3) V ̇
__________
ϕ ⋅ π ⋅ R m+3 ]
1/m
(3) (3) Δ P ring = L [ 2 m+1 (m + 2) V ̇
_____________
φ⋅ π ⋅ D ⋅ H m+2 ]
1/m
(4) (4) Δ P square = L [ 2 m+1 (m + 2) V ̇
__________
φ⋅ B ⋅ H m+2 ]
1/m
(5) (5) These equations have broad applications because the flow path in a small segment of many
extrusions dies and adaptors can be approximated by a circular tube or a slit for the purpose
of calculating pressure drop and flow rate. For a zero value of ΔP, the volumetric flow rate is
maximized. Thus, for screw speed equal to 100 rpm, the maximum volumetric flow rate can
be calculated equal to 7.9 × 10−6 m3/s. These equations have broad applications because the flow path in a small segment of many
extrusions dies and adaptors can be approximated by a circular tube or a slit for the purpose
of calculating pressure drop and flow rate. For a zero value of ΔP, the volumetric flow rate is
maximized. Thus, for screw speed equal to 100 rpm, the maximum volumetric flow rate can
be calculated equal to 7.9 × 10−6 m3/s. For this flow rate, the total drop of the pressure in the die ΔPT, including all five different
zones, is: Δ P T = Δ P Zone1 + Δ P Zone2 + Δ P Zone3 + Δ P Zone4 + Δ P Zone5 = 17.9 bar + 9.55 bar
+ 1.58 bar + 27.34 bar + 35.8 = 92.17 bar
(6) (6) If ΔPT is the total drop of the die pressure and ρ and Cp are the density and the specific heat,
respectively, then the average temperature increase at the die output (outlet), which is based
on the assumption that adiabatic conditions occur throughout the whole process, can be
expressed by Eq. (7) [2]. Δ T analytical = ΔP
_____
ρ ⋅ C p = 4.72 Κ
(7) (7) 3. Mathematical model In order to determine the pressure drop in the extrusion die, the power-law exponential
model was used, according to which the volumetric flow rate V ̇ of a non-Newtonian fluid is
described by Eqs. (1) and (2) [1, 2]. V ̇ = K ′ ⋅ ϕ ⋅ Δ P m
(1) (1) where ϕ is the fluidity, m is the flow exponent, ΔP is the pressure drop and K ′ is a die shape
constant. where ϕ is the fluidity, m is the flow exponent, ΔP is the pressure drop and K ′ is a die shape
constant. For the present work, a single-screw Johnson Plastics extruder was used to drive the flowing
high-density polyethylene into the spider die. For this kind of extruder, the volumetric flow
rate, as it has been described previously [2], is V ̇ = α ⋅ Ν −
β
__
μ ΔP
___
L
(2) (2) where α = 0.5 ⋅ π 2 ⋅ D 2 ⋅ H ⋅ sin φ⋅ cos φ , N is the screw speed, β = π
__
12 D ⋅ H 3 ⋅ sin 2 φ , μ is the melt viscosity,
L is the axial length of the screw, D is the inner barrel diameter, H is the depth of the channel
and ϕ is the helix angle of flight [10]. where α = 0.5 ⋅ π 2 ⋅ D 2 ⋅ H ⋅ sin φ⋅ cos φ , N is the screw speed, β = π
__
12 D ⋅ H 3 ⋅ sin 2 φ , μ is the melt viscosity,
L is the axial length of the screw, D is the inner barrel diameter, H is the depth of the channel
and ϕ is the helix angle of flight [10]. In the five different zones of the extrusion die, three shapes of the cross section can be found:
tube, ring shape and square shape. For these cross sections, the pressure drop is described by
Eqs. (3)–(5), respectively [1, 10]. 4. Design of the extrusion die The extrusion die was designed to produce high-density polyethylene pipes of 32 mm inner
nominal diameter and 2.4 mm thickness. The material used for the body of the extrusion die
was IMPAX steel. The extrusion die was assembled in five stages. Progressive views of the
assembly process are presented in Figure 2. A 3D view of the die along with the screw type used is given in Figure 3. Firstly, the spider head was combined with the male middle mandrel. Subsequently, the outer
mandrel and the torpedo were placed in the initial assembly. In stage 3, the die housing was
added, and in the following stage (stage 4), the middle ring was adapted to the back side of
the spider head. In the last stage (stage 5), the die ring was combined with the middle ring of
the previous stage. Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
18 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 Figure 2. Progressive view of the complete assembly process. Figure 2. Progressive view of the complete assembly process. Figure 3. 3D view of the spider extrusion die. Figure 3. 3D view of the spider extrusion die. Extrusion of Metals, Polymers, and Food Products 186 5.1. CFD analysis A three-dimensional conjugate heat transfer model, which has been developed for non-New-
tonian materials, was processed in the extrusion die. For the numerical solution, the follow-
ing consideration had been made: a homogeneous and isotropic high-density polyethylene
(HDPE) melt with a uniform temperature of T = 469 K is flowing into the spider die. The
temperature of the die surface was kept constant at the value Tw = 469 K, and the volumetric
flow rate of the polymer melt was fixed at V ̇ max = 7.9 × 10 −6 m 3 / s. In most polymer processes, the elastic memory effects can hardly be observed and, there-
fore, it can be ignored. Since this chapter is concentrated on a qualitative analysis of the flow
regimes, the inelastic model was selected as the most appropriate in terms of describing the
melt flow. Due to the polymer melts flow characteristics when it takes place in an extrusion die channel
while in steady state, the following assumptions have been made: Due to the polymer melts flow characteristics when it takes place in an extrusion die channel
while in steady state, the following assumptions have been made: • Incompressible steady laminar flow. • Since the Reynolds number of the melts’ flow is extremely low, inertial and gravitational
forces are neglected. • Since the Reynolds number of the melts’ flow is extremely low, inertial and gravitational
forces are neglected. • No slipping on the wall interface. • No slipping on the wall interface. • Uniform and constant die temperature, equal to 469 K. • Constant volumetric flow rate, equal to 7.9 × 10−6 m3/s. The inlet (input), the outlet (output) and the die wall are presented in Figure 4. Figure 4. Inner die model used in numerical analysis. Figure 4. Inner die model used in numerical analysis. Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 187 Since the polymer melts are non-Newtonian fluids, the Carreau-Yasuda model was selected
to describe dependence of the viscosity on the shear rate and temperature [3]. This model is
presented in Eq. (8). Since the polymer melts are non-Newtonian fluids, the Carreau-Yasuda model was selected
to describe dependence of the viscosity on the shear rate and temperature [3]. This model is
presented in Eq. (8). 5.1. CFD analysis n = a T ⋅ n 0 ( T R ) [1 + ( a T ⋅ λ ̇ ( T R ) γ ̇ )
α
]
(n−4) /α
(8) (8) where a T is the shift factor and n 0 , λ, α and n are model’s fitting parameters. If ∇ is the Hamilton differential operator, u is the velocity vector, T is the temperature, C p is the
heat capacity and Q is the total source term, the governing equations of the model used can be
written in the form below [2, 3]: Continuity equation: ∇u = 0
(9) (9) Motion equation: ∇σ = 0
(10) (10) Energy equation: ρ ⋅ C P ⋅ u ⋅ T = − ∇q + Q
(11) (11) The Cauchy stress vector is given in Eq. (12). The Cauchy stress vector is given in Eq. (12). The Cauchy stress vector is given in Eq. (12). The Cauchy stress vector is given in Eq. (12). σ = − p ⋅ I + S
(12) (12) where p, S and I are the hydrostatic pressure, the extra stress tensor and the Kronecker delta,
respectively. The CFD code of COMSOL 4.3b, using Carreau-Yasuda viscosity model, was used to solve
the governing equations. For this model, the effect of the viscous dissipation, that is, the shear
heating effect, which is responsible for the fluid temperature increase, was taken into account. This is quite important in polymer extrusion processes and their design. In order to create the fluid domain, the flow simulation add-in of SolidWorks was used and
two lids were created, one in the inlet and another in the outlet of extrusion die as shown in
Figure 5(b) and (c). Then, the fluid body assembly was created, choosing all the parts of the
extrusion die as shown in Figure 5(d). Finally, after deleting all the unneeded subparts, the
fluid domain was taken as shown in Figure 5(e). The mesh model used for the simulations is presented in Figure 5(f). This model included
95,215 tetrahedral finite elements, and the minimum and maximum element sizes were
5.56 × 10−4 and 13 × 10−3 mm, respectively. This mesh was created using automated unstruc-
tured mesh generator. The finite element analysis results for the temperature distribution are presented in Figure 6(a). These results were obtained using the energy balance equations in different positions of the
die domain. 5.1. CFD analysis The input temperature, that is, the temperature of the polymer melt when it enters
the die domain, was 469 K. Due to the viscous dissipation, this temperature progressively Extrusion of Metals, Polymers, and Food Products
188 Figure 5. Steps for the geometrical model design (a)–(e) and mesh model of the domain (f). Figure 5. Steps for the geometrical model design (a)–(e) and mesh model of the domain (f). Figure 6. Finite element analysis results for the die domain. Figure 6. Finite element analysis results for the die domain. Figure 6. Finite element analysis results for the die domain. Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 189 increases, while the flow is moving toward the outlet. The average temperature of the poly-
mer melt is given by Eq. (13) [2], and its increase calculated is equal to 1.53 K. T =
∫
s T ⋅ u ⋅ ds
_________
∫
s u ⋅ ds
(13) (13) The respective value for the temperature increase calculated using the mathematical model,
that is, 4.72 K, was considerably greater in comparison with the finite element analysis results. i The respective value for the temperature increase calculated using the mathematical model,
that is, 4.72 K, was considerably greater in comparison with the finite element analysis results. i This is due to the simplification assumption used for the mathematical analysis which indi-
cated that the die walls are adiabatic. In practice, this consideration of the adiabatic wall
facilitates the solving process of the mathematical model, but it considerably affects the tem-
perature increase value. The results of the finite element analysis performed as regards the pressure distribution on
the die domain are presented in Figure 6(b). It is obvious that the pressure follows a reduction
trend while moving from the extrusion die inlet to its outlet. The pressure drop calculated
using finite element analysis was 97.24 bar. This value was similar to the one calculated using
the mathematical model, that is, 92.17 bar (Eq. (6)). 5.2. Fluid-structure interaction analysis of the spider head The most crucial parts of such an extrusion die, as regards its failure under pressure, are the
spider legs. The so-called parts of the spider head are the thickest parts of the whole struc-
ture, and consequently, these are the most possible points for failure onset under pressure. Therefore, if a single spider leg is able to perform under a specific pressure without failure, it
is safe for the whole structure to perform under this pressure. In order to investigate if the spider legs are able to perform under the pressure occurred using
the maximum flow rate provided by the single-screw extruder of this study, a stress analy-
sis was conducted on a single spider leg. This fluid-structure interaction (FSI) problem was
solved using the COMSOL Multiphysics software. The solid mechanics continuum equations
(Eq. (14)), together with the fluid mechanics Navier-Stokes equations (Eq. (15), were solved
using the arbitrary Lagrangian-Eulerian (ALE) method. The deforming geometry dynamics
were applied on the boundaries of the moving grid (mesh), and new mesh coordinates were cal-
culated on the channel for each moving step of the boundaries. These moving mesh coordinates
were applied on the Navier-Stokes equations. Fixed coordinates were used for the structural
parts of the model, that is, for the nonfluid parts, since the strain of these parts was calculated
by the COMSOL Multiphysics using structural mechanics. The calculation of the deformed
coordinates using ALE formulation was based on the calculated strain of the structural parts. − ∇ ⋅ u = 0
(14)
ρ ∂ u
___
∂ t − ∇ ⋅ [− p ⋅ I + η (∇ u + (∇ u) T ) ] + ρ (u ⋅ ∇) u = F
(15) − ∇ ⋅ u = 0
(14) (14) ρ ∂ u
___
∂ t − ∇ ⋅ [− p ⋅ I + η (∇ u + (∇ u) T ) ] + ρ (u ⋅ ∇) u = F
(15) (15) t diagonal matrix and F is the volume force which affects the fluid. In Eq. (15), I is the unit diagonal matrix and F is the volume force which affects the fluid. Extrusion of Metals, Polymers, and Food Products 190 Eq. (15), that is, Navier-Stokes, if solved for the velocity u and the pressure p, describes the
fluid flow in the channel. Gravitation is not taken under consideration in this model. 5.2. Fluid-structure interaction analysis of the spider head The
same goes for the volume force which affects the fluid. Therefore, the value of the force F in
Eq. (15) is equal to zero (F = 0). These equations are applied on the deformed coordinates. The fluid flow at the inlet is described by a fully developed laminar flow and at the outlet
is described by a noncompressible flow (p = 0). No-slipping conditions, that is, u = 0, were
applied on the rest of the boundaries. An elastic and nonlinear model was used in order to apply the large displacement method on
the structural domain. Fixed support was applied on the lower and upper spider head bound-
aries (ring geometry), which indicates an ability lack for movement toward any direction. The spider head examined is presented in Figure 7. In the same figure, the meshing types for
the spider leg analysis can be found. The mesh of a single spider leg was unstructured, and
it was constituted by 8732 tetrahedral elements. The minimum and maximum element sizes
were 1.01 × 10−4 and 2.36 × 10−3 mm, respectively. The flow mesh was also unstructured and
was constituted by 62,412 tetrahedral elements. The minimum and maximum element sizes
for this type of mesh were 1.17 × 10−4 and 2.03 × 10−3 mm, respectively. After the mesh-generating process, the solution of the numerical model took place. Figure 8
presents the finite element analysis results for equivalent stress and total displacement. As can
be observed in Figure 8(a), the maximum flow rate of the extruder used leads to the develop-
ment of a stress equal to 14.79 × 10−2 MPa, which is the maximum stress that can be achieved
for HDPE flow with the specific extruder. On the other hand, the yield stress of the IMPAX
tool steel used for the die parts is 8 × 102 MPa. Since the maximum Von Misses equivalent Figure 7. Spider head and mesh types of spider leg, fluid and their combination. Figure 7. Spider head and mesh types of spider leg, fluid and their combination. Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
19 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
191 Figure 8. Von misses equivalent stress (a) and total displacement (b) developed on a single spider leg of the extrusion
die’s spider head. Figure 8. 5.2. Fluid-structure interaction analysis of the spider head Von misses equivalent stress (a) and total displacement (b) developed on a single spider leg of the extrusion
die’s spider head. stress is considerably lower than the yield stress of the spider leg material, the designed spi-
der head, and consequently the entire die, is effective for extrusion processes with the specific
single-screw extruder. stress is considerably lower than the yield stress of the spider leg material, the designed spi-
der head, and consequently the entire die, is effective for extrusion processes with the specific
single-screw extruder. 6. Manufacturing The G-code used for the cutting processes applied for the production of each part was gen-
erated using SolidCAM software (CAM package). It was transmitted to the CNC cutting Figure 9. 3D simulation of the cutting process for the production of the spider head in SoliCAM environment. Figure 9. 3D simulation of the cutting process for the production of the spider head in SoliCAM environment Extrusion of Metals, Polymers, and Food Products
192 Figure 10. Johnson Plastics single-screw extruder together with the mounted extrusion die. Figure 10. Johnson Plastics single-screw extruder together with the mounted extrusion die. machines used, an OKUMA MX-45VE CNC milling cutting center and an OKUMA LB10II
CNC lathe, with DNC technology. The 3D simulation of the cutting process for the produc-
tion of the spider head in SoliCAM environment is presented in Figure 9. Since the spider die was intended to be used for the production of HDPE tubes, it was
mounted on a single-screw Johnson Plastics extruder with characteristics: length/diameter
ratio 24.1, screw diameter 38 mm and compression ratio 2.75. The extruder with the mounted
extrusion die is presented in Figure 10. 7. Results and discussion A summary of the analytical, experimental and numerical results reported in the current
chapter including the pressure drop and temperature rise in the die is presented in Table 1. The calculated by the mathematical model pressure drop was approximately 3.3% lower than
the actual (experimental) result [2]. This can be explained by the fact that the analytical model
simplifies the pressure drop calculation in cases of complex geometries. However, the diver-
gence of the calculated value is quite low. On the other hand, the maximum average tempera-
ture rise data show 174% divergence compared with the corresponding temperature obtained
experimentally. This is due to the simplification assumption used for the mathematical analy-
sis which indicated that the die walls are adiabatic. The comparison between the experimental and non-Newtonian die flow simulations seems
to reveal the expected good agreement with the overall pressure drop as well as with the con-
stant wall temperature boundary conditions. The pressure data calculated by the numerical Carreau-Yasuda model (97.24 bar) show a
fairly good agreement with the experimental results (95.15 bar), whereas the average tem-
perature rise of the molten HDPE was T = 1.53 K, which is approximately 11% higher than the
experimental temperature value. This was the value calculated for the boundary conditions Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211
193 of wall with constant temperature. In the case of adiabatic boundary conditions, the average
temperature rise of the molten HDPE was considerably higher than the experimental one. Mathematical model
Carreau-Yasuda (Numerical)
Constant wall temperature
Carreau-Yasuda
(Numerical)
No heat flux
Experimental
dP (bar)
92.17
97.24
97.24
95.15
dT (K)
4.72
1.53
6.66
1.72
Error dP (%)
3.13
2.19
2.19
Error dT (%)
174.42
11.05
287.21
Table 1. Analytical, numerical and experimental data. http://dx.doi.org/10.5772/intechopen.72211 of wall with constant temperature. In the case of adiabatic boundary conditions, the average
Mathematical model
Carreau-Yasuda (Numerical)
Constant wall temperature
Carreau-Yasuda
(Numerical)
No heat flux
Experimental
dP (bar)
92.17
97.24
97.24
95.15
dT (K)
4.72
1.53
6.66
1.72
Error dP (%)
3.13
2.19
2.19
Error dT (%)
174.42
11.05
287.21
Table 1. Analytical, numerical and experimental data. Table 1. Analytical, numerical and experimental data. of wall with constant temperature. In the case of adiabatic boundary conditions, the average
temperature rise of the molten HDPE was considerably higher than the experimental one. of wall with constant temperature. 8. Conclusions Summarizing the main features of the results reported, it may be concluded that there is a
significant difference comparing the numerical and analytical models with regard to the tem-
perature developed in the fluid during the extrusion process. This can be explained by the fact
that the analytical model is based on the assumption that adiabatic conditions occur, which
means that there is no heat transfer between the wall and the polymer material as described
above. Finally, it was demonstrated by the stress analysis that the die construction is strong
enough to withstand the pressure developed during the die operation and that the stresses do
not exceed the material yield strength in any case. 7. Results and discussion In the case of adiabatic boundary conditions, the average
temperature rise of the molten HDPE was considerably higher than the experimental one. Figure 11 presents the pressure drop throughout all the die zones and the total pressure drop
(expressed as percentage) calculated using the mathematical model (Figure 11(a, b)) and
finite element analysis (Figure 11(c, d)). It is obvious that the majority of the pressure drop is
observed along the zone V, at the exit of extrusion die. Figure 11 presents the pressure drop throughout all the die zones and the total pressure drop
(expressed as percentage) calculated using the mathematical model (Figure 11(a, b)) and
finite element analysis (Figure 11(c, d)). It is obvious that the majority of the pressure drop is
observed along the zone V, at the exit of extrusion die. Figure 11. Pressure drop throughout the die zones and total pressure drop calculated using the mathematical model (a, b)
and finite element analysis (c, d). Figure 11. Pressure drop throughout the die zones and total pressure drop calculated using the mathematical model (a, b)
and finite element analysis (c, d). Extrusion of Metals, Polymers, and Food Products 194 The calculated, using finite element analysis, Von Misses equivalent stresses are significantly
lower than the yield stress of the die material, and therefore, it may be concluded that the
abovementioned tool steel is suitable for manufacturing spider dies for polymer extrusion
applications. The calculated, using finite element analysis, Von Misses equivalent stresses are significantly
lower than the yield stress of the die material, and therefore, it may be concluded that the
abovementioned tool steel is suitable for manufacturing spider dies for polymer extrusion
applications. Author details G.N. Kouzilos*, G.V. Seretis, C.G. Provatidis and D.E. Manolakos *Address all correspondence to: gkouzilosb@yahoo.com *Address all correspondence to: gkouzilosb@yahoo.com *Address all correspondence to: gkouzilosb@yahoo.com National Technical University of Athens, School of Mechanical Engineering, Athens, Greece References [1] Michaeli W. Extrusion Dies for Plastics and Rubber: Design and Engineering Compu-
tations. 3rd ed. Munich: Carl Hanser Verlag GmbH & Co. KG; 2003. p. 362. DOI:
10.3139/9783446401815 [2] Kouzilos GN, Markopoulos AP, Manolakos DE. Manufacturing and modeling of an
extrusion die spider head for the production of HDPE tubes. Journal of Manufacturing
Technology Research. 2015;6(1-2):1-15 [2] Kouzilos GN, Markopoulos AP, Manolakos DE. Manufacturing and modeling of an
extrusion die spider head for the production of HDPE tubes. Journal of Manufacturing
Technology Research. 2015;6(1-2):1-15 [3] Mamalis AG, Kouzilos G, Vortselas AK. Design feature sensitivity analysis in a numerical
model of an extrusion spider die. Journal of Applied Polymer Science. 2011;122(6):3537-
3543. DOI: 10.1002/app.34762 [3] Mamalis AG, Kouzilos G, Vortselas AK. Design feature sensitivity analysis in a numerical
model of an extrusion spider die. Journal of Applied Polymer Science. 2011;122(6):3537-
3543. DOI: 10.1002/app.34762 [4] Choudhary MK, Kulkarni JA. Modeling of three-dimensional flow and heat transfer in
polystyrene foam extrusion dies. Polymer Engineering & Science. 2008;48(6):1177-1182. DOI: 10.1002/pen.20990 Design of Polymer Extrusion Dies Using Finite Element Analysis
http://dx.doi.org/10.5772/intechopen.72211 195 [5] Lebaala N, Schmidtb F, Puissanta S. Design and optimization of three-dimensional
extrusion dies, using constraint optimization algorithm. Finite Elements in Analysis and
Design. 2009;45(5):333-340. DOI: 10.1016/j.finel.2008.10.008 [6] Huang GQ, Huang HX. Optimizing Parison thickness for extrusion blow molding by
hybrid method. Journal of Materials Processing Technology. 2007;182(1-3):512-518. DOI:
10.1016/j.jmatprotec.2006.09.015 [7] Yilmaz O, Gunes H, Kirkkopru K. Optimization of a profile extrusion die for flow bal-
ance. Fibers and Polymers. 2014;15(4):753-761. DOI: 10.1007/s12221-014-0753-3 [8] Gonçalves ND, Teixeira P, Ferrás LL, Afonso AM, Nóbrega JM, Carneiro OS. Design and
optimization of an extrusion die for the production of wood–plastic composite profiles. Polymer Engineering & Science. 2015;55(8):1849-1855. DOI: 10.1002/pen.24024 [9] Mamalis AG, Vortselas AK, Kouzilos G. Tube extrusion of polymeric materials:
Optimization of the processing parameters. Journal of Applied Polymer Science. 2012;126(1):186-193. DOI: 10.1002/app.36555 [10] Rauwendaal C. Polymer Extrusion. 5th ed. Munich: Carl Hanser Verlag GmbH & Co. KG; 2014. p. 950. DOI: 10.3139/9781569905395
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Analysis of the interaction of calcitriol with the disulfide isomerase ERp57
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Scientific reports
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cc-by
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Analysis of the interaction of
calcitriol with the disulfide
isomerase ERp57
OPEN received: 26 January 2016
accepted: 21 October 2016
Published: 29 November 2016 Elisa Gaucci1, Domenico Raimondo2, Caterina Grillo1, Laura Cervoni1, Fabio Altieri1,3,
Giulio Nittari1,†, Margherita Eufemi1,3 & Silvia Chichiarelli1 Elisa Gaucci1, Domenico Raimondo2, Caterina Grillo1, Laura Cervoni1, Fabio Altieri1,3,
Giulio Nittari1,†, Margherita Eufemi1,3 & Silvia Chichiarelli1 Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to
the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-
associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our
research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We
first studied the interaction through bioinformatics and fluorimetric analyses. Subsequently, we
focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’-
ERp57, containing the first three domains, and a’-ERp57, the fourth domain only. To consolidate the
achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow
us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other
potential binding sites to the dissociation constant, calculated as a Kd of about 10−9 M. The endoplasmic reticulum (ER) protein ERp57 is a member of the disulfide isomerase family and is involved in
the folding and reshuffling of disulfide bonds in nascent glycoproteins, acting in cooperation with the lectins cal-
reticulin and calnexin. All the disulfide isomerases share a thioredoxin fold and have catalytic and non-catalytic
domains, called respectively a-type or b-type domains. ERp57 has four thioredoxin-like domains, with the a and
a’ catalytically active domains in the N- and C-termini. Most of ERp57 is located in the ER lumen, but unusual
locations have been reported too, such as nucleus, cell membrane, cytosol and mitochondria1, even though the
functions outside the ER remain elusive. STAT3, member of the signal transducers and activators of transcription
(STAT) family, is a known interactor of ERp57 in the cytosol, cell membrane and nucleus, where the two proteins
bind together to the C-reactive protein (CRP) gene promoter2. ERp57 may also directly bind DNA, as revealed by
in vitro3 and in vivo studies4. ERp57 has been found to interact strongly with a number of small ligands, such as
antibiotics5,6 and polyphenols7, as well as to macromolecules8–10. received: 26 January 2016
accepted: 21 October 2016
Published: 29 November 2016 Analysis of the interaction of
calcitriol with the disulfide
isomerase ERp57
OPEN According to Doroudi et al.19, calcit-
riol interacts with ERp57 in caveolae, in complex with phospholipase A and caveolin 1 (Cav-1), leading then to
the activation of phospholipase A2 (PLA2) and protein kinase C (PKC). In calcitriol-stimulated leukemia cells,
ERp57 appears to be redistributed from plasma membrane and cytosol towards the nucleus, together with the
transcription factor NFkB, and to act in the differentiation pathway20. f
Recently some studies described a synergic action between ERp57 and VDR but not their direct interaction;
in fact, the authors hypothesize a synergic action for the mineralization of pre-osteoblasts in 3D culture21 and for
the Wnt5 calcium-dependent signaling mediated through Pdia3/ERp57, PLAA, and VDR22. However, another
study describes a Pdia3/ERp57-mediated but VDR-independent vitamin D rapid response in osteoblasts with an
increase in CaMKII (calcium/calmodulin-dependent protein kinase II) activity23. p
p
y
Our studies on HeLa cells have shown that the stimulation with calcitriol causes the exit of ERp57 from the ER
towards other cell compartments, such as the cytosol and the nucleus24. Currently, little is known about the role of
ERp57 in non-ER localizations. It is possible to hypothesize the involvement in signal transduction processes in
response to different extracellular stimuli and the trafficking through the cell. In fact, early studies on ERp57 have
misidentified this protein as phospholipase C alpha, but they have shown that ERp57 is able to interact with the
angiotensin II receptor25 and the vasopressin receptor26. Moreover, its association with the angiotensin receptor
is likely to possess functional significance, as suggested by its phosphorylation following angiotensin binding27. Zhu et al. have been reported a full description of the interaction of ERp57 with the all-trans retinoic acid recep-
tor α in Sertoli cells. ERp57 is associated with the receptor in the cytosol and is required for the transport of the
ligand-receptor complex into the nucleus, and subsequently into the ER to allow the receptor degradation by the
ERAD (ER-associated protein degradation). The role of ERp57 in the receptor activity has been ascribed to the
conformational changes of the receptor, in order to facilitate the binding of the ligand.28. Sehgal and collaborators
found STAT3 in the lipid raft fraction of cell membrane, associated with ERp5729. The involvement of ERp57
in signal transduction processes has been demonstrated for STAT3-involving pathways as described above2. Analysis of the interaction of
calcitriol with the disulfide
isomerase ERp57
OPEN p yp
ERp57 has been unexpectedly revealed as the membrane-associated receptor for calcitriol, the biologically
active form of vitamin D3, responsible for the rapid nongenomic response to the hormone11. Th
h h
f
d
h
k
ft
l h
d
h d
l The vitamin D3, which is formed in the skin after exposure to sunlight, needs two hydroxylation reactions
to become the active form 1,25-dihydroxyvitamin D3, also known as calcitriol. Its mechanism of action is sim-
ilar to other steroid hormones and involves the binding to the intracellular receptor VDR12. After this interac-
tion, calcitriol/VDR heterodimerizes with the retinoid X receptor (RXR) and the heterodimer binds specific
response elements, leading to either the activation or repression of gene transcription. The transcription pro-
cess proceeds through the interaction of VDR with coactivators and with the transcription machinery13. In this
way, calcitriol stimulates calcium and phosphate transport from intestine and kidney to the blood, but it has
also anti-proliferative and pro-differentiating effects. In addition to the regulation of gene expression, calcitriol
can exert rapid, nongenomic actions, which are performed by modulating the transmembrane transport of cal-
cium and chloride ions and activating signal transduction pathways, such as those involving protein kinase C
(PKC) and MAP kinases14. Among the MAP kinases, not only ERK1/2 is involved, but also ERK5, which partic-
ipates in calcitriol-induced cell differentiation in acute myeloid leukemia15. More recently, it has been found that 1Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185,
Rome, Italy. 2Stem Cell Lab - Department of Molecular Medicine - Sapienza Università di Roma, Viale Regina Elena
324, 00161, Rome, Italy. 3Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Piazzale Aldo
Moro 5, 00185, Rome, Italy. †Present address: International School of Advanced Studies - University of Camerino -
Piazza Cavour 19/f, 62032, Camerino (MC) – Italy. Correspondence and requests for materials should be addressed to
S.C. (email: silvia.chichiarelli@uniroma1.it) or D.R. (email: domenico.raimondo@uniroma1.it) Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 1 www.nature.com/scientificreports/ calcitriol inhibits Wnt/β-catenin signalling pathway in non-malignant murine colon cells16, while the inhibition
of Hedgehog pathway is responsible for the anti-tumour effect of calcitriol in basal cell carcinoma17. The two pro-
teins that could mediate the calcitriol-initiated signalling are VDR, which has been identified also in caveolae18,
and a membrane-associated protein, which has been revealed as ERp5711. Results
ER 57 ERp57-calcitriol interaction prediction. Blind docking experiments, i.e. a single docking experiment
carried out on the whole ERp57 protein surface, were performed in order to identify putative druggable cavi-
ties that calcitriol can explore. This approach for finding the putative binding site has already been successfully
applied to other biological targets31–33. Binding modes of calcitriol molecule with the most favorable energies
were evaluated and clustered. Results obtained clearly show that a’ and bb’ domains of ERp57 are preferred by
calcitriol (see Fig. 1a–f in which each docked conformation has been represented as a sphere whose center is
at the average position of all the atoms in that conformation). These data suggest that it is very likely that these
regions correspond to the binding portions explored by the calcitriol molecule. Two out of three regions predicted
by blind docking approach were also identified as potentially establishing favorable interactions with small lig-
ands by computational solvent mapping analysis34 (see Material and Methods), thus improving the reliability of
our hypothesis that these regions are compatible with calcitriol binding. In fact, as shown in Fig. S7, the organic
probes clusters are all in the b, b’ and a’ domains. Therefore, in order to refine our results, we performed focused
docking experiment increasing the number of energy evaluations and varying the docking box resolution. The
search space was restricted to the vicinity of the binding sites both predicted by blind docking and confirmed by
the FTsite program on a’, b and b’ domains, discarding a domain. Focused docking experiment consisted of three independent runs, with the docking box centered on the
predicted druggable hot spots. Figure 2 shows results of the dockings experiments by exploring cavities in the b
domain (Fig. 2a,b), between the b and b’ domains (Fig. 2c,d) and in the a’ domain (Fig. 2e,f). We selected the low-
est energy conformer belonging to the most populated cluster as the most likely calcitriol pose. It can be noticed
that in the cavity in the bb’ domains calcitriol is involved in two hydrogen bonds with Glu238 and Lys258, and in
the a’ domain with Glu388 and Asn392. Collectively, these results strongly support hypothesis that calcitriol binding region can be formed by well
defined pockets in the bb’ and a’ domains, thus putting the basis for a structural interpretation of the binding of
the calcitriol molecule to the ERp57 protein surface. Purification of recombinant mutant abb’-ERp57. Analysis of the interaction of
calcitriol with the disulfide
isomerase ERp57
OPEN Moreover, it has been found that the ERp57 silencing affects the internalization and phosphorylation of the EGF
receptor (EGFR) after EGF binding30.hi p
(
)t
g
The identification of the calcitriol binding site in the ERp57 structure can help to elucidate the role of ERp57
in its mechanism of action, in order to clarify the intracellular trafficking of the complex. In this context, our
investigation workflow has involved different approaches. We have performed a bioinformatic analysis of the
ERp57 structure and of the interaction with calcitriol, as well as a spectrofluorimetric analysis of their interaction. After these results, we have focused our study on specific deletion mutants of the protein. The mutants have been
chosen on the basis of the in silico results. In particular, one mutant is composed by the first three domains, which
are the catalytically active a domain and two adjacent b-type domains (abb’-ERp57), while the other one is the
fourth domain only (a’-ERp57). Moreover, for the abb’-ERp57 mutant we have refined a specific expression and
purification protocol. In order to consolidate the data obtained from the bioinformatic and fluorimetric analyses,
we performed the isothermal titration calorimetry of the whole protein and its two mutants. Results
ER 57 To analyse the interaction between calcitriol and
the abb′-ERp57 mutant, we expressed and purified for the first time the recombinant protein consisting of abb′
domains without GST-fusion, and tested its binding activity (see Material and Methods). Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 2 www.nature.com/scientificreports/ Figure 1. Docking analysis performed on the entire ERp57 protein (a,b), or at the level of the bb’ domains (c,d)
or a’ domain (e,f). The protein is depicted in grey as surface. Each docked conformation is represented by a
sphere placed at the average position of the coordinates of all the atoms in that conformation. The radii of the
spheres are 0.3 Å. Figure 1. Docking analysis performed on the entire ERp57 protein (a,b), or at the level of the bb’ domains (c,d)
or a’ domain (e,f). The protein is depicted in grey as surface. Each docked conformation is represented by a
sphere placed at the average position of the coordinates of all the atoms in that conformation. The radii of the
spheres are 0.3 Å. SDS–PAGE of different protein fractions during purification of abb’-ERp57 mutant are shown in Fig. S1 and
Fig. S2 (see Supplementary data). Calcitriol interacts with ERp57 and its deletion mutants – fluorimetric assays. We have con-
ducted in vitro studies with the aim of determining the affinity constant of the interaction between ERp57 and
calcitriol. Fluorimetric assays were performed to measure the variation of the protein intrinsic fluorescence, due
to three triptophane residues in the ERp57 sequence, after adding increasing concentrations of calcitriol. These
assays were performed both on oxidized and reduced protein. In the former case, the recombinant ERp57 was
oxidized with H2O2, and the binding was analyzed at the spectrofluorimeter, by measuring the emission at 336 nm
wavelength. There was a quenching of the fluorescence emission of the protein by increasing concentrations of
the ligand, in a large excess, but unfortunately the formation of both monomeric and dimeric species in solution,
due to both intra and intermolecular disulfide bridges, did not enable us from deriving the dissociation constant. Results
ER 57 In the case of totally reduced rERp57, with the use of TCEP as reducing agent, the affinity constant could be
obtained, since only the monomeric form is possible.l y
p
Considering the highly hydrophobic nature of calcitriol, another fluorimetric assay, in which the ligand
was not in large excess but was in nearly equimolar concentration with the protein in the reduced form, was Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 3 www.nature.com/scientificreports/ Figure 2. Docking analysis on the cavities located in the b domain of ERp57 (a,b), between the b and b’
domains (c,d), on the a’ domain (e,f). (a,c,e) The lowest energy conformer of the most populated cluster is
shown in black sticks on the surface of the protein. (b,d,f) detail of the interactions between the lowest ener
conformers and the surrounding residues. The hydrogen bonds are shown in wireframe and with dashed lin
The protein is depicted as cartoon. Figure 2. Docking analysis on the cavities located in the b domain of ERp57 (a,b), between the b and b’
domains (c,d), on the a’ domain (e,f). (a,c,e) The lowest energy conformer of the most populated cluster is
shown in black sticks on the surface of the protein. (b,d,f) detail of the interactions between the lowest energy
conformers and the surrounding residues. The hydrogen bonds are shown in wireframe and with dashed lines. The protein is depicted as cartoon. erformed to avoid the possible adsorption of calcitriol to the chamber of the quartz fluorimeter cuvette. The
rotein was finally saturated with an excess of ligand.ll i
In Fig. S3, the graph shows a plot of the fluorimetric data, in which the variation of the intrinsic fluorescence
of the protein is plotted as a function of the ligand concentration. The graphical display of the data allowed the
determination of the dissociation constant, which was calculated with the following formula, according to Cogan
et al.35. =
−
−
a
(Fluo
Fluo
)/(Fluo
Fluo
)
sat
o
sat ×
=
×
−
−
n
n
Protein
a
(1/ )[Ligand
a/(1
a)]
Kd/
tot
tot where Fluosat is referred to the fluorescence of the protein totally saturated with calcitriol, while Fluo0 to that of
the reduced rERp57 in absence of the ligand.h where Fluosat is referred to the fluorescence of the protein totally saturated with calcitriol, while Fluo0 to that of
the reduced rERp57 in absence of the ligand. Results
ER 57 Th
d
d
f
d
d h
d
d
d f
h Three independent experiments were performed and the mean dissociation constant, derived from the equa-
tion assuming y equal to zero, was calculated as 10−9 M, comparable with that of the nuclear VDR. n, which
corresponds to the number of ligand molecules bound to one single protein molecule, was nearly two, consistent
with the hypothesis of two binding sites in the ERp57 protein in the reduced form. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 4 www.nature.com/scientificreports/ ntificreports/
Figure 3. Isothermal Titration Calorimetry of abb’-ERp57 (20 μM) with calcitriol (100 μM). Both the
protein and the ligand were in the same buffer (see Materials and Methods). (Upper panel) heat evolved
upon injection of calcitriol into abb’-ERp57, plotted as a function of injection order. (Lower panel) Integrated
heats of reaction plotted against the molar ratio of total ligand concentration to total protein concentration. The solid line shows the best fit to the data for n = 0.394 (±0.00721) sites/mole, Ka = 5.98 (±1.4) × 106 M−1,
ΔH = 7.140 ± 0.183 × 103 kcal/mol. Figure 3. Isothermal Titration Calorimetry of abb’-ERp57 (20 μM) with calcitriol (100 μM). Both the
protein and the ligand were in the same buffer (see Materials and Methods). (Upper panel) heat evolved
upon injection of calcitriol into abb’-ERp57, plotted as a function of injection order. (Lower panel) Integrated
heats of reaction plotted against the molar ratio of total ligand concentration to total protein concentration. The solid line shows the best fit to the data for n = 0.394 (±0.00721) sites/mole, Ka = 5.98 (±1.4) × 106 M−1,
ΔH = 7.140 ± 0.183 × 103 kcal/mol. Three independent experiments were performed, also, with ERp57 mutants (abb’-ERp57 and a’-ERp57). The data are consistent with a direct interaction between a’-ERp57 and calcitriol (Fig. S4); this evidence could
be justified by a site-specific interaction of calcitriol with the a’ domain with a Kd ∼10−8 M and a stoichiometry
of ± 1.2 for the number of ligands bound.fi Three independent experiments were performed, also, with ERp57 mutants (abb’-ERp57 and a’-ERp57). The data are consistent with a direct interaction between a’-ERp57 and calcitriol (Fig. Discussionhi The disulfide isomerase protein ERp57, which mainly resides in the ER, is known to participate in a number of
well-studied processes, such as the quality control of newly synthesized glycoproteins37 and in the assembly of
MHC class I. In other subcellular compartments, where it is present in small amount, it is involved in a variety of
mostly unexplored processes.h The disulfide isomerase protein ERp57, which mainly resides in the ER, is known to participate in a number of
well-studied processes, such as the quality control of newly synthesized glycoproteins37 and in the assembly of
MHC class I. In other subcellular compartments, where it is present in small amount, it is involved in a variety of
mostly unexplored processes. y
p
p
In the present research, the interaction of ERp57 with calcitriol has been explored. The biologically active form
of vitamin D3 can exert its function of regulation of gene expression through the binding to the nuclear recep-
tor VDR, but it is also able to act through a membrane-associated receptor, displaying a wide variety of rapid,
nongenomic actions, such as the rapid activation of signalling cascades. The membrane associated receptor was
revealed to be ERp5711, which was originally called membrane-associated rapid response steroid binding protein
(1,25D3-MARRS). Furthermore, it has been demonstrated that the ERp57-calcitriol complex, which has been
found in the matrix vesicles of chondrocytes, is implicated in the reorganization of extracellular matrix38. In this
context, the expression of ERp57 was found to be decreased in the osteogenic differentiation of mouse embryoid
bodies, contrarily to VDR, leading to the hypothesis of a differential expression of the two proteins at diverse
stages of cell commitment39. In this work is shown, for the first time, the direct interaction between ERp57 and
calcitriol using the purified recombinant protein and its deletion mutants. g
pi
p
To date, a lot of studies have investigated the function of the interaction of ERp57 with calcitriol40–45. Moreover,
in a review on vitamin D analogues as potential therapeutics in melanoma, the 1,25-dihydroxy-lumisterol, locked
in the 6-cis configuration, was cited as a vitamin D analogue which activates the rapid-response pathways and
competes with 1α,25-dihydroxyvitamin D3 for MARRS receptor binding, but not for VDR binding43. In addi-
tion, Tohda et al. Results
ER 57 S4); this evidence could
be justified by a site-specific interaction of calcitriol with the a’ domain with a Kd ∼10−8 M and a stoichiometry
of ± 1.2 for the number of ligands bound.fi g
On the other hand, the data obtained for the abb’-ERp57 mutant (Fig. S5) were of difficult interpretation and
overall are not consistent with a line that brings out a direct interaction between the mutant and calcitriol. These
unpredictable data may be due to the fact that the two triptophane residues in the abb’-ERp57 mutant could be
partially quenched in the folding of the protein without the fourth a’ domain. Calcitriol interacts with ERp57 and its deletion mutants – calorimetric assays. In order to iden-
tify the domain(s) of ERp57 responsible for the interaction, the two deletion mutants of the protein were tested
by Isothermal Titration Microcalorimetry (ITC) (Fig. 3 for abb’-ERp57 and Fig. 4 for a’-ERp57). Before the assay,
the mutants were reduced with TCEP.hi The microcalorimetric titration curve fitted a possible model of interaction and the same model was matched
with the results of fluorescence measurements. A value of Kd of ∼10−9 M was obtained, consistent with the fluori-
metric results.hh The thermodynamic data were processed as described above. The values of ΔH were measured for each titra-
tion and the system also gave information on the change in entropy (ΔS). The binding free energy (ΔG) and the
dissociation constant (Kd) were calculated from the experimentally determined values of ΔH and Ka, using eqs 1
and 2 (see Materials and Methods). The in vitro binding of calcitriol to ERp57 was studied also by competition
ligand binding by displacement ITC (Fig. 5a,b).h The apparent binding constant, in the competition experiments, depends on the concentration of free inhibi-
tor, which changes during the experiment. The binding constant was calculated as described in Sigurskjold36 (see
Materials and Methods).h The calorimetric data are reported in Table 1.h h
The evidences indicated that the binding to each mutant was entropically driven. The value of TΔS is 295 kcal/
mol and 17.2 kcal/mol for the a’-ERp57 and abb’-ERp57 mutants respectively; the ΔH values are 283 kcal/mol and
8.00 kcal/mol. Moreover, we observe that calcitriol binds ERp57 with a ratio approximately of 1:1 for the a’-ERp57
mutant. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 5 www.nature.com/scientificreports/ Figure 4. Isothermal Titration Calorimetry of a′-ERp57 (0.05 μM) with calcitriol (0.5 μM). Results
ER 57 Both the protein
and the ligand were in the same buffer (see Materials and Methods). (Upper panel) heat evolved upon injection
of calcitriol into a’-ERp57, plotted as a function of injection order. (Lower panel) Integrated heats of reaction
plotted against the molar ratio of total ligand concentration to total protein concentration. The solid line shows
the best fit to the data, according to a model that assumes a single set of identical sites, for n = 1.15 (±0.00892)
sites/mole, Ka = 1.68 (±0.231) × 109 M−1, ΔH = 2.813 ± 0.032 105 kcal/mol. Figure 4. Isothermal Titration Calorimetry of a′-ERp57 (0.05 μM) with calcitriol (0.5 μM). Both the protein
and the ligand were in the same buffer (see Materials and Methods). (Upper panel) heat evolved upon injection
of calcitriol into a’-ERp57, plotted as a function of injection order. (Lower panel) Integrated heats of reaction
plotted against the molar ratio of total ligand concentration to total protein concentration. The solid line shows
the best fit to the data, according to a model that assumes a single set of identical sites, for n = 1.15 (±0.00892)
sites/mole, Ka = 1.68 (±0.231) × 109 M−1, ΔH = 2.813 ± 0.032 105 kcal/mol. Discussionhi in 201246 have presented a docking simulation between ERp57/1,25D3-MARRS and diosgenin
which is very similar in structure to calcitriol; in particular in this study they found that the exogenous stimula-
tor diosgenin activates the 1,25D3-MARRS pathway, which may be a very critical signalling target for anti-AD
(Alzheimer’s disease) therapy.i (
)
py
Our study analyses in details, for the first time, the direct interaction between calcitriol and ERp57 by means
of in vitro techniques and we have, also, hypothesized the probable interaction sites.l q
yp
p
By means of fluorimetric assays (Fig. S3), we have derived the dissociation constant of the complex between
recombinant ERp57, in the reduced form, and calcitriol, which was calculated as 10−9 M. Above all, our data con-
firm the Kd value of this interaction, indicated previously in the literature47. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 6 www.nature.com/scientificreports/ Figure 5. (a) Isothermal Titration Calorimetry of ERp57 (5 μM)/Silibinin (50 μM) with calcitriol (10 μM). Both the protein and the ligands were in the same buffer (see Materials and Methods). (Upper panel) heat
evolved upon injection of calcitriol into ERp57/Silibinin, plotted as a function of injection order. (Lower
panel) Integrated heats of reaction plotted against the molar ratio of total ligand concentration to total protein
concentration. The solid line shows the best fit to the data, according to a model that assumes a single set
of identical sites (for Kd see Table 1) (b) Isothermal Titration Calorimetry of ERp57 (5 μM) with Silibinin
(50 μM). The integrated heat of reaction is plotted against the molar ratio of total ligand concentration to total
protein concentration. The solid line shows the best fit to the data for n = 0.776 (±0.0183) sites/mole, Ka = 2.07
(±0.273) × 106 M−1, ΔH = 9.565 ± 0.312 103 kcal/mol. Figure 5. (a) Isothermal Titration Calorimetry of ERp57 (5 μM)/Silibinin (50 μM) with calcitriol (10 μM). Both the protein and the ligands were in the same buffer (see Materials and Methods). (Upper panel) heat
evolved upon injection of calcitriol into ERp57/Silibinin, plotted as a function of injection order. (Lower
panel) Integrated heats of reaction plotted against the molar ratio of total ligand concentration to total protein
concentration. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 Discussionhi The solid line shows the best fit to the data, according to a model that assumes a single set
of identical sites (for Kd see Table 1) (b) Isothermal Titration Calorimetry of ERp57 (5 μM) with Silibinin
(50 μM). The integrated heat of reaction is plotted against the molar ratio of total ligand concentration to total
protein concentration. The solid line shows the best fit to the data for n = 0.776 (±0.0183) sites/mole, Ka = 2.07
(±0.273) × 106 M−1, ΔH = 9.565 ± 0.312 103 kcal/mol. N
Kd
ΔH (cal/mol)
TΔS
a’-ERp57
0.720 ± 0.609
6.47 ± 0.74 × 10−10
2.83 ± 0.02 × 105
2.95 × 105
abb’-ERp57
0.483 ± 0.077
1.81 ± 0.13 10−7
8.00 ± 0.53 × 103
1.72 × 104
*ERp57 (silib)
—
1.76 ± 0.35 × 10−9
5.36 ± 3.00 × 104
—
Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence of
calcitriol. N
Kd
ΔH (cal/mol)
TΔS
a’-ERp57
0.720 ± 0.609
6.47 ± 0.74 × 10−10
2.83 ± 0.02 × 105
2.95 × 105
abb’-ERp57
0.483 ± 0.077
1.81 ± 0.13 10−7
8.00 ± 0.53 × 103
1.72 × 104
*ERp57 (silib)
—
1.76 ± 0.35 × 10−9
5.36 ± 3.00 × 104
— Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence o
calcitriol. The Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 7 www.nature.com/scientificreports/ results of the docking in the a’ domain were then re-clustered at 10 Å, to see if it was possible to obtain a single
conformation (Fig. S9). The clusters were reduced to 2, from 11 deriving from the 2.0 Å clustering. In the second
cluster (16 poses out of 100), the A ring of calcitriol points at the α helix of ERp57, in the first one (84 poses out
of 100) not. In both cases, the two fused rings are partially superimposed to the hot spot found by FTMap. The
in silico study has therefore highlighted the potential binding sites, present on the a’ domain, and bb’ domains.ii y
g
g
p
g
p
In the present work we also described in detail, for the first time, the protocol of expression and purification of
the abb’-ERp57 mutant, which has been used for the in vitro binding studies with calcitriol.hli p
g
The spectrofluorimetric and calorimetric binding experiments have confirmed the direct interaction between
ERp57 and calcitriol and also defined the Kd of the interaction. In particular, the analysis of the in vitro bind-
ing between the purified recombinant protein and calcitriol, conducted by spectrofluorimetry, has shown a Kd
rather low, ≈10−9 M, hence a rather high affinity, comparable with that of calcitriol against its canonical nuclear
receptor (VDR). The spectrofluorimetric assays performed on the two mutants of the protein (abb’-ERp57 and
a’-ERp57), did not allow us to make assumptions about the interaction, probably because of the low sensitivity
of the method. The spectrofluorimetric analysis is based on the intrinsec fluorescence of tryptophans, on the
other hand this value depends on the position of the residues inside the protein. In the abb’-ERp57 mutant, the
tryptophan residue is localized in a hydrophobic pocket and although it can potentially be part of the binding
site, this specific localization could affect the fluorescence variation, which may be minimal and therefore could
not be well detected.l For what concerns the spectrofluorimetric data of the calcitriol/a’-ERp57 interaction, we could hypothesize
a direct interaction with a Kd of ≈10−8 M. This result, however, presents high variability, as highlighted by the
standard deviation of the single points of the correlation graph. Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence o
calcitriol. Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence of
calcitriol. The first step of our bioinformatic analysis has been the detection of pockets or cavities in the ERp57 structure
(Fig. S6 and Table S1). Among the available programs, FTSite34 has been chosen. The prediction algorithm relies
on the experimental evidence that small organic molecules are able to bind ligand binding sites. In this way we
detected three possible binding sites in the ERp57 structure: one bigger cavity between the b and b’ domains, and
two smaller cavities, very close to each other, buried in the b domain. Then, the FTMap server48 has been used,
to sample the entire protein surface and identify “hot spots”, which can be defined as locations in the protein that
contribute to the ligand binding free energy. As shown in Fig. S7, the organic probes cluster are all in the b and
b’ domains, some of them overlapping with the cavities detected with FTSite, with the exception of one, which
is located in the a’ domain. The normal mode analysis, to study the dynamic properties especially in the hinge
regions, did not show a substantial difference in the three cavities, while the a’ domain was revealed as very flex-
ible (Fig. S8 and Table S2). g
Subsequently, docking analysis of calcitriol into the X-ray crystal structure of ERp57 was performed with
Autodock, to investigate the direct interaction between the ligand and the protein. After a first blind docking
(Fig. 1), analyses were performed centring the grid box in the bb’ and a’ domains. Regarding the bb’ domains, the
conformations are mainly distributed in the cavities identified by FTSite, while in the a’ domain, the majority is
found in the same region already identified by FTMap. Consequently, a more detailed analysis, increasing the
energy evaluations, was performed in these cavities (Fig. 2). In all cases, calcitriol is able to establish not only
hydrophobic interactions, but also hydrogen bonds, both as donor and acceptor, due to its three alcoholic moi-
eties. In addition to the main chain, the residues that could form hydrogen bonds with calcitriol are Glu238 and
Lys258 in bb’, Glu388 and Asn392 in a’ domain. All clusterings were performed at 2.0 Å RMSD tolerance. Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence o
calcitriol. In this case, also, the low sensitivity of the method
may have contributed to the experimental variability, in fact the a’-ERp57 mutant has only one triptophane
residue.h The binding experiments between ERp57, or its mutants, and calcitriol were also repeated by titration cal-
orimetry. Through this method, which is able to detect enthalpy and heat changes during a reaction, a very
strong interaction with the ligand has been put in evidence. In particular, it has been possible to show an
abb’-ERp57-calcitriol interaction with a Kd of ≈10−6 M, which was not possible to derive from the fluorimetric
data. The calorimetric analysis with the a’-ERp57 mutant and calcitriol has highlighted a Kd constant of ≈10−9 M. This indicates the presence of a strong interaction site, validated by the potential binding site highlighted by the
in silico study; more than ever these data demonstrate for the first time that the ERp57 protein structure contains
a domain that interacts directly with calcitriol. y
In order to study the interaction of ERp57 with calcitriol by titration calorimetry, we proceeded with competi-
tion experiments. We used the displacement titration calorimetry technique because our data, obtained by fluori-
metric assays, indicate that the binding constant between ERp57 and calcitriol should be near 10−8 M. Usually the
direct measurement of very large binding constants would require so low concentrations that the signals become
too small; this problem can be solved by using the displacement titration calorimetry, in which a less strongly
bound ligand is competitively inhibiting the binding of the stronger ligand. To perform this kind of experiment
we chose silibinin as a competitor, because it has an affinity of at least two orders of magnitude lower compared
to calcitriol49. Thus, it was found that the constant calculated with the fluorimetric data was compatible and con-
firmed by the calorimetric data. The results of the displacement titration calorimetry technique, using ERp57/
silibin/calcitriol, show that the affinity of the ligand corresponds to the interaction between calcitriol and the
a’-domain alone. In a previous work, we have found that silibinin binds to the aa’ domains49 and now the results
presented here substantiate that the interaction site of calcitriol and silibinin is the same. p
On the basis of these results, we can hypothesize that ERp57 and calcitriol interact directly and their Kd is
about 10−9 M. Table 1. Data obtained by calorimetric analysis of ERp57 and ERp57 selected domains in the presence o
calcitriol. In addition, these data recognize the a’-ERp57 domain as the site with the highest affinity, contrib-
uting to a greater extent than the other potential binding sites to the dissociation constant.hl g
g
p
g
The results obtained by fluorescence measurements and the titration curve obtained by microcalorim
indicate a binding reaction reaching an equilibrium.hl g
g
q
The evidences performed by spectrofluorimetry experiments with FITC-Insulin5, show that there is not any
alteration of the oxidoreductase activity of protein in the presence of calcitriol (Fig. S10 and Table S3).hfi y
p
p
g
The ERp57 domains involved in different interactions have been identified, i.e. calreticulin binds the bb’
domains50 and vancomycin may hinder the interaction between calreticulin/ERp576. Other proteins interact spe-
cifically with a and/or a’, such as REF-1/APE9 and also tapasin51. Moreover, different molecules interact with the
same domains, i.e. DNA interacts with the a’ domain9 and silibinin binds to the a/a’ domains49.h The interaction of ERp57 with ligands such as silibinin49, the EGF receptor30 and calcitriol could induce the
internalization of the protein. This event is the beginning, presumably, of specific processes of the signal trans-
duction that could be deeply studied in order to understand more clearly the roles of ERp57 on the plasma
membrane. Recently it was shown that the disruption of vitamin D/ERp57 pathway mimics amyloid pathology52;
moreover Sugimoto et al.53 demonstrate that Denosomin-Vitamin D3 hybrids, used as anti-Alzheimer’s disease
agents, exhibit nerve re-extension activity in Aβ-damaged neurons via the ERp57 (1,25D3-MARRS) pathway. Considering also the role of ERp57 in important cellular functions and the promising clinical use of vitamin D
analogues in prevention or therapy in several types of malignancies54, the interaction between these two mole-
cules is of sure interest and would need further investigations in the cellular context. Materials and Methods The protein was further purified on a heparin column using a narrow NaCl gra-
dient, dialyzed, and finally concentrated by using a Vivaspin concentrator (VivaScience). FITC-insulin reduction assay. Bovine insulin (Sigma-Aldrich) was labeled with fluorescein
(Sigma-Aldrich) as described in Heuck and Wolosiuk56. FITC-insulin emission intensity was followed at 519 nm
for 30 min at 25 °C, setting the excitation wavelength at 495 nm. For each reaction, 0.7 μM FITC-insulin was used,
in a final volume of 2 ml. The baseline was derived from the addition of 10 μM DTT, in 50 mM Tris-HCl, pH 8.0,
1 mM EDTA. Subsequently, fluorescence enhancement was obtained by adding 0.1 μM ERp57. Calcitriol was then
used at the following concentrations: 0.1, 1, 5 and 10 μM. Fluorescence quenching. Human recombinant ERp57, prepared as previously described, was oxidized for
1 h with 0.2 mM H2O2, and then extensively dialyzed with 20 mM Tris-HCl, pH 8.0, 20 mM NaCl. Alternatively,
rERp57 was reduced with 1.25 μM TCEP (tris-(2-carboxyethyl)-phosphine) for 20 min at room temperature. The
binding was measured by adding increasing concentrations of calcitriol (from 0.41 μM to 2.4 μM final concen-
tration) to a 34 nM solution of oxidized or reduced rERp57, as described above, in 10 mM Tris-HCl, pH 8.0. The
fluorescence of the protein was analyzed in a spectrofluorimeter (FluoroMax, Spex), thermostated at 25 °C, with
an excitation wavelength of 280 nm and an emission of 336 nm. In another experiment, nearly equimolar concen-
trations of ligand were used, from 20 nM to 70 nM, and the protein was finally saturated with 2.4 μM calcitriol. The experiment with mutants (abb’-ERp57and a’-ERp57) was performed using 1.5 μM solution of reduced
ERp57-mutants and increasing concentrations of calcitriol from 0.9 μM to 3.105 μM final concentration. Finally 20μM calcitriol was added as saturation concentration. Calorimetric assay. ITC experiments were performed at 25 °C using a MicroCal ITC200 microcalorimeter
(MicroCal Inc., Northampton, MA, USA). ERp57 and its deletion mutants were extensively dialyzed against the
buffer of choice (0.2 to 1 mM NaCl; 20 mM Tris-HCl, pH 8.0) with Amicon Ultra filters, and the final exchange
buffer was then used to dilute the silibinin stock solution (5 mM in DMSO) and the calcitriol stock solution
(12 mM in ethanol and the percentage of the ethanol was below 0.84%); the DMSO was added to the protein
solution at the same percentage of the ligand solution (below 1%). Materials and Methods Cloning, expression, and purification of recombinant ERp57 and deletion mutants. Human
recombinant ERp57 was cloned and expressed in E. coli BL21 strain using the expression vector pET21 (Novagen)
as described previously7. The recombinant protein was purified by ammonium sulphate fractionation and 8 Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 www.nature.com/scientificreports/ chromatography steps using a procedure similar to the one employed for the purification of pig liver ERp579. Also
the a’-ERp57 mutant was obtained as illustrated previously7. p
p
y
For the preparation of abb’-ERp57 mutant, a plasmid pGEX-2T vector containing the deletion mutant of
human abb’-ERp57 fused to the glutathione S-transferase protein (GST), was used. The coding sequence of
abb’-ERp57 was extracted from pGEX-2T vector by means of a specific restriction reaction (BamHI and EcoRI,
Thermo Scientific). The same digestion was conducted on pET21a vector. The restriction fragments with the
abb’-ERp57 coding sequence, without GST-protein, were purified with the Gel Extraction Kit (Qiagen) and then
ligated into BamHI and EcoRI sites of a pET21a vector (LigaseT4, Thermo Scientific). Cloning was performed
in DH5α E. coli by standard procedures55, while the protein was expressed in BL21 E. coli. The transformed cells
were grown in 2YT medium containing 0.03 mg/ml ampicillin at 37 °C with shaking until the A600 reached
0.6–0.8 OD and then induced with 0.8 mM IPTG at 16 C overnight. Cells were harvested by centrifugation and
resuspended in NEN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, and 0.5 mM EDTA) containing 0.25%
Triton X-100, 5 mM DTT and 0.2 mM PMSF. Cell suspension was lysed by sonication (Ultrasonic homogenizer
UP100H) and cleared by centrifugation at 12,000 g for 10 min at 4 °C. The supernatant was fractionated by ammo-
nium sulphate precipitation. The sonicated supernatant was incubated 2 h at 4 °C with 50% ammonium sulphate
and then centrifuged at 12,000 g for 15 min; the resulting supernatant was added of 75% ammonium sulfate and
incubated 2 h at 4 °C. After a centrifugation at the same speed, the pellet (with deletion mutant) was dissolved in
20 mM Tris-HCl, pH 8.0, 20 mM NaCl and dialyzed against the same buffer. Proteins were loaded onto a heparin
column (Affi-Prep Heparin, Bio-Rad) and eluted with 15 volumes of a linear 40–1,000 mM NaCl gradient in
20 mM Tris-HCl, pH 8.0. Fractions containing the recombinant protein were pooled and dialyzed against 10 mM
Tris-HCl, pH 8.0, 10 mM NaCl. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 ∆
= ∆
−∆
×
+
H app
H ligand
H inhib
(Kinhibitor[inhibitor]]/1
Kinhibitor[inhibitor]) [Ligand:calcitriol, Inhibitor: silibinin]
In Table 2 the concentrations of ERp57 and its mutants are reported with the amounts of the ligand. Bioinformatics analysis. The FTSite server (http://ftsite.bu.edu)34 was used for binding site prediction on
the experimental structure of ERp57. The strategy of FTSite consists in exploring the potential interactions of the
surface regions of a protein with 16 small organic molecules, which vary in size and shape, and it has been shown
to be effective in detecting ‘hot spots’ involved in binding to drug-size ligands Blind docking experiments were
performed using AutoDock4.2 software57. We performed blind docking in order to find the binding sites of calci-
triol onto ERp57 without any prior knowledge of its location. The initial coordinates of ERp57 have been obtained
from the 2.6 Å resolution structure of tapasin-ERp57 heterodimer51 (PDB code: 3F8U). For the study presented
here, we selected the coordinates of chain A, containing the residues 25–493 of ERp57 with the exclusion of the
N-terminal signal peptide and of the residues 494–501 at the C-terminus. Input coordinates of calcitriol were
extracted from the crystal structure of the nuclear receptor for vitamin d (VDR) complexed to calcitriol58 (PDB
code: 1DB1). Blind docking runs including 100 runs each were set up in the following way. Briefly, the target and
ligand molecules were equipped with Gasteiger charges using AutoDock Tools (ADT)57. All torsion angles of
the calcitriol were left free to vary during the minimization. The ERp57 coordinates were kept fixed during the
docking simulations. The dimensions of the box were large enough to include the whole protein, and the grid
spacing was set to 0.375 Å. Grid searching was performed using the Lamarckian genetic algorithm. The number
of energy evaluations was 2.5 × 106 (25 × 106 for focused dockings) and a population size of 200 was applied. All
other parameters were set at their default values. All poses of calcitriol were subsequently clustered. Docking runs
were started with a random ligand position and orientation. The docking poses were analyzed with ADT and
the images were generated with Pymol (The PyMOL Molecular Graphics System, Version 0.99rc6 Schrödinger,
LLC)59 and ADT. References 1. Turano, C., Gaucci, E., Grillo, C. & Chichiarelli, S. ERp57/GRP58: a protein with multiple functions. Cell. Mol. Biol. Lett. 16, 539–563
(2011). 2. Chichiarelli, S. et al. Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line. Arch. Biochem
Biophys. 494, 178–183 (2010).i 2. Chichiarelli, S. et al. Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line. Arch. Biochem
Biophys. 494, 178–183 (2010). 3 F
A
t l Bi di
f h
i di
lfid i
i
f
ER 60
h
l
i
i
d
i
f DNA J C ll p y
3. Ferraro, A. et al. Binding of the protein disulfide isomerase isoform ERp60 to the nuclear matrix associated regions of DNA. J. Cell
Biochem. 72, 528–539 (1999).hi 4. Chichiarelli, S. et al. The stress protein ERp57/GRP58 binds specific DNA sequenze in HeLa cells. J. Cell. Physiol. 210, 343–351
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5. Gaucci, E. et al. The binding of antibiotics to ERp57/GRP58. J. Antibiot. (Tokyo) 61, 400–402 (2008).f h
6. Frasconi, M. et al. Interaction of ERp57 with calreticulin: Analysis of complex formation and effects of vancomycin. Biophys. Chem. 160, 46–53 (2012). 7. Trnkovà, L., Ricci, D., Grillo, C., Colotti, G. & Altieri, F. Green tea catechins can bind and modify ERp57/PDIA3 activity. Biochim
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8. Dick, T. P., Bangia, N., Peaper, D. R. & Cresswell, P. Disulfide bond isomerization and the assembly of MHC class I-peptide
complexes. Immunity 16, 87–98 (2002). y
9. Grillo, C. et al. Cooperative activity of Ref-1/APE and ERp57 in reductive activation of transcription factors. Free Radic. Biol. Med. 41, 1113–1123 (2006).i 10. Sciandra, F. et al. Dystroglycan is associated to the disulfide isomerase ERp57. Exp. Cell. Res. 18, 2460–2469 (2012). 11. Nemere, I. et al. Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane bind
phosphate uptake in intestinal cells. Proc. Natl. Acad. Sci. USA 101, 7392–7397 (2004). 11. Nemere, I. et al. Ribozyme knockdown functionally links a 1,25(OH)2D3 membrane binding protein (1,25D3-MARRS) and
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p
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12. Carlberg, C. et al. Two nuclear signalling pathways for vitamin D. Nature 361, 657–660 (1993). 3. Christakos, S., Dhawan, P., Liu, Y., Peng, X. & Porta, A. New insights into the mechanisms of vitamin D action. J. Cell. Biochem. 88
695–705 (2003). Materials and Methods Samples were centrifuged before the exper-
iments to eliminate possible aggregates. Protein and ligand solutions were degassed before use. Titrations were
performed at 25 °C. The protein solution was placed in the sample cell, and each ligand solution was loaded into
the syringe injector. The titrations involved 19 injections of 2 μL at 180 s intervals. The syringe stirring speed was
set at 1,000 rpm. The reaction was very fast, as shown by the immediate appearance of an endothermic sharp
peak following the addition of ligand to the protein and its deletion mutants solution in the microcalorimetric
titration experiment. Reference titrations of ligand into buffer were used to correct for heats of dilution. The
thermodynamic data were processed with Origin 7.0 software provided by MicroCal. The values of ΔH were
measured for each titration, and fitting the binding isotherms with a one-site binding model yielded the values of
the association constant (Ka). The system also gave information of the change in entropy (ΔS). The binding free
energy (ΔG) and dissociation constant (Kd) were calculated from the experimentally determined values of ΔH
and Ka, using eqs 1 and 2: ∆
= −
= ∆
−
∆S
G
RT ln(Ka)
H
T
(1) (1) (2) =
Kd
1/Ka where R is the gas constant (1.987 cal•mol−1•K−1), and T is the working temperature (298 K).h where R is the gas constant (1.987 cal•mol−1•K−1), and T is the working temperature (298 K).h g
g
p
The in vitro binding of calcitriol and ERp57 was studied also by competition ligand binding by displacement
isothermal titration calorimetry. The apparent binding constant, in the competition experiments, depends on the
concentration of free inhibitor, which changes during the experiment36. Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 9 www.nature.com/scientificreports/ [protein or mutants]
Vitamin D
abb’- ERp57
20 μM
100 μM
a’- ERp57
0.05 μM
0.5 μM
ERp57 (silibinin)
5 μM (50 μM)
10 μM
Table 2. ERp57 and ERp57 selected domains concentration and ligands amount used in the calorimetric
analysis. [protein or mutants]
Vitamin D
abb’- ERp57
20 μM
100 μM
a’- ERp57
0.05 μM
0.5 μM
ERp57 (silibinin)
5 μM (50 μM)
10 μM
Table 2. ERp57 and ERp57 selected domains concentration and ligands amount used in the calorimetric
analysis. Table 2. ERp57 and ERp57 selected domains concentration and ligands amount used in the calorimetric
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The authors thank Prof. Carlo Turano for fruitful discussions. This work was supported by grants of the Istituto
Pasteur‐Fondazione Cenci Bolognetti and Sapienza Università di Roma. Author Contributions S.C. and E.G. conceived the experiments; E.G. and D.R. designed the in silico experiments; E.G. carried out the in
silico experiments and analyzed the data; E.G. and G.N. conducted the fluorimetric experiments; L.C. and G.N. conducted the calorimetric experiments; C.G. and G.N. prepared the protein and recombinant mutants; L.C. and
S.C. analyzed the fluorimetric and calorimentric data; S.C. and E.G. prepared the manuscript; D.R., F.A., and M.E. contributed to a critical revision. All authors reviewed the manuscript. www.nature.com/scientificreports/ Why vitamin D in Alzheimer’s disease? The hypothesis. J Alzheimers Dis. 40, 257–269 (2014) 52. Gezen-Ak, D., Yılmazer, S. & Dursun, E. Why vitamin D in Alzheimers disease? The hypothesis. J Alzheimers Dis. 40, 257–269 (2014). 53. Sugimoto, K., Yajima, H., Hayashi, Y., Minato, D., Terasaki, S., Tohda, C. & Matsuya, Y. Synthesis of Denosomin-Vitamin D3 Hybrids
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9. Sanner, M. F. Python: A Programming Language for Software Integration and Development. J. Mol. Graphics Mod. 17, 57–61 (1999) Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 11 www.nature.com/scientificreports/ Acknowledgementsh Scientific Reports | 6:37957 | DOI: 10.1038/srep37957 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Gaucci, E. et al. Analysis of the interaction of calcitriol with the disulfide isomerase
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Relevance of student and contextual school variables in
explaining a student’s severity of violence experienced Mooij, T. (2015). Relevance of student and contextual school variables in explaining a student’s severity of
violence experienced. Teachers and Teaching: Theory and Practice, 21(8), 926-940. https://doi.org/10.1080/13540602.2015.1005864 Mooij, T. (2015). Relevance of student and contextual school variables in explaining a student’s severity of
violence experienced. Teachers and Teaching: Theory and Practice, 21(8), 926-940. https://doi.org/10.1080/13540602.2015.1005864 Mooij, T. (2015). Relevance of student and contextual school variables in explaining a student’s severity of
violence experienced. Teachers and Teaching: Theory and Practice, 21(8), 926-940. https://doi.org/10.1080/13540602.2015.1005864 DOI:
10.1080/13540602.2015.1005864 Document status and date:
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www.ou.nl Relevance of student and contextual school variables Relevance of student and contextual school variables in explaining a
student’s severity of violence experienced Ton Mooij1,2 Abstract Teachers conceptualise and interpret violent behaviour of secondary students in different
ways. They also differ in their estimates of the relevance of student and contextual school
variables when explaining the severity of violence experienced by students. Research can
assist here by explicating the role of different types of contextual school variables. The
research question is twofold: 1) Do contextual school variables, in addition to a student’s
personal, family, and educational variables, explain a student’s violent behaviour? 2) If so,
what is the role of student composition variables compared with variables indicating the social
cohesion of the school? A hypothetical model was developed in which personal, family-
related, educational, and school variables of different types simultaneously explain the
severity of violence experienced by a student. The method used to test the model empirically
is secondary analysis of data collected in a Dutch national survey on school safety in
secondary education (N students = 78,840; N schools = 219). Severity of violence
experienced is assessed by the Mokken Scale on Severity of Violence Experienced (MSSVE). Multiple regression analyses reveal that a student who is older, a young male, born in the
country of residence, feels at home in another country, does not have an intact family, is not
religious, is enrolled in the highest educational track, and is achieving lower marks in the
school subjects of language and mathematics, experiences more severe violence than other
students (explained variance 3.4%). Simultaneously, different types of contextual school
variables are differently relevant. Mean severity of violence experienced by students at school
indicates clearly more variance (2.3%) than the combination of student composition variables
(0.4%). The conclusion is that the theoretical model is empirically supported, which also
underlines the validity of the MSSVE. The discussion focuses on a comprehensive multilevel
approach to stimulate and check improvement of social cohesion at school. 2 Open Universiteit in The Netherlands (Welten Instituut), Heerlen 2 Open Universiteit in The Netherlands (Welten Instituut), Heerlen 1 Relevance of student and contextual school variables Keywords: school safety; student violence; Mokken Scale on Severity of Violence
Experienced (MSSVE); secondary teachers; multiple regression analysis; Internet-based
monitoring Introduction Teachers and school leaders therefore need adequate and timely information about their eachers and school leaders therefore need adequate and timely information about their 2 Relevance of student and contextual school variables students’ social behaviour which does not seem to be generally or systematically available or
provided. Another complicating issue is that researchers assess violent behaviour within differing
behaviour areas and by means of differing procedures. These instruments and methods
include, for example, self-reporting instruments like the Olweus Bully/Victim Questionnaire
(Solberg & Olweus, 2003) or variations thereof (Wang, Iannotti, Luk, & Nansel, 2010), multi-
dimensional psychometric scales describing various types of peer victimisation (Mynard &
Joseph, 2000), clustering procedures to identify students with similar victimisation
experiences (Felix, Furlong, & Austin, 2009), and approaches that scale students according to
the severity of violence the victims have experienced (Michie & Cooke, 2006; Nylund,
Bellmore, Nishina, & Graham, 2007). One of the approaches to tackle the diversity of assessment procedures can be based
on Item Response Theory (IRT). IRT models generally clarify item functions on a latent
scale, relative to one another, with each item situated on a continuum of the underlying
construct from low to high difficulty or severity (cf. Schafer, 1996). Van Schuur (2003) treats
‘Mokken Scale Analysis’ as a probabilistic IRT model in which the probability of a positive
response to a dichotomous item depends on one or more respondent parameters and one or
more item parameters. Both types of parameters can be isolated and estimated separately
(Mokken, 1997; Molenaar & Sijtsma, 2000). For example, Regan, Bartholomew, Kwong,
Trinke, and Henderson (2006) evaluated the structure of the physical violence scale, part of
the Conflict Tactics Scales (CTS). They determined the ordering of items used to assess 14
acts of physical violence within heterosexual relationships in a sample of women and men. Nitschke, Osterheider, and Mokros (2009) developed a Mokken scale based on 11 items to
discriminate sexual sadists from sexual non-sadists. Furthermore, Mooij (2012) used data on
secondary school students concerning six types of violence to construct a Mokken scale. In
the unidimensional results, 25 items reflect an increase in the severity of violence experienced
by students: from verbal and mild physical violence, which occurs most frequently, to
combinations of social, material and severe physical violence, to very severe and serious
sexual violence, which occurs least frequently. Introduction Teachers perceive and interpret misbehaviour and the violent behaviour of students in different
ways (cf. Hong, 2012; Salvano-Pardieu, Fontaine, Bouazzaoui, & Florer, 2009). Teachers perceive and interpret misbehaviour and the violent behaviour of students in different
ways (cf. Hong, 2012; Salvano-Pardieu, Fontaine, Bouazzaoui, & Florer, 2009). Characteristics of teachers, the school and the students seem to play a role here. For example,
teachers who are younger, female, or working in low-attainment educational settings, use
more curricular differentiation and collaborate more with students on disciplinary issues than
other teachers (Mooij, 2011a). Furthermore, teachers in low-attainment schools, schools
situated in cities, or who are homosexual/lesbian, experience more violent behaviour as a
victim or witness than their respective counterparts. At the school level, social discrimination
and corresponding behavioural processes between teachers and students indicate the quality of
the social cohesion in a school (cf. Beauvais & Jenson, 2002; Carbines, Wyatt, & Robb, 2006;
Peschar, 2005). Social cohesion of a school reflects the degree of connectivity between
individual teachers or students and between groups of teachers or students. In this respect
research in secondary schools revealed the existence of school-based social mirroring
processes between teachers and students (Mooij, Sijbers, & Sperber, 2006). Teachers’
different perceptions and interpretations of the misbehaviour and violent behaviour of students
may thus be correct and indeed correspond with real differences in student characteristics and
violence motives and behaviour (cf. Mooij, 2011b). However, different interpretations of student behaviour between teachers in the same
school may hinder the adequate recognition and timely reduction or prevention of student
violence, which may increase the problem of violence for both teachers and students. This
delay and the lack of precision seem to be one of the reasons why violence in school is a major
concern for teachers and other professionals in countries such as the United States of America
(American Psychological Association, 1993; Mayer & Furlong, 2010; National Education
Association, 1994), New Zealand (Office of the Children’s Commissioner, 2010) and the
Netherlands (Ministry of Education, Culture and Science, 2011). Schools are complex
organisations in which professionals such as teachers and school leaders are expected to
collaborate to promote school safety and to assist students in their learning processes. Introduction The reliability of this ‘Mokken Scale on
Severity of Violence Experienced’ (MSSVE) is relatively high (rho=0.94), which was
confirmed in a cross-validation study. This scale result on the MSSVE can be used to clarify more of the variables that are
relevant in the explanation of students’ violent behaviour. Valid empirical information could,
for example, assist teachers to develop a more unified picture of what is going on in their 3 Relevance of student and contextual school variables school and, therefore, to develop strategies to address the problem. This teacher relevance
aligns with the need to know more about the meaning and potentials of Mokken scaling in this
field (cf. Siu, 2011; You, Ritchey, Furlong, Shochet, & Boman, 2011). In this respect the
score of a student on the MSSVE (Mooij, 2012) can be theoretically related to various types
of explanatory variables, followed by empirical assessment in practice, to check the validity
of the MSSVE. At an individual level, personal variables, like age and gender, and specific
family or educational characteristics, seem relevant (Armstrong, 2011; Kettler, 2011). At the
same time, various types of contextual school variables seem to be applicable, for example,
means of personal or family variables of the students at school (‘student composition
variables’), or variables characterising social school policy and school cohesiveness
(Cronbach, 1983; Henry, Farrell, Schoeny, Tolan, & Dymnicki, 2011; Hong, 2012; Mooij,
Smeets, & De Wit, 2011). As the research of Beauvais and Jenson (2002) and Carbines et al. (2006) makes clear,
a student’s integration in a school’s social, cultural, and curriculum processes is dependent on
many characteristics that operate more or less simultaneously in practice. In addition to the
relevance of personal and family variables, one main issue for teachers and researchers is the
relevance of these different types of contextual school variables for the students’ experience
of violence. In particular teachers and school leaders will be supported by adequate and timely
information about their students’ social behaviour and knowing about the possible influences
of other variables on that behaviour. To provide for this information, the question for research
is formulated as: 1) Do contextual school variables, in addition to a student’s personal, family,
and educational variables, explain a student’s violent behaviour? 2) If so, what is the role of
student composition variables compared with variables indicating the social cohesion of the
school? Introduction The present goal is to answer this question by elaborating a theoretical explanatory
model and checking the model empirically by relating the MSSVE score to other individual
student variables and different types of contextual school variables. The outcome will
determine both the validity and usability of the MSSVE and the possibly differentiated
relevance of personal, family, educational, and school contextual variables to the students’
violent behaviour. Theoretical model 4 4 Relevance of student and contextual school variables Research on violence demonstrates some consistent findings about relationships between
individual or personal and other variables, and a student’s violence score. Figure 1 presents an
overview in a theoretical explanatory model that elaborates the hypothesised influences of
specific student level and school level variables on the severity of violence experienced by a
student. Figure 1 about here At the student level, traditionally relevant variables are personal background variables such as
age and gender (see Figure 1). Being a young male, and being older, is to experience more
severe types of violence than being a young female or being younger (Farrow & Fox, 2011;
Loeber, Slot, Van der Laan, & Hoeve, 2008; Marshall, 1992; Regan et al., 2006). In addition,
variables such as country of birth, and feeling at home in the country of actual residence, are
important for a student’s functioning and sense of well-being in school (Beauvais & Jenson,
2002; Carbines et al., 2006; Felix et al., 2009). Being foreign-born, and not feeling at home in
the country of residence, appear to be related to less than optimal functioning in education,
which may imply that a student has experienced more violence compared with counterparts
who were born in, and feel at home in, the country of residence (Mooij, De Wit, & Fettelaar,
2011). Relevant family-related variables are whether or not the student’s family is intact and
whether or not he or she is religious (see Figure 1). Having an intact family and being
religious are both related to living in a more harmonious or socially cohesive environment,
which is assumed to involve less experience of violence (Beauvais & Jenson, 2002; Carbines
et al., 2006; Felix et al., 2009). Other types of individual student variables are educational
variables such as educational track and school marks. The higher the educational track, and
the better a pupil’s educational performance as expressed in higher marks, the less violence
the pupil is expected to experience at school (Loeber et al., 2008; Marsh & Martin, 2011). Attending a school means being part of many complex and different social, emotional,
and curriculum processes that are simultaneously active in different multilevel ways
(Cronbach, 1983; Felix et al., 2009). At the school level, various types of variable, which
indicate the school’s social composition, may be relevant to the severity of violence
experienced by a student (see Figure 1). A first category is based on personal student
characteristics. It may be that, at schools that have relatively more older students, or more
young males than young females, and so on (see the student level in Figure 1), each student in 5 Relevance of student and contextual school variables the school experiences more violent behaviour than students attending schools with other or
opposite characteristics (cf. Figure 1 about here also Lee, Borden, Serido, & Perkins, 2009; Matjasko, 2011). Students’ mean educational track and their school marks may also play a role (American
Psychological Association, 1993): achieving higher marks will motivate learning and thus
prevent violent behaviour. A second category of school variables supposed to be relevant concerns the social
cohesion or social atmosphere of the school. Such contextual school variables may be more
important to each student’s social and violent behaviour than, for example, contextual school
characteristics based on indicators of the school’s student composition (Beauvais & Jenson,
2002; Carbines et al., 2006; Henry et al., 2011). In this respect, mean level of severity of
violence experienced by the students at school can function as an indicator of the school’s
degree of social cohesion. Table 1 about here The information in Table 1 about educational track reflects the differentiation in the Dutch
secondary educational system, which varies from special education (category for lowest
attainment) to pre-university education (category for highest attainment). The distribution
across educational tracks is generally in line with national distributions (Mooij et al., 2008). Mean school marks are satisfactory (school marks usually vary from 1 (lowest) to 10
(highest)). Violence experienced by a student was assessed by 29 items reflecting antisocial or
aggressive activities typical of verbal, material, social, mild physical, severe physical, and
sexual types of violence. Each student indicated whether he or she had experienced each
antisocial activity since the 2007 summer vacation (August) and the date of data collection in
early 2008. Scoring of each item was performed by the student choosing one of seven answer
alternatives (from ‘never’ to ‘always’). For technical reasons (see Molenaar & Sijtsma, 2000),
the scores obtained for each item were dichotomised (never=0, once or more=1). Mokken
scale analysis resulted in a reliable scale (MSSVE: reliability rho=0.94) consisting of 25 items
(see Table 2, and Mooij, 2012). Secondary analysis In 2005 an Internet-based survey was developed to assist the Dutch Ministry of Education,
Culture and Science record and evaluate trends in school safety among students, school staff,
and school leadership. Starting in 2006, every second year both national government and
participating schools should be informed about cross-sectional and longitudinal results
concerning school safety and the corresponding feelings, experiences, and incidents of
violence. Moreover, government and schools were interested in school measures to reduce
and prevent violence (see also Mooij, Smeets, & De Wit, 2011). The model in Figure 1
contains part of the variables integrated in the digital national survey. Therefore, the
theoretical model can be checked empirically by performing secondary analysis on part of
Dutch national data on school safety (cf. Mooij, De Wit, & Polman, 2008). The secondary analysis uses the national data collected in 2008. In this year, a total of
78,840 students participated in the survey at 219 secondary schools located throughout the
Netherlands. The students completed the Internet-based questionnaire in their classrooms,
under teacher supervision (see further Mooij, 2011a, 2011b). Univariate and multivariate data
analyses were carried out with the Statistical Package for the Social Sciences (SPSS, version
17.0). 6 Relevance of student and contextual school variables Relevance of student and contextual school variables Student-level variables The variables at the student level are operationalised as illustrated in Table 1. The variables
are shown in combination with their univariate results. The mean age of the secondary school
students is 14; 50% are young males. About 6% of the students were not born in the country
of residence, whereas 10% feel at home in a country other than the country of residence. Some 79% have an intact family. About 45% are non-religious; 18% are religious but do not
attend church, mosque or temple; and 37% are religious and do attend one of these religious
buildings. Table 2 about here The ordered items in Table 2 reflect a unidimensional latent Mokken Scale construct
characterised by local independence, or respondent and item measurement invariance. For
each item it is true that the more the student can be described in terms of the construct, the
greater the chance that the response to the item will be positive (the scale is ‘monotone
homogeneous’) (Molenaar & Sijtsma, 2000; Sijtsma & Molenaar, 2002). Furthermore, 7 Relevance of student and contextual school variables MSSVE is characterised by ‘double monotonicity’ which implies that the ordering of items is
uniform across groups of respondents or, in other words, item response functions do not
intersect. Mooij (2012) confirmed that the Mokken scale of 25 items is invariant between
groups based on gender (young males versus young females), country of birth (country of
residence versus another country), and feeling at home in the country (country of residence
versus another country). The invariance supports the existence of the Mokken scale both
within and across each subcategory, for example for young males, young females, and young
males and young females simultaneously. This invariance result does not imply that, for
example, young males and young females obtain the same total score. Given these outcomes,
the sum score of a student on the MSSVE can be used to indicate each student’s scale score
on severity of violence experienced (see Table 2). School-level variables In line with the modelling in Figure 1, a first category of contextual school variables consists
of student composition variables. These variables are indicated by per school aggregated
personal student variables such as mean age, mean percentage of young females, and so on. Table 3 presents descriptive results of these mean or composite variables; the number of
schools is either 209 or 210. The second category of school-level variable concerns the
indicator of the school’s social cohesion, which is based on the mean score on the MSSVE for
all students per school who completed this instrument. Table 3 about here Relevance of student and contextual school variables Relevance of student and contextual school variables Model 3 informs the relevance of each of these two categories of contextual school variables,
taking Model 1 as the same base model. Results Table 4 presents the results on Models 1-3 in terms of B coefficients, standard errors (SE),
statistical significances, and percentages of variance explained. The analyses are carried out
on data of 70.791 students representing 208 secondary schools. Under Model 1, all student
variables are entered simultaneously in one step to assess their relevance. The results in Table
4 illustrate that, compared with younger students and young females, older students and
young males experience more severe violence. Also, students experience more severe
violence when they are born in the country of residence, feel at home in a country other than
the country of residence, and do not have an intact family. Compared with being non-
religious, which is the category of reference, students who are religious experience less severe
violence, regardless of whether or not they attend church, mosque, or temple. The reference
category for educational track is the highest track for secondary education, that is, pre-
university education. The simultaneous regression results of Model 1 illustrate that, in
addition to the other outcomes, attending pre-university education and achieving lower marks
in the subjects of Dutch language and mathematics increase a student’s score on severity of
violence experienced. The total percentage of explained variance is 3.4. Analysis The scores on the school variables in Table 3 were assigned to each student within each
school. In other terms, each student in a school received disaggregated or contextual scores
based on the mean characteristic of the school. This procedure facilitates the carrying out of
three direct or simultaneous multiple regression analyses at an individual level of analysis. Figure 1 serves to analyse and compare three models. Model 1 combines the student level
variables of Figure 1, to check their simultaneous relevance in one step to explain the
individual score on the MSSVE. Model 2 adds the student composition variables as
contextual school variables to the student level variables of Figure 1, whereas Model 3
combines the disaggregated indicator of the school’s social cohesion with the student level
variables. In particular the comparison of the relative variance explained by Model 2 versus 8 Table 4 about here The results under Model 2 in Table 4 demonstrate that including the first category of
contextual school variables in the multiple regression analysis adds another 0.4% variance to
the explained variance of Model 1. Furthermore, ‘being religious but not church-going’ no
longer differs from not being religious, and enrolment in special education no longer differs
from pre-university education in terms of explaining severity of violence experienced. Student
composition variables that are statistically relevant in increasing the severity of violence
experienced by a student are: a higher percentage of younger students at school, a higher
percentage of foreign-born students, a higher percentage of students who do not feel at home
in the country, a higher percentage of students with an intact family (which differs from the
effect at the student level), a higher percentage of students who are not religious, and a higher
percentage of students achieving lower school marks in mathematics. 9 Relevance of student and contextual school variables The results under Model 3 in Table 4 demonstrate that including only the mean score
on the MSSVE as a contextual school variable clearly raises the percentage of explained
variance. This percentage increases from 3.4% (Model 1) to 5.7% (Model 3), which is more
than the performance of Model 2 (3.8%). Relevance of student and contextual school variables The interpretation of these results emphasises the relevance of explanatory variables at
both the student level and the school level (see Figure 1). Moreover, the importance of
personal, family, and educational variables at the student level is generally in line with the
expectations based on the research literature as expressed above in the theoretical design of
Figure 1. The role of pre-university education is contrary to expectations, however. This may
be because, in the present design and analyses, the outcome is statistically controlled for
student age, school marks of school subjects that are important in the school career, and the
other variables. This issue deserves more research, in particular regarding the role of the other
personal, family, educational, and school mean variables. In this respect the mean MSSVE
variable acts as a contextual school variable indicating rather strong aspects of the social and
cultural environment of a student. The present outcome therefore seems to confirm the
qualitative research of Beauvais and Jenson (2002) and Carbines et al. (2006). Their research
suggests that, when students at a school show a low degree of pro-social behaviour, each
student has to adjust in order to function relatively adequately. On the other hand, at schools
where pro-social behaviour and corresponding social cohesion are relatively high, each
student can more easily participate or join in. In other terms: a student in a school
characterised by a relatively high degree of severity of violence has to adapt and become
rather violent too, or become isolated, in order to survive in this school environment. In particular the indicator of the social cohesion of the school, that is the students’
mean level of severity of violence experienced, then seems the most relevant for teachers and
school leaders in evaluating social safety and students’ violent behaviour at school. This is
also the case because measures to change student characteristics like age, gender, or intactness
of family, are hardly or not possible to realise. Therefore, differentiated proposals and
procedures to develop, implement, and empirically check pro-social discrimination and social
behaviour processes within the whole school are required, to effectively prevent and reduce
the incidence of antisocial behaviour of students at the school, class, and individual level. Discussion The aim of this study is to elaborate the relevance of different types of variables concerning
the explanation of severity of violence experienced by a secondary student, to inform teachers
and school leaders about opportunities to promote the degree of social cohesion at school. The
research question to be answered is whether contextual school variables, in addition to a
student’s personal, family, and educational variables, explain a student’s violent behaviour
and, if so, what the role is of student composition variables compared with variables
indicating the social cohesion of school. The hypothetical relevance of different types of
variables has been illustrated in the theoretical model in Figure 1. Secondary analysis of data
of a national study on school safety has been carried out to present an empirical answer to the
research question. In addition to student personal, family, and educational variables, student
composition and social cohesive contextual school variables were used in successive multiple
regression analyses. Statistical significance of B coefficients and relative percentages of
explained variance were used to answer the research question. The results with respect to a first empirical Model 1 show that a student who is
relatively older, a young male, born in the country of residence, who feels at home in a
country other than the country of residence, does not have an intact family, is not religious, is
enrolled in the highest educational track, and is achieving lower school marks in the subjects
of language and mathematics, experiences more severe violence than a student who does not
fit this description. Introducing a first category of student composite variables disaggregated
from the school level causes only minor changes in the individual student level effects (see
Table 4, Model 2). Inclusion of these contextual school variables in the student level variables
of Model 1 raises the percentage of explained variance from 3.4% to 3.8%. Including only the
students’ mean level of severity of violence experienced as a social cohesive contextual
variable demonstrates the contributory effect of this mean school variable to be relatively
large: the explained variance is increased from 3.4% (Model 1) to 5.7% (Model 3). 10 Relevance of student and contextual school variables Probably the best way is to combine pedagogical, social, curricular, and disciplinary measures
in a comprehensive and explicit school culture that offer teachers specific support in their
daily work and effectively integrate students’ social responsibilities since their first day in
school (Alschuler, 1980; Mooij, 1999a, 1999b; Salmivalli, Kaukiainen, & Voeten, 2005;
Stevens, De Bourdeaudhuij, & Van Oost, 2000). At the class level, teachers should refer to the
school-wide culture and rules of social cohesion in pro-social and preventative ways. Teachers
know students best and can get along with them in differentiated ways, either in line with the
normative culture or more individually where needed. At the individual level, all teachers and 11 Relevance of student and contextual school variables students should experience that their own cognitive and other interests, social behaviour, and
feelings of safety are promoted best when all persons in school are functioning in a socially
responsible way (cf. Chapman & Harris, 2004; Sørlie, Hagen, & Ogden, 2008). To realise a whole-school improvement it is thus necessary to design a comprehensive
multilevel approach to promote pro-social behaviour and reduce or prevent violent and other
forms of socially problematic behaviour (Chapman, Buckley, Sheehan, Shochet, &
Romaniuk, 2011; Henry et al., 2011; Mooij & Smeets, 2009). Longitudinal assessment of
curricular, instructional, social, and social policy characteristics is required to estimate causal
effects on the development of each student’s social and learning behaviour (Astor, Guerra, &
Van Acker, 2010; Glover, Gough, Johnson, & Cartwright, 2000; Mooij, 2013; Mooij &
Fettelaar, 2013). Digital information collection and feedback procedures such as sketched in
the Method section can also produce differentiated benchmarks to reliably and validly
evaluate the development of social cohesion from differentiated diagnostic points of view
(Mooij, 2013; Mooij & Fettelaar, 2013). In this way schools can get and use their own
feedback scores including school trends over time, to formulate and evaluate own school
policy goals and own systematic support strategies for both teachers and students. The overall conclusion of this study is that a combination of specific personal, family,
and educational student variables, and in particular the school-based mean variable on
severity of violence experienced, plausibly explains the severity of violence experienced by a
student. Therefore, a multilevel approach to understand and reduce violent behaviour of
students is indicated. Relevance of student and contextual school variables Given the verification carried out with respect to theoretically expected
relationships, it is also concluded that the results provide evidence of the validity of the
MSSVE. These outcomes encourage further exploration of the meaning and relevance of the
Mokken scale. As noted above it would, for example, be very instructive to use other
categories of explanatory variables, like curricular or social characteristics assessed by school
personnel or school leaders at multiple levels (cf. Kuusisaari, 2013; Mitchell, Finkelhor,
Wolak, Ybarra, & Turner, 2010; Mooij et al., 2011; Mooij & Fettelaar, 2013). Relevance of student and contextual school variables Relevance of student and contextual school variables Acknowledgements The national school safety monitor survey for secondary education was developed at the request of the
Dutch Ministry of Education, Culture and Science, which consented to secondary analyses of the data
and publication of the results. I am grateful to Renske Maas for her assistance in performing the
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latent structure of the psychological sense of school membership scale. Journal of
Psychoeducational Assessment, 29(3), 225-237. doi:10.1177/0734282910379968 17 Relevance of student and contextual school variables Relevance of student and contextual school variables Table 1. Univariate results of individual student variables
Variable
N
Min. Max. Mean or %
SD
Age (in years)
78,297
7
25
14.31
1.48
Gender (0=young male; 1=young female)
78,297
0
1
0.50
0.50
Country of birth (0=residence; 1=other)
78,089
0
1
0.06
0.23
Feel at home country (0=resid.; 1=other)
75,587
0
1
0.10
0.29
Have intact family (0=no; 1=yes)
78,297
0
1
0.79
0.41
Religiosity: categories
78,297
Not religious=1
35,187
0
1
44.9%
Religious, do not attend church, etc=2
13,902
0
1
17.8%
Attend church, mosque etc=3
29,208
0
1
37.3%
Educational track: categories
78,297
Special education=1
2576
0
1
3.3%
Practical education=2
2645
0
1
3.4%
Preparatory secondary vocation. educ.=3
43,576
0
1
55.7%
Senior gen. sec. educ./pre-univ. educ.=4
14,286
0
1
18.2%
Pre-university education=5
12,832
0
1
16.4%
Other=6
2382
0
1
3.0%
Mean school marks in
Dutch language
77,682
2
9
6.82
0.97
English language
76,970
2
9
6.86
1.26
mathematics
75,360
2
9
6.73
1.26 Table 1. Univariate results of individual student variables 18 Relevance of student and contextual school variables Table 2. Mokken Scale score assessing severity of violence experienced by secondary
students Table 2. Mokken Scale score assessing severity of violence experienced by secondary
students Table 2. ** Number of students with zero sumscore. * Number of students included = 77,005; number missing = 1292. * Number of students included = 77,005; number missing = 1292. ** Number of students with zero sumscore. Relevance of student and contextual school variables Mokken Scale score assessing severity of violence experienced by secondary
students
Severity
Order and items in Mokken scale
Violence type
N*
%
Cum. %
Most
25 rape
Sexual
1068
1.4
100.0
24 using a weapon
Severe physical
360
0.5
98.6
23 sexually molesting someone
Sexual
489
0.6
98.1
22 threatening someone with a weapon
Severe physical
648
0.8
97.5
21 feeling someone up
Sexual
894
1.1
96.7
20 spray-painting or dirtying something
Material
1318
1.7
95.5
19 stealing
Material
1359
1.7
93.8
18 beating or roughing someone up
Severe physical
1503
1.9
92.0
17 threatening
Social
1802
2.3
90.1
16 intimidating
Social
2071
2.6
87.7
15 destroying things
Material
2339
3.0
85.1
14 blackmailing
Social
2488
3,2
82,0
Middle
13 scratching or damaging something
Material
2908
3.7
78.8
12 sexual gestures
Sexual
3241
4.1
75.0
11 hiding or mislaying something
Material
3668
4.7
70.8
10 making sexual comments
Sexual
3842
4.9
66.0
9 spreading false rumours
Social
4057
5.2
61.0
8 hitting
Mild physical
4400
5.6
55.8
7 tripping someone on purpose
Mild physical
4464
5.7
50.1
6 pushing or kicking someone on purpose
Mild physical
4534
5.8
44.3
5 striking or hurting someone on purpose
Mild physical
4644
5.9
38.4
4 making a lot of noise on purpose
Verbal
5454
7.0
32.3
3 bothering someone on purpose
Verbal
4017
5.1
25.3
2 talking in an extra loud voice
Verbal
3948
5.0
20.0
1 calling someone names
Verbal
4556
5.8
14.9
Least
0
6933**
8.9
9.0
* Number of students included = 77,005; number missing = 1292. 19 Relevance of student and contextual school variables Relevance of student and contextual school variables Table 3. Univariate results of two types of contextual school variables
Variable
N
Min. Max. Relevance of student and contextual school variables Mean
SD
Student composition variables
Mean age of students
210
11
16.33
14.28
0.63
Mean % of young females
210
0
1
47
0.14
Mean % students born in country other than residence
209
0
1
7
0.09
Mean % students feeling at home in other country
209
0
1
12
0.12
Mean % students with intact family
210
0
1
74
0.14
Mean religious (not=1, not church=2, attend church=3)
210
1
3
1.87
0.42
Mean educational track
210
1
5.02
3.30
0.64
Mean school marks in Dutch language
209
6.00
7.83
6.89
0.31
Mean school marks in English language
209
5.98
7.80
6.91
0.30
Mean school marks in mathematics
209
5.50
7.79
6.78
0.32
Indicator of social cohesion
Mean level of severity of violence experienced
209
3.41
25
8.48
2.24 Table 3. Univariate results of two types of contextual school variables 20 Relevance of student and contextual school variables Table 4. Multiple Regression results of severity of violence experienced predicted by
individual variables (model 1) and individual and contextual school variables (models 2 and
3)
Model 1
Model 2
Model 3
B
SE1
B
B
B
SE1
Constant
6.84
0.35**
6.60
1.70**
-1.51
0.40**
Explanatory student variables
Age
.38
0.02**
0.40
0.02**
0.36
0.02**
Gender (0=young male; 1=young female)
-1.30
0.05**
-1.30
0.05**
-1.27
0.05**
Country of birth (0=residence; 1=other)
-0.41
0.11**
-0.37
0.11**
-0.41
0.11**
Feel at home in country (0=resid.; 1=other)
0.57
0.08**
0.75
0.09**
0.69
0.08**
Have intact family (0=no; 1=yes)
-0.69
0.06**
-0.78
0.06**
-0.74
0.06**
Religiosity: Not religious (ref. category)
ref
ref
ref
ref
ref
ref
Religious, do not attend church, mosque
-0.21
0.06**
-0.09
0.07
0.10
0.06
Religious, attend church, mosque, temple
-0.59
0.05**
-0.35
0.06**
-0.32
0.05**
Student’s educational track
Special education
-0.33
0.14*
-0.26
0.14
-0.04
0.14
Practical education
-1.71
0.15**
-1.24
0.17**
-0.34
0.15*
Preparatory secondary vocational educ. -1.27
0.06**
-1.01
0.09**
-0.62
0.07**
Senior general sec. educ. / pre-un. educ. -0.65
0.08**
-0.60
0.08**
-0.44
0.08**
Pre-university education (refer. 1 *: .01 ≤ p ≤ .05; **: p < .01. Relevance of student and contextual school variables categ.)
ref
ref
ref
ref
ref
ref
Other
-0.29
0.15*
-0.17
0.15
-0.06
0.14
Mean school marks in Dutch
-0.09
0.03**
-0.09
0.03**
-0.08
0.03**
Mean school marks in English
-0.02
0.02
-0.01
0.02
-0.01
0.02
Mean school marks in mathematics
-0.12
0.02**
-0.10
0.02**
-0.11
0.02**
Explanatory contextual school variables
Mean age of students at school
--
--
-0.14
0.06*
--
--
Mean % of young females at school
--
--
-0.42
0.30
--
--
Mean % born in country other than residence
--
--
3.65
1.09**
--
--
Mean % who feel at home in other country
--
--
-3.77
0.64**
--
--
Mean % who have intact family
--
--
3.20
0.60**
--
--
Mean % students not religious
--
--
1.62
0.16**
--
--
Mean % students attending pre-univ. educ. --
--
-0.09
0.17
--
--
Mean marks in Dutch language at school
--
--
-.04
0.13
--
--
Mean marks in English language at school
--
--
0.12
0.12
--
--
Mean marks in mathematics at school
--
--
-0.30
0.11**
--
--
Mean level of severity violence experienced
--
--
--
--
0.91
.02**
Percentage of variance explained
3.4
3.8
5.7
1 *: .01 ≤p ≤.05; **: p < .01. Table 4. Multiple Regression results of severity of violence experienced predicted by
individual variables (model 1) and individual and contextual school variables (models 2 and
3) 1 *: .01 ≤ p ≤ .05; **: p < .01. 21 Relevance of student and contextual school variables Relevance of student and contextual school variables Figure 1 Theoretical model of individual and contextual school variables relevant to severity of
violence experienced by a secondary student 22 Relevance of student and contextual school variables
23
Level
Types of independent variables
Dependent variable
Indicator of social cohesion:
School
4. Mean severity of violence experienced
variables
Student composition variables (cf. student variables):
3. Mean educational variables
2. Mean family variables
1. Mean personal background variables
Personal student variables:
Severity of violence
Student
3. Education (track, school marks)
experienced
variables
2. Family (family intact, religion)
1. Personal backgr. (age, gender, country birth, at home country)
Assumed causal relationship
Figure 1 Relevance of student and contextual school variables
Level
Types of independent variables
Dependent variable
Indicator of social cohesion:
School
4. Mean severity of violence experienced
variables
Student composition variables (cf. student variables):
3. Mean educational variables
2. Mean family variables
1. Mean personal background variables
Personal student variables:
Severity of violence
Student
3. Education (track, school marks)
experienced
variables
2. Family (family intact, religion)
1. Personal backgr. (age, gender, country birth, at home country)
Assumed causal relationship Relevance of student and contextual school variables Dependent variable Severity of violence
experienced Figure 1 23
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OLSVis: an animated, interactive visual browser for bio-ontologies
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* Correspondence: vercruys@nt.ntnu.no
Department of Biology, Norwegian University of Science and Technology,
Trondheim, Norway SOFTWARE Open Access © 2012 Vercruysse et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. OLSVis: an animated, interactive visual browser
for bio-ontologies Steven Vercruysse*, Aravind Venkatesan and Martin Kuiper Abstract Background: More than one million terms from biomedical ontologies and controlled vocabularies are available
through the Ontology Lookup Service (OLS). Although OLS provides ample possibility for querying and browsing
terms, the visualization of parts of the ontology graphs is rather limited and inflexible. Results: We created the OLSVis web application, a visualiser for browsing all ontologies available in the OLS
database. OLSVis shows customisable subgraphs of the OLS ontologies. Subgraphs are animated via a real-time
force-based layout algorithm which is fully interactive: each time the user makes a change, e.g. browsing to a new
term, hiding, adding, or dragging terms, the algorithm performs smooth and only essential reorganisations of the
graph. This assures an optimal viewing experience, because subsequent screen layouts are not grossly altered, and
users can easily navigate through the graph. URL: http://ols.wordvis.com Conclusions: The OLSVis web application provides a user-friendly tool to visualise ontologies from the OLS
repository. It broadens the possibilities to investigate and select ontology subgraphs through a smooth visualisation
method. Keywords: Bio-ontologies, Visualisation, Browsing, Web application Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Background [11]. Some of these ontology viewers are text-based, i.e. they use a folder/subfolder-interface to explore hierarch-
ies (e.g. AmiGO [10], MGI GO Browser [12]). However,
many ontologies feature multiple-inheritance: they have
terms that are linked to more than one parent. This mul-
tiple-inheritance is more clearly visualised in a two-di-
mensional
display,
with
nodes
and
connectors
in
between. For instance, the Ontology Lookup Service
(OLS) offers static images that clarify better how terms
are positioned and related to adjacent terms in the hier-
archy, and it provides this unified interface for the
browsing of 79 bio-ontologies [13]. Also, the NCBO Bio-
Portal features the graph browser FlexViz, which draws
subgraphs from 293 ontologies and allows clicking on
terms to bring up its local environment (e.g. child or par-
ent terms) [11]. FlexViz is one of the most powerful
viewers currently available. But despite the added flexi-
bility
and
user-interaction
support,
this
graphical
browser may feel rigid and sometimes confusing, because
it only shifts between static, pre-calculated, and often
sub-optimal configurations. The addition of new terms
may therefore result in large graph reorganisations that
are often hard to follow. Ontologies constitute an increasingly important know-
ledge resource. In the biomedical domain the engineering
of ontologies is predominantly organised by the Open
Biomedical Ontology (OBO) Foundry [1]. Ontologies ar-
range terms hierarchically, connected by relationships in
directed acyclic graphs. OBO ontologies represent forma-
lised biological knowledge and are broadly used in the
analysis and interpretation of experimental results, e.g. by
linking Gene Ontology (GO) terms [2] to gene sets [3,4]. Ontologies provide also an important resource to find ac-
curate terms for use in scientific reports. Many tools are available for browsing ontologies (see
[5,6]). Several of them are integrated in systems dedi-
cated to analyse specific data sets (e.g. calculating overre-
presented GO categories in a gene list: GOrilla [7],
agriGO [8], and GOTermFinder [3]). Other tools are
designed for more general-purpose ontology exploration,
such as QuickGO [9], AmiGO [10], or NCBO’s FlexViz Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Vercruysse et al. Use case I: Browsing ontologies in OLSVis
h
f
ll
h Use case I: Browsing ontologies in OLSVis
The first use case illustrates how OLSVis can make
ontology browsing more intuitive: A user is interested in
the placement of the term ‘mitochondrion’ in the Gene
Ontology hierarchy. She can proceed in two ways: a) se-
lect the ontology of choice and then search for the
chosen term, or b) do a direct search for the GO term
‘mitochondrion’. Autosuggestion enables her to perform
a quick selection of the term from the autosuggest list. Autosuggestion also highlights the occurrence of the
chosen term in other ontologies. The user selects the
GO entry, in this case ‘mitochondrion’ (GO:0005739)
and OLSVis shows the official GO term centered in the
visualiser, along with its child terms and all paths of an-
cestor terms up to a GO root term (Figure 1). The dis-
play of the local environment of the term is dynamic and
the visualiser allows the use of various features to further
refine the display (see the toolbar). For instance, the
‘Eraser’ tool can be used to hide unnecessary terms from
the display panel. In some cases the relation names are
abbreviated for a clearer view and displayed in full by
mouse-hovering. Parts of the graph can be made less/
more compact by increasing/decreasing the length of
connectors. Also, similar to modern map-applications,
OLSVis supports moving the graph by dragging its back-
ground and zooming by mouse scrolling. Furthermore, a
‘filters’ panel is provided to assist the user in narrowing
or broadening the search space. In Figure 1 both ‘is_a’
and ‘part_of’ relations are shown. Although FlexViz constitutes an interesting first step
towards a fully flexible and user-friendly browsing experi-
ence, it leaves room to explore alternative approaches to
ontology visualisation. We therefore investigated if the
use of a fundamentally different layout method would
give a better user-experience for the general-purpose
browsing of ontologies. We chose to implement a real-
time, force-based layout algorithm, which can organise
nodes and connections globally and dynamically. First,
it uses a ‘minimum energy’ principle, ensuring that
nodes and connection-structures are distributed opti-
mally relative to each other in the available screen
space. Second, it immediately responds when (and as
long as) the user interacts with the graph, updating the
nodes’ positions continuously until a new optimal con-
figuration is reached. Background BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Page 2 of 9 One can easily experience why we consider the NCBO
Bioportal’s FlexViz not an optimal viewer even in simple
use scenarios, by trying for example the following exer-
cise in FlexViz: open the ontology ‘Gene Ontology’,
search for ‘mitochondrion’, and then expand some terms
upward towards the root, e.g. ‘intracellular membrane-
bound organelle’ and then ‘intracellular organelle’. When
doing so, one is confronted with terms moving all over
and far out of the viewport, with the viewport shifting
over large distances. This is caused by many terms being
placed next to each other on a too wide hierarchy level. Much of the overview is lost, and an attempt to regain
some of it back by zooming out will leave the node labels
too small to read. Using other layout algorithms than the
default
one
(‘tree
layout’)
seems
also
less
than
satisfactory. applied the concept of a term’s local environment (child
terms and path to the root [13]) as the basic viewing unit
for the visualiser. We illustrate the advantages of OLSVis
through three use cases, exemplifying both the added
functionalities and the enhanced user-experience that
OLSVis brings to ontology visualisation. Use Case I
demonstrates a general overview of the features of OLS-
Vis, highlighting its interactive environment using the
Gene Ontology. Use Case II illustrates an approach to
view common ancestor terms shared between two Gene
Ontology terms; and Use Case III demonstrates the visu-
alisation of the local neighbourhood of a protein. Results and discussion Clicking any term
launches a new search focused on that term. The left panel shows ontology details for the displayed term. connections. Additionally, for customised visualisation,
shared terms could be repositioned and fixed by using
the ‘Anchor node’ functionality. Non-anchored terms will
slide to new optimal positions. This example demon-
strates the potential of OLSVis in displaying environ-
ments for multiple terms which is currently not available
in any other visualiser. the user to save the local environment in XGMML for-
mat that may be imported in network building tools such
as Cytoscape [16,17]. Alternately, the user can obtain the
list of nodes and relationships in the current view in a
tab-delimited file. Results and discussion Web-based ontology visualisers are largely used for
browsing and to analyse the placement of a given term
in an ontology. They help to get a grasp of the local en-
vironment of a term of interest or to view terms that
form the connection to the root term (path to the root). As
bio-ontologies
are
getting
increasingly
complex,
browsing through them requires a visualiser that offers
more intuitive functionalities such as the autosuggestion
of terms, an ‘undo’ function, filtering for relationships
and additional functions that facilitate smooth and user-
friendly browsing. The visualisers that are currently
available only have some of these characteristics, and
often show limitations with respect to browsing speed,
scalability issues, context-based display of a term’s envir-
onment,
or
overall
user
interaction
support. This
prompted us to create the web application OLSVis: a fast,
interactive visualiser to explore OLS ontologies based on
minimal and smooth relayouts. OLSVis exploits the
speed
and
ease-of-use
of
the
WordVis
application
[14,15]. Inspired by the Ontology Lookup Service, we Other improvements that OLSVis provides concern
the animation and presentation of terms after specific
user actions. For instance, clicking on ‘cytoplasm’ will
shift the display into cytoplasm’s local environment (the
children and all ancestors of the term ‘cytoplasm’). The
algorithm switches between local environments by gently
pushing out terms and inserting new terms, which allows
a user to easily keep track of the changing display. A but-
ton on the toolbar may be used to prevent automatic re-
moving of nodes. Its dynamic layout algorithm and the
additional graph interaction tools all contribute to the
user-friendliness of OLSVis. Furthermore, OLSVis allows Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Page 3 of 9 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Figure 1 Screenshot of the OLSVis web application. OLSVis has been used to search the Gene Ontology term ‘mitochondrion’ (see search
box). The graph panel on the right shows the term’s child, parent and ancestor terms linked by various relationship types. Clicking any term
launches a new search focused on that term. The left panel shows ontology details for the displayed term. Figure 1 Screenshot of the OLSVis web application. OLSVis has been used to search the Gene Ontology term ‘mitochondrion’ (see search
box). The graph panel on the right shows the term’s child, parent and ancestor terms linked by various relationship types. Use case III: Visualising the local neighbourhood of a
protein Suppose a user wants to identify the common ancestry
between two different terms, in order to assess their re-
latedness. Use case II shows an example based on the
cellular components ‘mitochondria’ and ‘sarcoplasm’. Here the user first selects ‘Gene Ontology’ from the
ontology list and then enters two terms in the search
box, separated by a comma. OLSVis reads both text
strings as separate terms, matches them to their respect-
ive terms in the selected ontology, GO, and then displays
a merged view of their local environments. Figure 2
shows the terms that hereby are displayed, linking ‘mito-
chondria’ and ‘sarcoplasm’ and showing their shared Biologists are often interested in understanding the vari-
ous attributes of a particular protein such as protein
modifications, biological functions, or protein interac-
tions. Use case III illustrates how OLSVis can be used
for visualising the local neighbourhood of a protein. In
this example the protein is cdc23 (H. sapiens). The user
enters the string ‘cdc23’ and the autosuggestion list
shows a number of matches from the Cell Cycle Ontol-
ogy (CCO) [18]. Selection of the term ‘cdc23_HUMAN
(CCO:B0002212)’ displays the local neighbourhood of Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Page 4 of 9 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Figure 2 OLSVis screenshot of use case II. The canvas shows the combined local environments of two search terms, their paths to the root
and thereby the relatedness between the terms. The search box in the left panel shows the two terms. Also a number of terms were ‘anchored’
by the user. eenshot of use case II. The canvas shows the combined local environments of two search terms, their paths to the root
edness between the terms. The search box in the left panel shows the two terms. Also a number of terms were ‘anchored’ Figure 2 OLSVis screenshot of use case II. The canvas shows the combined local environments of two search terms, their paths to the root
and thereby the relatedness between the terms. The search box in the left panel shows the two terms. Also a number of terms were ‘anchored’
by the user. JavaScript and HTML5 make it possible to create anima-
tions that are fully connectable with other elements on
the web page, and that require no extra browser plugins. Layout engine We applied our GraphVis layout module to the explor-
ation of ontologies in OLSVis, and upgraded it among
others with hierarchical layout for parent/ancestor terms,
see Figure 1. When the user searches for an ontology
term, OLSVis will by default show it together with its
child terms and parent terms up to the ontology root(s),
see Figure 1. After initial placement of ontology terms,
OLSVis uses a real-time force-based layout algorithm
that gently moves the terms towards more optimal posi-
tions. The algorithm is explained in [14] and [15]. It Use case III: Visualising the local neighbourhood of a
protein HTML5 defines the < canvas > HTML-element, basically
a rectangular empty space on the web page, onto which
JavaScript code (which is included in the web page)
draws basic shapes like lines, circles, text, etc. Note that
the older SVG (Scalable Vector Graphics) technology
requires
computationally
expensive
(slow)
DOM-
updates; therefore only canvas is appropriate for smooth
animation of large graphs. Because a sufficiently power-
ful JavaScript library for animated graph browsing did
not exist yet, we wrote one from scratch: GraphVis. We
first applied GraphVis in the webapp WordVis [14,15],
which visualises WordNet, a lexical database of English
[19]. this term whilst providing a warning message that alerts
the user as to the large number of terms associated with
the chosen protein. When browsing large ontologies (e.g. CCO), a user usually has to deal with performance issues
as the visualiser may actually fail to load the subgraph
due to its size. Instead, OLSVis loads up to 500 terms
smoothly and if more it gives a notification to the user. The user is suggested to use the filter panel to narrow
the search space for improved performance and viewing. For example, clicking on ‘parents only’ will update the
current view with a simplified graph (Figure 3). Alterna-
tively, a number of relation types could be filtered away. Here we note that CCO includes bidirectional relation-
ships, so leaving some out can clarify the intended par-
ent–child hierarchy. The user may then choose to save
the current display in formats provided by OLSVis. For
instance, biologists to a large extent still work on spread-
sheets where they periodically associate a particular pro-
tein of interest with an ontological term. In such cases,
saving the current view in a tab-delimited format makes
it easier for them to use the terms associated with a pro-
tein in their annotation work. Implementation For the client-side of the software, we used the modern
web technologies of JavaScript and the new HTML5
standard. In contrast to traditional Flash-objects or Java-
applets, which are isolated objects in the web page, Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Page 5 of 9 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Figure 3 Screenshot illustrating use case III. The canvas shows the local neighbourhood of a CCO term. The left panel shows the term name
in the search box and the pop-out panel with filter settings. The filter setting enables the display of only parent terms. Figure 3 Screenshot illustrating use case III. The canvas shows the local neighbourhood of a CCO term. The left panel shows the term name
in the search box and the pop-out panel with filter settings. The filter setting enables the display of only parent terms. OLS web-service to retrieve data proved to be painfully
slow, because each mouse click required several web-ser-
vice queries, which typically resulted in total query times
of several tens of seconds. EBI updates the OLS database
weekly by polling its ontology providers through the
CVS and SVN version control systems. OLSVis detects
OLS’ updates automatically and then updates its local
copy. In addition, a number of table-indexes and pre-
calculated fields are added to enable the speed of OLS-
Vis. On the server-side of OLSVis, PHP scripts translate
client-side requests into custom queries on the local
MySQL database. Note that the web-application’s front-
end is designed independent from the database back-
end. Given software that would be able to calculate
node-environments (filterable paths-to-roots) in reason-
ably short times, the visualiser would be usable also for
other semantic resources. models nodes as repelling, electrically charged rectan-
gles. This distributes them over the screen, prevents
them from occupying the same space (if possible), and
prevents term labels from overlapping. Connections are
modelled as mechanical springs, which hold nodes to-
gether and which may be given a specific preferred-
length in order to create a certain global structure in the
graph. Connections may also have a preferred orienta-
tion (e.g. down-to-up for ‘is a’ links). This layout is fully
interactive: each time the user makes a change (such as
focusing on a new term, hiding, adding or dragging
terms, changing connection lengths), it smoothly yet
minimally reorganises the graph. Implementation This assures an optimal
viewing experience that minimises each operation’s effect
on graph reorganisation, and maximises the user’s ability
to keep track of changes and comprehend the new lay-
out. More features A ‘roots’ link appears next to the search box after select-
ing a specific ontology. Clicking it shows and expands
this ontology’s root term(s) (if defined in the OLS data), i. e. showing them and their child terms. This enables easy
top-down ontology exploration. Multiple terms and iden-
tifiers can be searched, separated by commas. Therefore
in-term commas must be preceded by a backslash, and
genuine backslashes doubled. First hits from autosugges-
tion are then expanded. When a term’s local environment
contains too many terms (this happens with application
ontologies such as the Cell Cycle Ontology [18]), OLSVis
will only show the first 500 terms and will suggest using
filters. OLSVis supports URL-shortcuts: (1)A term or identifier can be expanded directly via
URLs like: ols.wordvis.com/q = GO:0005739, or . . ./q
= mito. The part after /q = will be put in the search
box and the first term that would have been
autosuggested will be expanded. (2)A specific ontology can be preselected via a URL
like: ols.wordvis.com/ont = GO. The part after /ont =
is the ontology’s short name from the selection list. This is a shortcut for users that are mainly interested
in a specific ontology. (3)‘q’ and ‘ont’ can be combined like: (3)‘q’ and ‘ont’ can be combined like:
. . ./ont = GO&q = mitochondrion,sarcoplasm , which
also illustrates a multi-term query. (3)‘q’ and ‘ont’ can be combined like:
. . ./ont = GO&q = mitochondrion,sarcoplasm , which
also illustrates a multi-term query. (4)Some ontologies use non-standard prefixes in term-
identifiers (GO has ‘GO:’, but ZFA may use ‘ZFS:’,
and NEWT has none), so identifiers may be
disambiguated by adding their ontology’s short name
as prefix, e.g. . . ./q = NEWT:1234, or . . ./q = ZFA:
ZFS:0000019. (4)Some ontologies use non-standard prefixes in term-
identifiers (GO has ‘GO:’, but ZFA may use ‘ZFS:’,
and NEWT has none), so identifiers may be
disambiguated by adding their ontology’s short name
as prefix, e.g. . . ./q = NEWT:1234, or . . ./q = ZFA:
ZFS:0000019. Basic visualisation
h
h The chosen term is then expanded: it is placed in the
centre of the graph panel, amidst its local environment of
child terms and parent terms, and connected with further
ancestors up to the ontology root(s). This configuration is
inspired by OLS’ static images [13]. Child terms are
ordered in a half circle under the expanded term; ances-
tors are put in hierarchical levels above it. Relations are
shown as labelled arrows; their lengths are adjusted for
good hierarchical positioning. After initial placement, the
visualiser slides terms to more optimal positions via real-
time animation; hereby the graph ‘feels’ and behaves as if
terms are repelling electric charges (or repelling magnets)
that are connected over mechanic/elastic springs. This
creates a layout that minimises term overlap. In addition,
the connecting arrows undergo a small north–south
orienting force to enhance a hierarchical alignment of
terms. The visible graph is fully customisable: see the
toolbar in Figure 1 or the online description for mouse/
keyboard shortcuts. It has undo/redo history, and terms
can be dragged and pushed around. Clicking on any dis-
played term will re-centre on that term and expand its
local neighbourhood. Hereby the graph is subtly reorga-
nised via real-time animation, and is transformed into the
new term’s local environment (by addition and removal
of terms). This enables easy and intuitive browsing
through ontologies. The automatic removal of already
visible terms can be turned off via the rightmost button
on the toolbar. Hovering over any term makes its defin-
ition pop up. For a relation arrow, its non-abbreviated
name pops up. Leaf terms (=without child terms) get a
slightly orange background. The three most common
relations (is_a, part_of, develops_from) get a coloured
arrow. In the left panel, data for the last expanded term
is shown: its identifier (hovering shows ontology’s full
name) and definition; its synonyms, annotations and
cross-references (as in the OLS database); and its child
terms (each clickable to expand), to make them easier
visible when there are many. When zooming in, OLSVis
increases distances faster than font sizes; this is more
useful and is an extra method (next to electrostatic re-
pulsion) to counteract overlapping terms. Term searching
h l
h OLSVis visualises the contents of the OLS database
[13,20,21], which holds around 80 bio-ontologies and
over 1 million concepts. OLSVis uses a local copy of
OLS‘ publicly available database, in order to provide a
smooth visualisation with fast response times. Only via a
local copy placed on OLSVis’ server can the node envir-
onments be calculated sufficiently fast. The use of the While the user types one (or several) terms or identifiers
(e.g. ‘mito’ or ‘PO:0009001’) in the search box, a selection
of best known matches is shown in a pop-out list. This
includes preferred terms as well as their synonyms. For
each autosuggested term, the ontology’s (short)name and
identifier is shown, and mouse-hovering shows its ontol-
ogy’s full name. Autosuggestions can be confined to a Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Page 6 of 9 Initially the three most common relation types are listed in
the panel; this list grows each time the visualiser encoun-
ters new types. Relation types that are currently in the
visualiser are highlighted. The filter that hides obsolete
terms also hides them in autosuggestion lists. Earlier
expanded terms and their environment are by default
automatically removed when clicking on a new term, but
can be kept in the visualiser by turning off the rightmost
toolbar button. Several toolbar tools enable further cus-
tomisation of the graph. Connections can be made longer
or shorter (also via Alt + scrolling up/down). Terms can
be anchored to a fixed position, and anything can be
removed manually via the Eraser tool. single specific ontology by selecting one from the drop-
down list. Pressing ‘Enter’ in the search box will display
the term that is selected in the autosuggestion list. If the
user has no term selected, OLSVis will take the first term
(also if the autosuggestion list has not appeared yet). Conclusions Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G: GO::
TermFinder–open source software for accessing Gene Ontology
information and finding significantly enriched Gene Ontology terms
associated with a list of genes. Bioinformatics 2004, 20(18):3710–3715. 4. Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess
overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005, 21(16):3448–3449. ,
(
)
5. Katifori A, Halatsis C, Lepouras G, Vassilakis C, Giannopoulou EG: Ontology
Visualization Methods - A Survey. ACM Comput Surv 2007, 39(4):Article10. 6. Lanzenberger M, Sampson J, Rester M: Ontology Visualization: Tools and
Techniques for Visual Representation of Semi-Structured Meta-Data. Journal of Universal Computer Science 2010, 16(7):1036–1054. 5. Katifori A, Halatsis C, Lepouras G, Vassilakis C, Giannopoulou EG: Ontology
Visualization Methods - A Survey. ACM Comput Surv 2007, 39(4):Article10. 6. Lanzenberger M, Sampson J, Rester M: Ontology Visualization: Tools and
Techniques for Visual Representation of Semi-Structured Meta-Data. Journal of Universal Computer Science 2010, 16(7):1036–1054. 7. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z: GOrilla: a tool for
discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinforma 2009, 10:48. 7. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z: GOrilla: a tool for
discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinforma 2009, 10:48. Comparison with other visualisation tools The authors declare that they have no competing interests. The utility and performance of OLSVis was assessed in
comparison to other tools commonly used for ontology
visualisation, including some biological data analysis tools
that have visualisation components integrated in them, as
listed in Table 1. The evaluation addressed a number of cri-
teria, including tool functionality (e.g. support of multiple
term searching); scalability (e.g. handling of large numbers
of terms); and some aspects that capture user-friendliness
and intuitiveness of
browsing (e.g. context-dependent
browsing). The table shows that some of OLSVis’ features
are not provided by any other visualiser, and that the other
tools only support a subset of what OLSVis offers. Clearly,
OLSVis offers the most interactive visualisation environ-
ment. FlexViz ranks well too, as it also provides a relatively
high level of user-interaction; however, OLSVis makes more
efficient and intuitive use of the available screen space. Acknowledgements SV was supported by Funksjonell Genomforskning (FUGE) Midt-Norge and
the Norwegian University of Science and Technology [grant number
81179100]. AV was supported by Faculty of Natural Sciences and Technology,
NTNU, Norway. We thank EBI for making the OLS database available and
keeping it up-to-date. Authors’ contributions
S
d
d
d SV designed and programmed the software and drafted the manuscript. AV
helped testing the software and draft the manuscript. MK helped conceive
the web application and draft the manuscript. All authors read and approved
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annotation data. Bioinformatics 2009, 25(2):288–289. Other requirements: Modern browser: recent
version of Firefox, Chrome, Opera, Safari or Internet
Explorer. (IE 8 not recommended; please upgrade to
IE 9, which supports ‘canvas’ and thus is much
faster). No browser plugin needed. 11. Noy NF, Shah NH, Whetzel PL, Dai B, Dorf M, Griffith N, Jonquet C, Rubin DL,
Storey MA, Chute CG, Musen MA: BioPortal: ontologies and integrated
data resources at the click of a mouse. Nucleic Acids Res 2009, 37
(Web Server issue):W170–W173. 11. Noy NF, Shah NH, Whetzel PL, Dai B, Dorf M, Griffith N, Jonquet C, Rubin DL,
Storey MA, Chute CG, Musen MA: BioPortal: ontologies and integrated
data resources at the click of a mouse. Nucleic Acids Res 2009, 37
(Web Server issue):W170–W173. License: The web-application is freely accessible for
use. 12. Blake JA, Bult CJ, Eppig JT, Kadin JA, Richardson JE, Mouse Genome
Database Group: The Mouse Genome Database genotypes::phenotypes. Nucleic Acids Res 2009, 37(Database issue):D712–D719. 12. Blake JA, Bult CJ, Eppig JT, Kadin JA, Richardson JE, Mouse Genome
Database Group: The Mouse Genome Database genotypes::phenotypes. Nucleic Acids Res 2009, 37(Database issue):D712–D719. Any restrictions to use by non-academics: No
specific restrictions. 13. Côté R, Reisinger F, Martens L, Barsnes H, Vizcaino JA, Hermjakob H: The
Ontology Lookup Service: bigger and better. Conclusions OLSVis was created to improve the exploration of bio-
ontologies. Other visualisers like FlexViz, may feel rigid
and sometimes confusing, because the addition of new
terms may result in largely rearranged term displays. OLSVis demonstrates that the user experience for ontol-
ogy exploration can be substantially improved by using
real-time animation of force-based graph relayout, and
by providing improved user interaction on the graph’s
structure. This new webapp provides the scientific com-
munity with a versatile and more user-friendly tool to
explore ontologies and to find related and more precise
ontology terms. g
,
(
)
2. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP,
Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A,
Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene
ontology: tool for the unification of biology. Nat Genet 2000, 25(1):25–29. 3. Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G: GO::
TermFinder–open source software for accessing Gene Ontology
information and finding significantly enriched Gene Ontology terms
associated with a list of genes. Bioinformatics 2004, 20(18):3710–3715. 4. Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess
overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005 21(16):3448–3449 g
2. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP,
Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A,
Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene
ontology: tool for the unification of biology. Nat Genet 2000, 25(1):25–29. 3. Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G: GO::
TermFinder–open source software for accessing Gene Ontology
information and finding significantly enriched Gene Ontology terms
associated with a list of genes. Bioinformatics 2004, 20(18):3710–3715. 4. Maere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess
overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005, 21(16):3448–3449. 2. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP,
Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A,
Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G: Gene
ontology: tool for the unification of biology. Nat Genet 2000, 25(1):25–29. 3. Customised visualisation Terms in the graph can be right-clicked for more
options. The visible graph can be exported to an XGMML
file (eXtensible Graph Markup and Modeling Language)
and can subsequently be imported in Cytoscape [16,17]
for further analysis. There, node labels will show the term A click on ‘filters’ (left of the search box) brings up a panel
to set filters that prune the expanded node’s environment. For instance, any relation type can be excluded; this means
that they are omitted when building e.g. the path-to-root. ontology visualisers
OntoViz
IsaViz
GOSurfer
GOMiner
OntoTrack
OBO-Edit
QuickGO
AmiG
✓(semi) 1
✓(semi) 1
✓(semi) 1
e
one-by-one
dragging
highlighting a
selected branch
one-by-one
dragging
one-by-one
dragging
ancestors +
children:
layered
graph-view or
radar-view
tree view
ancestors +
children:
layered
ancestors +
children:
layered
ancestors +
children:
layered
ancestors
subfo
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓4
✓4
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
ogies)
OWL
ontologies
RDF graph
visualiser
GO (1)
GO (1)
OWL
ontologies
OBO
ontologies
GO (1)
GO (1
–(Protégé)
–(Java)
–(.exe)
–(Java)
–(Java)
–(Java)
✓(HTML)
✓(HT
functions (which are displayed on OLSVis’ toolbar). 3: Each with their own shortcomings, see ‘Background’. 4: Browser’s back button. 5
e’s “environment”. GOMiner [25], OntoTrack [26], OBO-Edit [27], QuickGO [9], AmiGO [10]). ontology visualisers
OntoViz
IsaViz
GOSurfer
GOMiner
OntoTrack
OBO-Edit
QuickGO
Ami
✓(semi) 1
✓(semi) 1
✓(semi) 1
e
one-by-one
dragging
highlighting a
selected branch
one-by-one
dragging
one-by-one
dragging
ancestors +
children:
layered
graph-view or
radar-view
tree view
ancestors +
children:
layered
ancestors +
children:
layered
ancestors +
children:
layered
ancestors
subf
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓4
✓4
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
ogies)
OWL
ontologies
RDF graph
visualiser
GO (1)
GO (1)
OWL
ontologies
OBO
ontologies
GO (1)
GO
–(Protégé)
–(Java)
–(.exe)
–(Java)
–(Java)
–(Java)
✓(HTML)
✓(H
functions (which are displayed on OLSVis’ toolbar). 3: Each with their own shortcomings, see ‘Background’. 4: Browser’s back button. e’s “environment”. GOMiner [25], OntoTrack [26], OBO-Edit [27], QuickGO [9], AmiGO [10]). Am
sub
✓
✓4
✓
✓
✓ ntology v
Onto
✓(se
one-
drag
ance
child
laye
✓
✓
✓
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✓
es)
OWL
onto
–(Pro
nctions (whic
“environme
Miner [25], O Vercruysse et al. Abbreviations
ll
l 14. WordVis [http://wordvis.com]. CCO: Cell Cycle Ontology; CVS: Concurrent Versions System; EBI: European
Bioinformatics Institute; GO: Gene Ontology; NCBO: National Center for 15. Vercruysse S, Kuiper M: WordVis: JavaScript and Animation to Visualize the
WordNet Relational Dictionary. In Proceedings of the Third International 15. Vercruysse S, Kuiper M: WordVis: JavaScript and Animation to Visualize the
WordNet Relational Dictionary. In Proceedings of the Third International Page 9 of 9 Page 9 of 9 Vercruysse et al. BMC Bioinformatics 2012, 13:116
http://www.biomedcentral.com/1471-2105/13/116 Conference on Intelligent Human Computer Interaction: 29–31 August 2011,
Prague. In press. Conference on Intelligent Human Computer Interaction: 29–31 August 2011,
Prague. In press. 16. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin N,
Schwikowski B, Ideker T: Cytoscape: a software environment for
integrated models of biomolecular interaction networks. Genome Res
2003, 13(11):2498–2504. 17. Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas
R, Avila-Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R,
Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A,
Wang PL, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I,
Schwikowski B, Warner GJ, Ideker T, Bader GD: Integration of biological
networks and gene expression data using Cytoscape. Nat Protoc 2007, 2
(10):2366–2382. 18. Antezana E, Egaña M, Blondé W, Illarramendi A, Bilbao I, De Baets B, Stevens
R, Mironov V, Kuiper M: The Cell Cycle Ontology: An application ontology
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Linguistics. 2nd edition. Edited by Brown K, et al. Oxford: Elsevier;
2005:665–670. 20. Côté RG, Jones P, Apweiler R, Hermjakob H: The Ontology Lookup Service,
a lightweight cross-platform tool for controlled vocabulary queries. BMC
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[http://protegewiki.stanford.edu/wiki/OntoViz] 22. Sintek M: OntoViz Tab: Visualizing Prot ég é Ontologies; 200 [http://protegewiki.stanford.edu/wiki/OntoViz] 23. IsaViz [http://www.w3.org/2001/11/IsaViz] 24. Zhong S, Storch KF, Lipan O, Kao MC, Weitz CJ, Wong WH: GoSurfer: a
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ll
l Zhong S, Storch KF, Lipan O, Kao MC, Weitz CJ, Wong WH: GoSurfer: a
graphical interactive tool for comparative analysis of large gene sets in
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S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein
JN: GoMiner: A Resource for Biological Interpretation of Genomic and
Proteomic Data. Genome Biol 2003, 4(4):R28. 25. Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan
S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein
JN: GoMiner: A Resource for Biological Interpretation of Genomic and
Proteomic Data. Genome Biol 2003, 4(4):R28. 26. Liebig T: OntoTrack: Fast browsing and easy editing of large ontologies,
Proceedings of The Second International Workshop on Evaluation of Ontology-
based Tools; 2003. 27. Day-Richter J, Harris MA, Haendel M: Gene Ontology OBO-Edit Working
Group, Lewis S: OBO-Edit–an ontology editor for biologists. Bioinformatics
2007, 23(16):2198–2200. 27. Day-Richter J, Harris MA, Haendel M: Gene Ontology OBO-Edit Working
Group, Lewis S: OBO-Edit–an ontology editor for biologists. Bioinformatics
2007, 23(16):2198–2200. doi:10.1186/1471-2105-13-116
Cite this article as: Vercruysse et al.: OLSVis: an animated, interactive
visual browser for bio-ontologies. BMC Bioinformatics 2012 13:116. Submit your next manuscript to BioMed Central
and take full advantage of:
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Submit your manuscript at
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and take full advantage of:
• Convenient online submission
• Thorough peer review
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• Immediate publication on acceptance
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• Research which is freely available for redistribution
Submit your manuscript at
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|
https://openalex.org/W2518684893
|
https://www.nature.com/articles/srep33261.pdf
|
English
| null |
High steady-state column density of I(2P3/2) atoms from I2 photodissociation at 532 nm: Towards parity non-conservation measurements
|
Scientific reports
| 2,016
|
cc-by
| 8,827
|
measurements G. E. Katsoprinakis1, G. Chatzidrosos2, J. A. Kypriotakis2, E. Stratakis1 & T. P. Rakitzis1,2 Steady-state column densities of 1017 cm−2 of I(2P3/2) atoms are produced from photodissociation of I2
vapour at 290.5 K using 5 W of 532 nm laser light. Recombination of the I(2P3/2) atoms at the cell walls is
minimized by coating the cell surface with a hydrophobic silane (dimethyldichlorosilane/DMDCS). Operation at room temperature, and at an I2 vapour pressure of ~0.2 mbar, without using a buffer gas,
allows relatively low Lorentz and Doppler widths of ~2π × 1.5 (FWHM) and ~2π × 150 (HW at 1/e2)
Mrad/s, respectively, at the
→
5 P
5 P
°
°
2
3 2
2
1 2
/
/ M1 transition of atomic iodine at 1315 nm. These high
column densities and low linewidths are favorable for parity nonconservation optical rotation
measurements near this M1 transition. Furthermore, as the cell is completely sealed, this method of
production of high-density 127I(2P3/2) atoms is also compatible with using iodine radioisotopes, such as
for the production of high-density 129I(2P3/2). The measurement of weak absorption or polarimetric signals, associated with forbidden transitions, requires large
effective atomic or molecular column densities, typically of around ~1015 cm−2 or higher. High atomic densities
are usually produced using high-temperature ovens (such as for most metals). For example, parity nonconserving
optical rotation was measured in Tl, Bi, and Pb1–4, using column densities of ~1019 cm−2, produced by heating
cells of length ~1 m to temperatures of about 1300 K (giving Doppler widths of ~2π × 250 Mrad/s). Discharge
lamps are used to produce atomic radicals from diatomic dissociation, such as ~2 × 1016 cm−2 O(3PJ) from dis-
charge lamps using O2
5, and ~1016 cm−3 I(2P3/2) atoms from I2
6–8. However, both oven and discharge lamp meth-
ods usually require precursor and/or buffer gases, with pressures of around 20–50 mbar (giving Lorentz widths
of about 2π × 20–200 Mrad/s). PNC optical rotation has only been measured for Tl, Bi, and Pb, which all satisfy the following important cri-
teria: strong and accessible M1 transitions (all happen to be near 1.28 μm); large values for the ratio
R = Im[E1PNC]/M1 (between 10−7–10−6); and can be produced at large column densities, ρc ~ 1019 cm−2. Together,
these factors allowed the production of PNC optical rotation signals of ~1 μrad, and their measurement with a
precision of about 1%. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 17 March 2016
accepted: 24 August 2016
Published: 15 September 2016 1Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas, 71110 Heraklion-Crete,
Greece. 2Department of Physics, University of Crete, 71003 Heraklion-Crete, Greece. Correspondence and requests
for materials should be addressed to T.P.R. (email: ptr@iesl.forth.gr) Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 High steady-state column
density of I(2P3/2) atoms from I2
photodissociation at 532 nm:
Towards parity non-conservation
measurements received: 17 March 2016
accepted: 24 August 2016
Published: 15 September 2016 Results The second term describes the three-body recombination reaction I + I + I2 → 2I2, where
two iodine atoms recombine in the presence of one iodine molecule. Other particles may act as third bodies in this
process, but molecular iodine is by far the most effective19 (in particular, the recombination rate for the 3I → I2 + I
is much smaller). The last term describes the sticking of iodine atoms on the container walls, i.e. I + wall → Iwall. This process eventually leads to recombination of two iodine atoms into a molecule, i.e. Iwall + Iwall → I2, and is
thus a source term for molecular iodine. The produced atomic iodine density depends on the photon flux of the
photodissociating laser, Φ, the temperature, which has an impact predominantly on the molecular iodine density,
[I2], but also on the kr and kw recombination parameters, and thus on the chemical properties of the cell surfaces
which come in contact with I and I2, and which determine the recombination rate, kw.h The steady state solution of Eq. (1) is: The steady state solution of Eq. (1) is: σ
= −
+
+
Φ
∞
k
k
k
k
[I]
4[I ]
2[I ]
(2)
w
w
r
r
2
2
2
2 (2) k
2[I ] r
2 In the absence of the photodissociating green photon flux, Φ = 0, at a given temperature and for a specific cell,
and after equilibrium has been reached, the molecular iodine density, [I2], is a constant determined by the vapour
pressure of the solid iodine pellet inside the I2 holder area, and the temperature of the coldest spot the iodine
vapour comes in contact with. We measure the absorption of a low-power blue led (center λ = 466 nm,
FWHM = 24 nm), to determine the density of the molecular iodine vapor inside the cell. We use the Beer-Lambert
law,
∫
λ
λ
=
σ λ
−
P
P
e
( )
d
l
B,OUT
B,IN
( )[I ]
2
, where PB,IN(OUT) is the incident (transmitted) blue led power, σ is the
absorption cross section of I2, and l = 123 cm is the length of the cell. The lineshape of PB,IN(λ) comes from our
own measurements of the spectrum of the blue led using an Ocean Optics USB4000 spectrometer, while the
wavelength dependence of σ(λ) is from20. Results We consider the simplest possible model for the production of atomic iodine, assuming all iodine is either in the
ground molecular state (denoted as I2) or in the ground atomic state (denoted as I). We assume that the popu-
lation of excited states of I2 and production of I3 molecules are negligible (such assumptions have been used in
previous studies to determine the rate constant kr for the recombination of iodine atoms16,17). In this case, the rate
equation governing the production rate of atomic iodine from photodissociating I2 is given by10,17: σ
=
Φ
−
−
d
dt
k
k
[I]
[I ]
[I] [I ]
[I]
(1)
r
w
2
2
2 (1) where [I] and [I2] are the atomic and molecular iodine densities, respectively, σ = 2.4 × 10−18 cm2 is the I2 photo-
dissociation cross section at 532 nm18, Φ the green laser photon flux, related to the power of the green laser,
P, through Φ =
λ
A
P
hc
1
/ , with A the cell cross sectional area, kr is the gas phase three-body recombination rate, and
kw is the rate at which atomic iodine sticks to the cell walls, where it will eventually recombine into molecular
iodine. We note that for the determination of Φ, the relevant area A is that of the cell, and not of the green laser
beam (as the I and I2 diffusion times are smaller than the time it takes the photodissociating laser to deplete the
molecular iodine). This is corroborated by the observation that the I-atom density is largely homogeneous (we
vary the overlap of the IR and green beams by parallel translation of the IR in the radial direction of the cell), and
insensitive to the size of the photodissociating green beam (maximal variations in the I-atom density of less than
20% are observed).hii The first term on the right-hand side of Eq. (1) quantifies the photodissociation reaction, I2 + hν → I + I, where
I ≡ I(2P3/2), and is the term which creates atomic iodine. It depends on the density of iodine molecules, on the flux
of photons from the 532 nm laser source, and on the photodissociation cross section. The second and third terms
are destruction terms. measurements The maximum PNC optical rotation angle φmax is proportional to R; it is proportional to ρc
for small optical depths
(OD
1), and proportional to (ρc)1/2 for large optical depths
(OD
1)9; finally, φmax
generally increases with decreasing Lorentz and Doppler widths9. Therefore, φmax can be increased by increasing
R (i.e. by choosing a transition with large R) and ρc (i.e. by maximizing the density and pathlength), while decreas-
ing the Lorentz width (e.g. decreasing pressure) and the Doppler width (e.g. decreasing temperature). Not all of
these changes can be performed simultaneously, and an optimum combination of these experimental conditions
must be found. Recently, our group has proposed a method for the measurement of parity nonconserving (PNC) optical
rotation using a novel bowtie cavity9–11 to enhance the effective column density by the number of cavity passes
(~1000), while also introducing signal reversals which allow the isolation of the PNC signals from backgrounds. 1Institute of Electronic Structure and Laser, Foundation for Research and Technology-Hellas, 71110 Heraklion-Crete,
Greece. 2Department of Physics, University of Crete, 71003 Heraklion-Crete, Greece. Correspondence and requests
for materials should be addressed to T.P.R. (email: ptr@iesl.forth.gr) Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 www.nature.com/scientificreports/ The operation of the cavity has been demonstrated through the measurement of chiral optical rotation of gases,
liquids, and thin films12,13. This signal enhancement, and the background suppression and subtraction proce-
dures, open the way for PNC optical rotation measurements in other atomic and molecular systems, which have
smaller values of R and/or can be produced at smaller column densities, such as I(2P3/2)10 and metastable Hg and
Xe11, molecular oxygen14, and other diatomic molecules15. For example, for the I(2P3/2) → I(2P1/2) M1 transition
at 1315 nm, R = 0.8 × 10−8, which is about 20 times smaller than that of the Tl(2P1/2) → Tl(2P3/2) M1 transition at
1.28 μm: R = 1.5 × 10−7. However, Katsoprinakis et al.10 proposed to compensate for this smaller value of R by
using large cavity-enhanced pathlengths (~1000 m) to achieve column densities of about 1020 cm−2 and higher,
but also by generating the I(2P3/2) atoms from the photodissociation of I2 molecules using 532 nm laser light, at
low pressure and near room temperature, so that the Lorentz and Doppler widths can be significantly reduced, to
~2π × 1 Mrad/s and ~2π × 150 Mrad/s, respectively. measurements In addition, the production of high-density atoms through
photodissociation at room temperature is more convenient and more compatible with an optical cavity experi-
ment than using a high-temperature oven.h g
g
p
The aim of this paper is to demonstrate the feasibility of producing I(2P3/2) atoms from I2 photodissociation, at
high single-pass column densities of at least 1017 cm−2, while also achieving low Lorentz and Doppler widths. This
method depends critically on minimizing the sticking and recombination of I(2P3/2) at the cell walls. We study
the dependence of the produced atomic I(2P3/2) density on the photodissociating laser power, and investigate the
effects of varying molecular iodine density and of various coatings of the iodine cell walls. Results To
this end we acquire transmission spectra of the IR laser as its frequency is scanned across the
°
→
°
5 P
5 P
2
3/2
2
1/2
transition of atomic iodine, for various powers of the photodissociating green laser, for various coatings of the
inner surface of the iodine cell, and for molecular iodine vapour densities corresponding to two different temper-
atures, 290.5 K and 273 K (for the same cell). The experimental setup used is described in the Methods section,
and is a typical pump-probe setup, with the CW photodissociating green laser (pump) and the IR laser (probe)
counter-propagating and overlapping. The produced steady-state density of atomic iodine, as well as the charac-
teristic widths of the two main broadening mechanisms, are obtained by fitting the acquired IR transmission
spectra using the atomic absorption theory briefly presented in the Methods section. Absorption at Room temperature. We vary the green laser power up to its maximum value of 5 W and
record the transmission spectra for the IR laser, as it is scanned across the
°
→
°
5 P
5 P
2
3/2
2
1/2 transition of I (see
Methods section). Each of the transmission peaks is fitted independently, as shown in Fig. 1a, to yield a value for
the density of atomic iodine. The produced atomic iodine density is plotted in Fig. 1b versus the incident photo-
dissociating laser photon flux, for a dimethyldichlorosilane (DMDCS) coated cell, at T = 290.5 K. The fitting of the
data in Fig. 1b is performed using Eq. (2).i g
g
For the fit, we use the literature value for kr at T = 290.5 K, kr|290.5K = 4.4 × 10−30 ml2 s−1 16, and the measu
molecular iodine density to obtain the wall-sticking rate, kw at 290.5 K: =
±
−
k
387
4s
(3)
w
1 =
±
−
k
387
4s
w
1 (3) From Fig. 1b we notice that the [I] − Φ curve is mostly linear for high green laser power. From Eq. (2) we see
that if the kr term dominates, then the steady state atomic iodine density goes as the square root of the green pho-
ton flux,
σ
=
Φ k
[I]
/ r , yielding a maximum atomic iodine density [I]max ~ 3.9 × 1015 cm−3 for our maximum
green laser power of 5 W. Results From the absorption measurements, we get [I2] = (5.45 ± 1.9) × 1015 cm−3. Assuming literature values21, the ambient temperature corresponding to the measured density is found to be
T = 290.5 ± 0.1 K. Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 2 www.nature.com/scientificreports/ 0
5
10
15
20
25
0
2
4
6
8
Photon flux, Φ [×1018 photons / sec cm2]
Iodine density [×1014 cm-3]
12
10
8
6
4
1.0
0.8
0.6
0.4
0.2
0.0
Frequency detuning (GHz)
IR Transmission
4
3
3
3
2
3
(a)
(b)
Figure 1. (a) Each F → F′ absorption peak is fitted independently, and an iodine density value is extracted. Colored, dotted features are experimental transmission data; the solid line is the fitted curve. (b) Atomic iodine
density vs photodissociating laser photon flux, for a DMDCS coated cell at 290.5 K. Eq. (2) is used to fit the data
(solid line). 0
5
10
15
20
25
0
2
4
6
8
Photon flux, Φ [×1018 photons / sec cm2]
Iodine density [×1014 cm-3]
12
10
8
6
4
1.0
0.8
0.6
0.4
0.2
0.0
Frequency detuning (GHz)
IR Transmission
4
3
3
3
2
3
(a)
(b)
Figure 1. (a) Each F → F′ absorption peak is fitted independently, and an iodine density value is extracted. Colored, dotted features are experimental transmission data; the solid line is the fitted curve. (b) Atomic iodine
density vs photodissociating laser photon flux, for a DMDCS coated cell at 290.5 K. Eq. (2) is used to fit the data
(solid line). Figure 1. (a) Each F → F′ absorption peak is fitted independently, and an iodine density value is extracted. Colored, dotted features are experimental transmission data; the solid line is the fitted curve. (b) Atomic iodine
density vs photodissociating laser photon flux, for a DMDCS coated cell at 290.5 K. Eq. (2) is used to fit the data
(solid line). We aim at determining the dependence of atomic iodine density on the power of the photodissociating green
laser, on the molecular iodine density, and on the different chemical coatings of the inner surface of the cell. Results On the other hand, if the kw term is dominant, the relation is linear, [I] = σ[I2]Φ/kw. The
experimental curve for the DMDCS coating doesn’t show too visible a curvature, implying that the loss of atomic
iodine occurs mostly as adsorption to the walls. For the rest of the coatings investigated, the wall adsorption was
even more dominant as a loss mechanism. For the DMDCS coated cell at 290.5 K, the steady state atomic iodine density achieved at peak green laser
power, [I] = (0.89 ± 0.06) × 1015 cm−3, is only a factor of ~4 lower than the maximum value in the absence of wall
recombination, [I]max, given above.i We note that a somewhat better fit to the data can be obtained by adding more detailed processes, such as
the formation of an I3 intermediate19, adding a dependence to kw on [I], or including the electronic excitation of
I2 molecules from absorption of 532 nm light. However, the study of such effects is left for future work, particu-
larly for measurements at higher [I] densities, where contributions that deviate from Eq. (2) should appear more
strongly. g y
Finally, we note that, although it is known that angular momentum of I(2P3/2) atoms are aligned due to the
photodissociation dynamics22, we observe no evidence of alignment (i.e. no difference in the absorption spectra
is observed, for the photodissocation and probe laser polarization directions being parallel, compared to being
perpendicular). This depolarization is caused by the thousands of collisions each atom suffers, with I2 molecules
and the cell surface, before recombination. Effect of surface coatings and [I2] on produced I density. Coatings. We measured I-atom density for
five different cell surfaces: paraffin, dichlorodimethylsilane (DMDCS/Si(CH3)2Cl2), perfluorodecyltrichlorosilane
(FDTS/C10H4Cl3F17Si), dilute phosphoric acid (H3PO4), and, of course, no coating. Paraffin coating has been
widely used in atomic physics for many decades23, acid coating has been used to prevent iodine atom cell-wall
recombination17,24, whereas DMDCS and FDTS coatings are used routinely on glass25. Absorption measurements Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 3 www.nature.com/scientificreports/ No coang
Acid coang
FDTS
DMDCS
0
0.2
0.4
0.6
0.8
1. 0
0.2
0.4
0.6
0.8
1. Atomic I peak absorpon lengths
Passivaon
Passivaon
Prolonged
exposure
PGreen = 5 W
0
5
10
15
20
25
0
2
4
6
8
Photon flux, Φ [×1018 photons / sec cm2]
Iodine density [×1014 cm-3]
(a)
(b)
Figure 2. Results We see that reducing the
temperature leads to the production of lower atomic iodine densities, due to the reduced molecular iodine vapour
pressure. Therefore, raising the temperature is expected to increase the production of atomic iodine, until the
photodissociating light is depleted. I2 density. For the DMDCS coated cell, we repeated the absorption measurement procedure with the I2 holder
immersed in ice. The temperature of the created cold spot (0 °C) determines the vapour pressure of I2 inside the
cell. However, this only affects the density of molecular iodine, and not the remaining parameters (cell tempera-
ture, recombination rates etc.), as the vapour is quickly thermalized. Varying the green laser power and repeating
the fitting process for each individual hyperfine absorption peak, we arrive at the results plotted in Fig. 2b, where
the room temperature results of Fig. 1b are plotted in the same figure for comparison. We see that reducing the
temperature leads to the production of lower atomic iodine densities, due to the reduced molecular iodine vapour
pressure. Therefore, raising the temperature is expected to increase the production of atomic iodine, until the
photodissociating light is depleted. Results •
The best wall-recombination suppression was achieved with the DMDCS coating. h
•
FDTS, which was applied similarly, did not exhibit the same levels of performance. Moreover, both the
DMDCS and the FDTS coatings started to deteriorate over a few days period, most probably due to passiva-
tion of the surface by molecular iodine.ifi y
•
Our first try in applying a paraffin coating on the cell walls resulted into thick layers with rugged surfaces,
which caused iodine to stick at very high rates. Performance was similar to no coating at all, if not worse. Applying into thiner and smoother layers might have resulted in better performance, but, since DMDCS
provided an excellent combination of ease of application and wall-sticking-prevention operation, paraffin
was not investigated further. g
•
Application of dilute phosphoric acid performed better than no coating, however it was not investigated fur-
ther, especially as the application of the DMDCS was much easier in comparison.h g
•
Application of dilute phosphoric acid performed better than no coating, however it was not investigated fur-
ther, especially as the application of the DMDCS was much easier in comparison.h p
y
pp
p
•
The uncoated cell exhibited the worst performance, yielding atomic iodine densities more than an order of
magnitude smaller than the ones obtained in the DMDCS-coated cell. Continued exposure to molecular
iodine, though, improved these values, possibly due to coating of the walls with solidified molecular iodine. •
The uncoated cell exhibited the worst performance, yielding atomic iodine densities more than an order of
magnitude smaller than the ones obtained in the DMDCS-coated cell. Continued exposure to molecular
iodine, though, improved these values, possibly due to coating of the walls with solidified molecular iodine. I2 density. For the DMDCS coated cell, we repeated the absorption measurement procedure with the I2 holder
immersed in ice. The temperature of the created cold spot (0 °C) determines the vapour pressure of I2 inside the
cell. However, this only affects the density of molecular iodine, and not the remaining parameters (cell tempera-
ture, recombination rates etc.), as the vapour is quickly thermalized. Varying the green laser power and repeating
the fitting process for each individual hyperfine absorption peak, we arrive at the results plotted in Fig. 2b, where
the room temperature results of Fig. 1b are plotted in the same figure for comparison. Results (a) Comparison of the peak absorptivities observed for various coatings of the inner glass cell walls. The DMDCS coating produced the best results, although passivation by molecular iodine seems to hinder its
long-term performance. (b) Comparison of atomic iodine densities produced at 0 °C (red) and 290.5 K (blue). Lowering the I2 pellet temperature, and therefore lowering the I2 vapour pressure, leads to lower atomic iodine
densities. No coang
Acid coang
FDTS
DMDCS
0
0.2
0.4
0.6
0.8
1. 0
0.2
0.4
0.6
0.8
1. Atomic I peak absorpon lengths
Passivaon
Passivaon
Prolonged
exposure
PGreen = 5 W
0
5
10
15
20
25
0
2
4
6
8
Photon flux, Φ [×1018 photons / sec cm2]
Iodine density [×1014 cm-3]
(a)
(b)
Figure 2. (a) Comparison of the peak absorptivities observed for various coatings of the inner glass cell walls. The DMDCS coating produced the best results, although passivation by molecular iodine seems to hinder its
long-term performance. (b) Comparison of atomic iodine densities produced at 0 °C (red) and 290.5 K (blue). Lowering the I2 pellet temperature, and therefore lowering the I2 vapour pressure, leads to lower atomic iodine
densities. Figure 2. (a) Comparison of the peak absorptivities observed for various coatings of the inner glass cell walls. The DMDCS coating produced the best results, although passivation by molecular iodine seems to hinder its
long-term performance. (b) Comparison of atomic iodine densities produced at 0 °C (red) and 290.5 K (blue). Lowering the I2 pellet temperature, and therefore lowering the I2 vapour pressure, leads to lower atomic iodine
densities. were performed for the various coated cells, and the corresponding peak absorption values, which are directly
proportional to the atomic iodine densities, are presented in the bar-chart of Fig. 2a. Although the performed
study was far from exhaustive and long term performance (over months) of the coatings was not determined,
from Fig. 2a we can draw some important conclusions: were performed for the various coated cells, and the corresponding peak absorption values, which are directly
proportional to the atomic iodine densities, are presented in the bar-chart of Fig. 2a. Although the performed
study was far from exhaustive and long term performance (over months) of the coatings was not determined,
from Fig. 2a we can draw some important conclusions: •
The best wall-recombination suppression was achieved with the DMDCS coating. Conclusions Our measurements demonstrate the steady-state production of I densities of about 1015 cm−3, and column densi-
ties of about 1017 cm−2, using 5 W of 532 nm light, and cells coated with DMDCS. Even higher I densities of about
1016 cm−3 should be possible by using higher 532 nm laser power (e.g. 50–200 W commercial lasers are available),
and by improving the cell coatings further. Increased photodissociation will also reduce the Lorentz width of the
1315 nm transition, which is beneficial for polarimetry applications10: indeed, collisional broadening by I2 is about
2π × 7.7 (Mrad/s)/mbar, and by I about 2π × 3.2 (Mrad/s)/mbar26. At our current I2 pressure of ~0.2 mbar, the
Lorentz linewidth is ~2π × 1.5 Mrad/s. Further photodissociation (e.g. decreasing I2 to less than 1 × 1015 cm−3, to
produce [I] close to ~5 × 1015 cm−3) can reduce the linewidth to below 2π × 0.5 Mrad/s. This iodine-atom source
can be used for various PNC measurements in iodine atoms, such as for the measurement of the weak nuclear
charge, Qweak, nuclear spin-dependent PNC effects (needed to resolve the inconsistencies between such meas-
urements in Cs and Tl1,2,27, and with theoretical calculations28–31), and for isotope-dependent PNC effects32,33. Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 4 www.nature.com/scientificreports/ Figure 3. (a) Schematic of the experimental setup used for the study of atomic iodine production from
photodissociation of molecular iodine at 532 nm. (b) The iodine glass cell. (c) The iodine cell with the green
photodissociating laser passing through it. Figure 3. (a) Schematic of the experimental setup used for the study of atomic iodine production from
photodissociation of molecular iodine at 532 nm. (b) The iodine glass cell. (c) The iodine cell with the green
photodissociating laser passing through it. This method seems promising for the production of high atomic densities from the photodissociation of other
diatomic molecules, such as from O2 or Br2. Methodsh The experimental setup used is shown in Fig. 3a. The probe IR laser (Toptica Photonics AG DL-Pro, P ~ 50 mW,
continuously tunable around 1315 nm) is coupled to an optical fiber, and the fiber output beam propagates
through the iodine cell. The beam of the photodissociating green laser (EKSMA Optics DPSS-532N-5000, P ~ 5 W
CW, λ = 532 nm, bandwidth ~2 nm) is collimated to about twice the size of the IR beam using the spherical mir-
rors M1 and M2, and is injected to the iodine cell, counter-propagating to and overlapping with the IR beam. The input and output powers of the IR (green) laser are monitored by the PDIR/IN (PDG/IN) and PDIR/OUT (PDG/OUT)
photodiodes (Thorlabs GmbH, models DET10N for the IR and DET36A for the green), respectively. The pho-
todiode readings on the oscilloscope are calibrated to correspond to the actual powers of the two lasers incident
to and transmitted from the iodine vapour, taking into account the transmission losses of the two windows. Mirrors M3, M4 and M5 are coated for 532 nm, and induce minimal losses to the power of the IR laser travers-
ing them. Mirror M4 is used to dump most of the output green power, so as to minimize injection of green light
into the IR fiber, and thus negative feedback to the IR laser, and to avoid saturating the PDG/OUT photodiode. The
cell, shown in Fig. 3b, was a home design; it was 123 cm long, with an inner diameter of 8 mm, and was made
of BK7. In the middle of the cell were the pump outlet and the molecular iodine holder, both with individual
controlling valves, while two uncoated windows seal both ends. The vacuum pump connected to the pump out-
let was an Agilent Technologies TriScroll 300, a dry-scroll pump not requiring oil for operation. The DMDCS
(Sigma-Aldrich > 98.5%) and FDTS (Sigma-Aldrich > 97%) coatings were applied in Hellmanex cleaned cells,
using the substance in liquid form and allowing it to evaporate overnight inside the cell, in a controlled, clean
environment, at ambient atmospheric pressure. Iodine in crystalline form was acquired commercially from
Sigma-Aldrich (99.999% trace metal basis; ID: 229695). The experimental procedure is outlined below: 1. Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 Methodsh Initially the cell is pumped down to ~10−2 mbar and care is taken to ensure that, after pumping, the leak
rate of the cell is insignificant within the time frame of the measurement (~1 min).hh 1. Initially the cell is pumped down to ~10−2 mbar and care is taken to ensure that, after pumping, the leak
rate of the cell is insignificant within the time frame of the measurement (~1 min).hh gi
2. The pump valve is closed and the iodine holder valve is opened. The vapour pressure of molecular iodine
reaches equilibrium (~0.22 mbar) within a few seconds, as confirmed by the transmitted green laser power
reaching a steady state value. gi
2. The pump valve is closed and the iodine holder valve is opened. The vapour pressure of molecular iodine
reaches equilibrium (~0.22 mbar) within a few seconds, as confirmed by the transmitted green laser power
reaching a steady state value. Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 5 www.nature.com/scientificreports/ -0.01
-0.02
-0.03
-0.04
1.00
0.95
0.90
0.85
0.80
0.75
0.70
Time (sec)
Transmission
-0.01
-0.02
-0.03
-0.04
0.11
0.10
0.09
0.08
0.07
PDIR,OUT voltage (V)
(a)
(b)
(c)
F = x → F΄ = 3
(x = 4, 3, 2, left to right)
1.00
0.95
0.90
0.85
0.80
0.75
0.70
10
5
0
-- 5
-- 10
Frequency detuning [GHz]
Transmission
3
2
2
2
1
2
4
3
3
3
2
3
Figure 4. (a) Typical IR laser scan over the F → F′ = 3 group of the
°
→
°
5 P
5 P
2
3/2
2
1/2 transition, with the
photodissociating green light ON (blue) and OFF (red/background measurement). (b) Resulting normalized
absorption profile. (c) Solid lines: Combined F → F′ = 2 (left) and F → F′ = 3 (right) absorption measurements. Dashed line: The simulated transmission spectrum of Fig. 5b. The horizontal axis has been converted to
frequency units for this figure. This is the highest atomic iodine density we have recorded, [I] ~ 0.89 × 1015 cm−3,
using the full power of the photodissociating laser (P ~ 5 W), and the DMDCS coated cell. Methodsh The green laser is switched off, and a minute-long, time averaged measurement of the transmitted IR light
is taken, in the absence of atomic iodine, as the laser is still scanned over the same frequency range. This is
to be used as a 100%-transmission reference measurement. A typical measurement pair (green ON/OFF) is shown in Fig. 4a for the F → F′ = 3 group of the
°
→
°
5 P
5 P
2
3/2
2
1/2 transition, and the normalized absorption profile, resulting from the division of the green
ON and OFF curves of (a), is shown in Fig. 4b. The combined measurements for the F → F′ = 2 and F → F′ = 3
transition groups is given in Fig. 4c, where the conversion of the horizontal axis from scan times to frequency
detunings relative to the line center has been performed, using the known hyperfine constants of 127I34,35.h h
6. The series of measurements described above is repeated for cells with different coatings and, for the same
cell, with the iodine holder at two different temperatures. h
6. The series of measurements described above is repeated for cells with different coatings and, for the same
cell, with the iodine holder at two different temperatures. A typical data set is obtained as follows: We vary the green laser power from zero up to its maximum value
of ~5 W, and we extract the atomic iodine density from the optical depth calculated at each of the six observed
absorption peaks (3 from the F → F′ = 2 and 3 from the F → F′ = 3 group). For each power value, we do this once
as the IR laser is scanned from lower to higher frequencies, and once as it is scanned from higher to lower ones. We repeat the process as we decrease the green laser power from its maximum power down to zero. Note: from
Fig. 1b we see that for relatively high green laser powers the measurement errors become significant. Methodsh -0.01
-0.02
-0.03
-0.04
1.00
0.95
0.90
0.85
0.80
0.75
0.70
Time (sec)
Transmission
-0.01
-0.02
-0.03
-0.04
0.11
0.10
0.09
0.08
0.07
PDIR,OUT voltage (V)
(a)
(b)
F = x → F΄ = 3
(x = 4, 3, 2, left to right) (c)
1.00
0.95
0.90
0.85
0.80
0.75
0.70
10
5
0
-- 5
-- 10
Frequency detuning [GHz]
Transmission
3
2
2
2
1
2
4
3
3
3
2
3 Frequency detuning [GHz] Figure 4. (a) Typical IR laser scan over the F → F′ = 3 group of the
°
→
°
5 P
5 P
2
3/2
2
1/2 transition, with the
photodissociating green light ON (blue) and OFF (red/background measurement). (b) Resulting normalized
absorption profile. (c) Solid lines: Combined F → F′ = 2 (left) and F → F′ = 3 (right) absorption measurements. Dashed line: The simulated transmission spectrum of Fig. 5b. The horizontal axis has been converted to
frequency units for this figure. This is the highest atomic iodine density we have recorded, [I] ~ 0.89 × 1015 cm−3,
using the full power of the photodissociating laser (P ~ 5 W), and the DMDCS coated cell. 3. The IR laser is switched on and scanned across the
°
→
°
5 P
5 P
2
3/2
2
1/2 atomic iodine transition. The basic
atomic absorption theory relevant to this transition is given at the end of this section. The transmission
spectrum is observed on the oscilloscope and is averaged for approximately 1 min.hf 3. The IR laser is switched on and scanned across the
°
→
°
5 P
5 P
2
3/2
2
1/2 atomic iodine transition. The basic
atomic absorption theory relevant to this transition is given at the end of this section. The transmission
spectrum is observed on the oscilloscope and is averaged for approximately 1 min.hf 3. The IR laser is switched on and scanned across the
°
→
°
5 P
5 P
2
3/2
2
1/2 atomic iodine transition. The basic
atomic absorption theory relevant to this transition is given at the end of this section. The transmission
spectrum is observed on the oscilloscope and is averaged for approximately 1 min.hf p
p
g
pp
y
4. Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 Methodsh This was
due to two main reasons: (a) instabilities of the IR power due to negative feedback of the green laser into the IR
laser, and (b) variation of the IR laser power transmitted through the cell during the green-OFF measurements,
as molecular iodine stuck to the windows forming an opaque layer (we note that with the green laser ON, the I2
layer at the windows would be removed via photodissociation). These two factors caused the ON and OFF curves
of Fig. 4a to not coincide, hence introducing significant errors in the calculation of [I]. Despite this, we see that
Eq. (2) describes the observed data well.h
2
2 q
Finally, a short description of the atomic theory formalism used in this work is given here. The
°
→
°
5 P
5 P
2
3/2
2
1/2
transition of atomic iodine is a magnetic dipole transition with a nominal frequency of 1315.27 nm, between the
ground 52P3/2 and the metastable 52P1/2 states, both of odd parity. The energy level breakdown of the transition is
shown in Fig. 5a, and a typical transmission spectrum in Fig. 5b. There are two distinct groups of hyperfine tran-
sitions, the F → F′ = 2 (with F = 1, 2, 3) and F → F′ = 3 (with F = 2, 3, 4), red and blue detuned, respectively, by a
few GHz from the nominal transition frequency. q
y
For intensities well below saturation, the transmission of the IR light follows the Beer-Lambert law: ω =
=
ρσ ω
−
T
I
I
e
( )
,
(4
l
OUT
IN
( ) ω =
=
ρσ ω
−
T
I
I
e
( )
,l
OUT
IN
( ) (4) Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 6 www.nature.com/scientificreports/ where T is the ratio of transmitted (I
) to incident (I ) intensity ρ the number density of the atomic iodine
1.00
0.95
0.90
0.85
0.80
0.75
0.70
10
5
0
-- 5
-- 10
Frequency detuning [GHz]
Transmission
3
2
2
2
1
2
4
3
3
3
2
3
F
3
2
4
321
1315 nm
52P1/2
o
o
52P3/2
(a)
(b)
Figure 5. (a) Energy level structure of the
°
→
°
5 P
5 P
2
3/2
2
1/2 M1 transition of atomic 127I. Methodsh q
y
× 1015 cm−3, Γ = 2π × 3 Mrad/s, and ΔωD = 2π × 148 Mrad/s (half-width at 1/e2 value, for T = 290.5 K). where T is the ratio of transmitted (IOUT) to incident (IIN) intensity, ρ the number density of the atomic iodine
vapour, σ the absorption cross section, and l the length of the cell. All frequency dependence of the transmission
is contained in the expression for the cross section, which is given by9: where T is the ratio of transmitted (IOUT) to incident (IIN) intensity, ρ the number density of the atomic iodine
vapour, σ the absorption cross section, and l the length of the cell. Methodsh All frequency dependence of the transmission
is contained in the expression for the cross section, which is given by9:
∑
σ ω
σ
ω
=
″
′
′
′
C
( )
( )
(5)
F F
FF
FF
o
, (5) where σo is the integrated absorption cross section, CFF′ are geometrical factors, and ″ is the absorptive part of the
Voigt profile: σ
πµ ω
=
+
′
c
J
M
1
2
1 3
(6)
JJ
o
o
1
2
=
+
′ +
+
′
′
′
{
}
C
F
F
I
J
J
F
I
F
(2
1)(2
1)
2
1
1
(7)
FF
2
ω
π
ω
″
=
′
∆
ω
ω
ω
ω
′
−
∆
Γ
∆
′
(
)
w
( )
,
(8)
FF
/ 2
D
FF
D
D
σ
πµ ω
=
+
′
c
J
M
1
2
1 3
(6)
JJ
o
o
1
2
(6) =
+
′ +
+
′
′
′
{
}
C
F
F
I
J
J
F
I
F
(2
1)(2
1)
2
1
1
(7)
FF
2 (7) ω
π
ω
″
=
′
∆
ω
ω
ω
ω
′
−
∆
Γ
∆
′
(
)
w
( )
,
(8)
FF
/ 2
D
FF
D
D
(8) In the equations above, ωJJ′ is the nominal transition frequency and ωFF′ the frequency of the F → F′ transition,
=
J
3
2, ′ =
J
1
2 and =
I
5
2, M1 ≡ 〈M1〉 ≡ 〈J||μ(1)||J′〉 = 1.15 μB is the reduced matrix element for the magnetic-dipole
operator μ(1)10, w′ is the real part of the Faddeeva function, Γ is the FWHM homogeneous linewidth, and
ω
ω
∆
=
′
k T
Mc
2
/(
)
JJ
D
B
2 is the Doppler width. g
g
8. Mikheyev, P. A., Shepelenko, A. A., Voronov, A. I. & Kupryaev, N. V. “Production of iodine atoms by dissociating CH3I and HI in a
dc glow discharge in the flow of argon”. J. Phys. D 37, 3202–3206, doi: 10.1088/0022-3727/37/22/024 (2004). References
1. Vetter, P. A., Meekhof, D. M., Majumder, P. K., Lamoreaux, S. K. & Fortson, E. N. “Precise Test of Electroweak Theory from a New
Measurement of Parity Nonconservation in Atomic Thallium”. Phys. Rev. Lett. 74, 2658–2661, doi: 10.1103/PhysRevLett.74.2658 (1995).
2. Edwards, N. H., Phipp, S. J., Baird, P. E. G. & Nakayama, S. “Precise Measurement of Parity Nonconserving Optical Rotation in
Atomic Thallium”. Phys. Rev. Lett. 74, 2654–2657, doi: 10.1103/PhysRevLett.74.2654 (1995).
3. Macpherson, M. J. D., Zetie, K. P., Warrington, R. B., Stacey, D. N. & Hoare, J. P. “Precise measurement of parity nonconserving
optical rotation at 876 nm in atomic bismuth”. Phys. Rev. Lett. 67, 2784–2787, doi: 10.1103/PhysRevLett.67.2784 (1991).
4. Meekhof, D. M., Vetter, P., Majumder, P. K., Lamoreaux, S. K. & Fortson, E. N. “High-precision measurement of parity
nonconserving optical rotation in atomic lead”. Phys. Rev. Lett. 71, 3442–3445, doi: 10.1103/PhysRevLett.71.3442 (1993).
5. Gupta, M., Owano, T., Baer, D. & O’ Keefe, A. “Quantitative determination of the O(3P) density via visible cavity-enhanced
spectroscopy”. Appl. Phys. Lett. 89, 241503, doi: 10.1063/1.2408655 (2006).
6. Azyazov, V. N., Mikheyev, P. A., Vorobyov, M. V. & Ufimtsev, N. I. “Properties of a DC glow discharge iodine atom generator”. Proc.
SPIE 7131, 71310A, doi: 10.1117/12.816461 (2008).
7. Mikheyev, P. A., Shepelenko, A. A., Voronov, A. I. & Kupryaev, N. V. “Atomic iodine production in a gas flow by decomposing methyl
iodide in a dc glow discharge”. Quantum Electron 32, 1–4, doi: 10.1070/QE2002v032n01ABEH002115 (2002).
8. Mikheyev, P. A., Shepelenko, A. A., Voronov, A. I. & Kupryaev, N. V. “Production of iodine atoms by dissociating CH3I and HI in a
dc glow discharge in the flow of argon”. J. Phys. D 37, 3202–3206, doi: 10.1088/0022-3727/37/22/024 (2004). 7. Mikheyev, P. A., Shepelenko, A. A., Voronov, A. I. & Kupryaev, N. V. “Atomic iodine production in a gas flow by decomposing methy
iodide in a dc glow discharge”. Quantum Electron 32, 1–4, doi: 10.1070/QE2002v032n01ABEH002115 (2002). p
y
y
4. Meekhof, D. M., Vetter, P., Majumder, P. K., Lamoreaux, S. K. & Fortson, E. N. “High-precision measurement of parity
nonconserving optical rotation in atomic lead”. Phys. Rev. Lett. 71, 3442–3445, doi: 10.1103/PhysRevLett.71.3442 (1993).
3 References
1. Vetter, P. A., Meekhof, D. M., Majumder, P. K., Lamoreaux, S. K. & Fortson, E. N. “Precise Test of Electroweak Theory from a New
Measurement of Parity Nonconservation in Atomic Thallium”. Phys. Rev. Lett. 74, 2658–2661, doi: 10.1103/PhysRevLett.74.2658 (1995).
2. Edwards, N. H., Phipp, S. J., Baird, P. E. G. & Nakayama, S. “Precise Measurement of Parity Nonconserving Optical Rotation in
At
i Th lli
” Ph
R
L tt 74 2654 2657 d i 10 1103/Ph
R
L tt 74 2654 (1995) h
y
y
Macpherson, M. J. D., Zetie, K. P., Warrington, R. B., Stacey, D. N. & Hoare, J. P. “Precise measurement of parity nonconserving
ptical rotation at 876 nm in atomic bismuth”. Phys. Rev. Lett. 67, 2784–2787, doi: 10.1103/PhysRevLett.67.2784 (1991). p ,
,
,
,
,
,
Q
(
)
y
y
spectroscopy”. Appl. Phys. Lett. 89, 241503, doi: 10.1063/1.2408655 (2006).
6. Azyazov, V. N., Mikheyev, P. A., Vorobyov, M. V. & Ufimtsev, N. I. “Properties of a DC glow discharge iodine atom generator”. Proc.
SPIE 7131, 71310A, doi: 10.1117/12.816461 (2008).l Methodsh The two distinct hyperfine
transition groups, F → F′ = 2 (left) and F → F′ = 3 (right), are indicated. The dashed lines mark the nominal transition
frequency of 1315.27 nm, and the center-of-gravity energies of the 2P3/2 and 2P1/2 states. (b) Calculated transmission
spectrum of an IR laser as its frequency is scanned across the resonance. For the calculation we assume
[I] ~ 0.89 × 1015 cm−3, Γ = 2π × 3 Mrad/s, and ΔωD = 2π × 148 Mrad/s (half-width at 1/e2 value, for T = 290.5 K). 1.00
0.95
0.90
0.85
0.80
0.75
0.70
10
5
0
-- 5
-- 10
Frequency detuning [GHz]
Transmission
3
2
2
2
1
2
4
3
3
3
2
3
(b) Figure 5. (a) Energy level structure of the
°
→
°
5 P
5 P
2
3/2
2
1/2 M1 transition of atomic 127I. The two distinct hyperfine
transition groups, F → F′ = 2 (left) and F → F′ = 3 (right), are indicated. The dashed lines mark the nominal transition
frequency of 1315.27 nm, and the center-of-gravity energies of the 2P3/2 and 2P1/2 states. (b) Calculated transmission
spectrum of an IR laser as its frequency is scanned across the resonance. For the calculation we assume
[I] ~ 0.89 × 1015 cm−3, Γ = 2π × 3 Mrad/s, and ΔωD = 2π × 148 Mrad/s (half-width at 1/e2 value, for T = 290.5 K). Figure 5. (a) Energy level structure of the
°
→
°
5 P
5 P
2
3/2
2
1/2 M1 transition of atomic 127I. The two distinct hyperfine
transition groups, F → F′ = 2 (left) and F → F′ = 3 (right), are indicated. The dashed lines mark the nominal transition
frequency of 1315.27 nm, and the center-of-gravity energies of the 2P3/2 and 2P1/2 states. (b) Calculated transmission
spectrum of an IR laser as its frequency is scanned across the resonance. For the calculation we assume
[I] 0 89
1015
3 Γ
2
3M d/
d Δ
2
148M d/ (h lf
idth t 1/ 2
l
f
T
290 5K) p
q
y
I] ~ 0.89 × 1015 cm−3, Γ = 2π × 3 Mrad/s, and ΔωD = 2π × 148 Mrad/s (half-width at 1/e2 value, for T = 290.5 K). Author Contributions G.E.K. conceived, constructed and performed the experiment, analysed the data, and wrote the manuscript. G.C. and J.A.K. performed the experiment. E.S. designed and provided the DMDCS and FDTS coatings. T.P.R. conceived and directed the experiment, and co-wrote the manuscript. G.E.K. conceived, constructed and performed the experiment, analysed the data, and wrote the manuscript. G.C. and J.A.K. performed the experiment. E.S. designed and provided the DMDCS and FDTS coatings. T.P.R. conceived and directed the experiment, and co-wrote the manuscript. www.nature.com/scientificreports/ “Least-squares analysis of overlapped bound-free absorption spectra and predissociation data in diatomics: The
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18. Tellinghuisen, J. “Least-squares analysis of overlapped bound-free absorption spectra and pre
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Measurement of Parity Nonconservation in Atomic Thallium”. Phys. Rev. Lett. 74, 2658–2661, doi: 10.1103/PhysRevLett.74.2658 (1995). h
3. Macpherson, M. J. D., Zetie, K. P., Warrington, R. B., Stacey, D. N. & Hoare, J. P. “Precise measurement of parity nonconserving
optical rotation at 876 nm in atomic bismuth”. Phys. Rev. Lett. 67, 2784–2787, doi: 10.1103/PhysRevLett.67.2784 (1991). 4. Meekhof, D. M., Vetter, P., Majumder, P. K., Lamoreaux, S. K. & Fortson, E. N. “High-precision measurement of parity
nonconserving optical rotation in atomic lead”. Phys. Rev. Lett. 71, 3442–3445, doi: 10.1103/PhysRevLett.71.3442 (1993). p
y
y
spectroscopy”. Appl. Phys. Lett. 89, 241503, doi: 10.1063/1.2408655 (2006). 6. Azyazov, V. N., Mikheyev, P. A., Vorobyov, M. V. & Ufimtsev, N. I. “Properties of a DC glow discharge iodine atom generator”. Proc. SPIE 7131, 71310A, doi: 10.1117/12.816461 (2008). Scientific Reports | 6:33261 | DOI: 10.1038/srep33261 7 Acknowledgementsh g
This research was supported by the ERC grant TRICEPS (grant no. 207542). Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Katsoprinakis, G. E. et al. High steady-state column density of I(2P3/2) atoms from I2
photodissociation at 532 nm: Towards parity non-conservation measurements. Sci. Rep. 6, 33261; doi: 10.1038/
srep33261 (2016). How to cite this article: Katsoprinakis, G. E. et al. High steady-state column density of I(2P3/2) atoms from I2
photodissociation at 532 nm: Towards parity non-conservation measurements. Sci. Rep. 6, 33261; doi: 10.1038/
srep33261 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ 8 Scientific Reports | 6:33261 | DOI: 10.1038/srep33261
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English
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<i>Photobacterium damselae</i> subspecies <i>damselae</i> Pneumonia in Dead, Stranded Bottlenose Dolphin, Eastern Mediterranean Sea
|
Emerging infectious diseases
| 2,023
|
cc-by
| 3,512
|
Author affiliations: University of Haifa, Haifa, Israel (D. Morick,
N. Davidovich, Z. Zemah-Shamir, E. Bigal, P. Itay, M. Roditi-Elasar,
Y. Zuriel, D. Tchernov, A.P. Scheinin); Hong Kong Branch of
Southern Marine Science and Engineering, Guangzhou, China
(D. Morick, D. Tchernov); Kimron Veterinary Institute, Bet Dagan,
Israel (S.E. Blum, M. Flecker); Israeli Veterinary Services,
Bet Dagan (N. Davidovich); Ministry of Health, Jerusalem, Israel
(A. Rokney, I. Nasie, N. Feldman); Hebrew University of
Jerusalem, Rehovot, Israel (K. Aharoni); Universidade Federal
do Paraná, Curitiba, Brazil (N. Wosnick) Photobacterium damselae
subspecies damselae Pneumonia
in Dead, Stranded Bottlenose
Dolphin, Eastern Mediterranean Sea
Danny Morick, Shlomo E. Blum, Nadav Davidovich, Ziv Zemah-Shamir, Eyal Bigal,
Peleg Itay, Assaf Rokney, Iris Nasie, Noa Feldman, Marcelo Flecker, Mia Roditi-Elasar,
Kobi Aharoni, Yotam Zuriel, Natascha Wosnick, Dan Tchernov, Aviad P. Scheinin, chronic suppurative pneumonia and splenic lym-
phoid depletion caused by this pathogen. Photobacterium damselae subspecies damselae, an
abundant, generalist marine pathogen, has been reported
in various cetaceans worldwide. We report a bottlenose
dolphin in the eastern Mediterranean Sea that was found
stranded and dead. The dolphin had a severe case of
chronic suppurative pneumonia and splenic lymphoid de-
pletion caused by this pathogen. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 The Study The Study
On January 29, 2021, a bottlenose dolphin was found
beached nearby Ashdod, Israel. The carcass under-
went a postmortem examination based on a widely
accepted protocol (5) with some modifications be-
cause the carcass was also sampled for several ana-
tomic and physiologic studies. Samples of the spleen,
liver, lung, kidney and brain were collected for quan-
titative PCR molecular detection of Toxoplasma gondii
(6) and canine distemper virus (7), and for PCR detec-
tion of Brucella spp. (8). Samples of spleen and lung
were fixed in 10% buffered formalin for routine histo-
logic evaluation. Samples of lungs and fluid from the
thoracic cavity were obtained by using sterile swabs
for lung samples and sterile syringes and needles for
fluid samples and inoculated onto tryptone soy agar,
blood agar (5% sheep blood enriched tryptone soy
agar), and MacConkey agar, and incubated for 24–48
h at 37°C. Confirmation of bacteria species was ini-
tially performed by using matrix-assisted laser de-
sorption/ionization time-of-flight mass spectrometry
according to the manufacturer’s protocol (Autoflex;
Bruker, https://www.bruker.com). T
he common bottlenose dolphin (Tursiops trunca-
tus) is perhaps the most common and widespread
dolphin species in the Mediterranean Sea (1). Photo-
bacterium damselae subspecies damselae is a pathogen
that produces wound infections and hemorrhagic
septicemia and high mortality rates and affects vari-
ous marine animals, such as fish, mollusks, crusta-
ceans, and cetaceans (2,3). Highly pathogenic P. dam-
selae subsp. damselae isolates have 2 major virulence
factors: the phospholipase D damselysin (Dly) and
the pore-forming toxin phobalysin P (initially called
HlyApl). Both toxins are encoded by the plasmid
pPHDD1 and produce hemolytic and cytolytic activi-
ties in a synergistic manner (4). We report a bottle-
nose dolphin in the eastern Mediterranean Sea that
was found stranded, dead, and had a severe case of T The dolphin weighed 200 kg, had a length of 263
cm, and was identified as a mature female that had a
moderate nutritional status (9). At external examina-
tion, a deep bruise was observed on the front of the
dorsal fin, and an old visible scar was observed on
the right side of the chest, which might have been the
result of an injury by a foreign body that might have
instigated the inflammation within the lung, lead-
ing to pneumonia (Figure 1, panel A). No additional
external signs of interaction with fishing gear were 179 DISPATCHES Figure 1. The Study Photobacterium
damselae subspecies damselae
pneumonia in a bottlenose
dolphin, eastern Mediterranean
Sea. Gross pathologic
examination of the dolphin
(Tursiops truncatus) showed
a scar (oval) at the right side
of the chest (A) that might be
a sign for a previous wound
that initiated the infection (B,
C). Four abscesses, 5–10 cm
in diameter, filled with purulent
fluid and necrotic debris were
observed in the right lung of the
animal. Hemolytic phenotype
of the P. damselae subsp. damselae isolate on sheep blood
agar (D) indicates the border of
the halo of 1 colony (arrow). A
weak hemolytic phenotype was
observed after culturing isolate
on blood agar plates for 24 h. observed. The carcass was at stage 3 on the decompo-
sition condition code scale (5). Internal examination
indicated 4 large, firm nodules, 5–10 cm in diameter,
replacing the cranial aspect of the right lung lobe. On cut sections, nodules were filled with purulent to
caseous, thick, granular, green-tinged exudate sur-
rounded by a dense fibrous capsule (abscess) (Figure
1, panels B, C). No other abnormalities were observed
in all other internal organs. susceptibility to amoxicillin/clavulanic acid; fluo-
roquinolones; and first-, second-, and third-genera-
tion cephalosporins. The isolate species was also characterized and
confirmed by using 16S rRNA gene primers and
Sanger sequencing of the 800-nt PCR product. Whole-
genome sequencing (WGS) was performed to obtain
the allelic multilocus sequence typing (MLST) profile
for sequence type determination and to analyze the
presence of the 2 P. damselae subsp. damselae major
virulence factor genes (dly and hlyApl). Pure bacterial colonies of spherical or ovoid
cocci, 1–2 µm in diameter, consistent with the ge-
nus Photobacterium, appeared on the blood agar
plates at 48-hours postinoculation. Matrix-assisted
laser desorption/ionization time-of-flight mass
spectrometry confirmed the initial identification of
Photobacterium damselae. The isolate was resistant
to ampicillin and susceptible to gentamicin, sulfa-
methoxazole/trimethoprim, florfenicol, amikacin,
and polymyxin B. The isolate also had intermediate pl
We extracted DNA by using the QIAsymphony
SP System and the QIAsymphony DNA Mini Kit
(QIAGEN, https://www.qiagen.com), according to
the manufacturer’s recommendations. We prepared a
DNA library by using the Nextera XT Library Prepa-
ration Kit (Illumina, https://www.illumina.com), fol-
lowed by WGS using the Illumina MiSeq and a 250-
bp paired-end read length. Reads were assembled by Table 1. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 llele 1 sequence of each gene (taken from P. damselae subsp. damselae PubMLST database).
s 372–390 bp (10). The toxR allele length of the isolate of this study is 390 bp, and the allele 1 reference length is 387 bp. Table 1. Similarity-based gene extraction of genes used in MLST scheme, including the obtained MLST allelic profile for
Photobacterium damselae subspecies damselae pneumonia, in bottlenose dolphin, eastern Mediterranean Sea*
Gene
Identity,† %
Coverage, %
Reference length, bp
No. mismatches
No. open gaps
MLST allelic profiles
glpF
99.58
100.00
480
2
0
26
gyrB
97.58
100.00
537
13
0
27
metG
99.53
100.00
429
2
0
7
pntA
97.98
100.00
396
8
0
23
pyrC
98.42
100.00
507
8
0
31
toxR
93.32
99.74‡
387
23
1
35
*MLST, multilocus sequence typing.
†The percentage of identity to allele 1 sequence of each gene (taken from P. damselae subsp. damselae PubMLST database).
‡The toxR alleles length range is 372–390 bp (10). The toxR allele length of the isolate of this study is 390 bp, and the allele 1 reference length is 387 bp. P. damselae Pneumonia in Bottlenose Dolphin P. damselae Pneumonia in Bottlenose Dolphin Table 2. PubMLST database of Photobacterium damselae subspecies damselae isolates from different marine animals, including a
bottlenose dolphin in the eastern Mediterranean Sea* Table 2. PubMLST database of Photobacterium damselae subspecies damselae isolates from different marine animals, including a
bottlenose dolphin in the eastern Mediterranean Sea*
Country
Host
Organ
Year
NA
2010
2012
2013
2014
2015
2016
2021
Australia
Seriola lalandi
Ot
2
0
0
0
4
2
0
0
Israel
Tursiops truncatus
Lu
0
0
0
0
0
0
1
0
Italy
Caretta caretta
Icc
0
0
3
0
0
0
0
0
Delphinus delphis
Ot
0
0
1
0
0
0
0
0
Physeter macrocephalus
Br
0
0
0
1
0
0
0
0
Mf
0
0
0
1
0
0
0
0
Sp
0
0
0
2
0
0
0
0
Ut
0
0
0
1
0
0
0
0
Stenella coeruleoalba
Br
0
0
14
3
0
0
0
0
Icc
0
0
1
0
0
0
0
0
In
0
0
3
0
0
0
0
0
Jf
0
0
3
0
0
0
0
0
Li
0
0
6
0
0
0
0
0
Ln
0
0
2
0
0
0
0
0
Lu
0
1
3
0
0
0
0
0
Ot
0
0
3
0
0
0
0
0
Sp
0
0
5
0
0
0
0
0
Stenella spp. P. damselae Pneumonia in Bottlenose Dolphin Br
0
0
3
0
0
0
0
0
In
0
0
1
0
0
0
0
0
Li
0
0
1
0
0
0
0
0
Ln
0
0
2
1
0
0
0
0
Lu
0
0
1
1
0
0
0
0
Sp
0
0
1
0
0
0
0
0
Tursiops truncatus
Br
0
1
3
0
0
0
0
0
Icc
0
0
1
1
0
0
0
0
Lu
0
0
1
0
0
0
0
0
Sp
0
0
1
0
0
0
0
0
Un
0
0
0
1
0
0
0
0
Japan
Labracoglossa argentiventris
Ot
0
0
0
0
0
0
0
1
Sardinops melanostictus
Ot
0
0
0
0
0
0
0
1
United States
Carcharhinus plumbeus
Li
0
0
0
0
0
0
0
1
Chromis punctipinnis
Ot
0
0
0
0
0
0
0
1
Total
2
2
59
12
4
2
1
4
*Database contained 86 isolates as of February 5, 2020 (https://pubmlst.org/organisms/photobacterium-damselae). Br, brain; Icc, intracardiac clot; In,
intestine; Jf, joint fluid; Li, liver; Ln, lymph node; Lu, lung; Mf, mesenteric fluid; NA, no year data in PubMLST database; Ot, other; Sp, spleen; Un,
unknown; Ut, uterus. *Database contained 86 isolates as of February 5, 2020 (https://pubmlst.org/organisms/photobacterium-damselae). Br, brain; Icc, intracardiac clot; In,
intestine; Jf, joint fluid; Li, liver; Ln, lymph node; Lu, lung; Mf, mesenteric fluid; NA, no year data in PubMLST database; Ot, other; Sp, spleen; Un,
unknown; Ut, uterus. using the BioNumerics 8.0 Platform SPAdes 3.13.1
(Applied Maths, https://www.applied-maths.com). halos on sheep blood agar plate (Figure 1, panel D). This phenotype is typical of P. damselae subsp. dam-
selae lacking the pPHDD1 plasmid and having the
chromosomal PhlyC gene (hlyAch). WGS of the he-
molytic genes dly and hlyApl yielded only the hlyA
sequence, which showed 99% identity to the hlyAch
sequences in GenBank. The assembly was deposited to the pubMLST P. damselae database under identification no. 91. We ob-
tained the allelic MLST profile by using the BioNu-
merics Sequence Extraction Tool (Applied Maths)
and according to the P. damselae scheme based on 6
housekeeping genes (glpF, gyrB, metG, pntA, pyrC,
and toxR) (10). This tool was also used for identifica-
tion of virulence factor gene sequences dly (GenBank
accession no. 9937366) and hlyApl (GenBank accession
no. ID 9937197). The Study Similarity-based gene extraction of genes used in MLST scheme, including the obtained MLST allelic profile for
Photobacterium damselae subspecies damselae pneumonia, in bottlenose dolphin, eastern Mediterranean Sea*
Gene
Identity,† %
Coverage, %
Reference length, bp
No. mismatches
No. open gaps
MLST allelic profiles
glpF
99.58
100.00
480
2
0
26
gyrB
97.58
100.00
537
13
0
27
metG
99.53
100.00
429
2
0
7
pntA
97.98
100.00
396
8
0
23
pyrC
98.42
100.00
507
8
0
31
toxR
93.32
99.74‡
387
23
1
35
*MLST, multilocus sequence typing. †The percentage of identity to allele 1 sequence of each gene (taken from P. damselae subsp. damselae PubMLST database). ‡The toxR alleles length range is 372–390 bp (10). The toxR allele length of the isolate of this study is 390 bp, and the allele 1 reference length is 387 bp. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 180 P. damselae Pneumonia in Bottlenose Dolphin Hemolysis was tested by culturing
the isolate on 5% sheep blood agar (#PD-005; Hylabs
Ltd, https://www.hylabs.co.il) for 24 h at 37°C.i The MLST allelic scheme extraction (Table 1) re-
sulted in a new profile that was submitted to the iso-
late collection of the PubMLST P. damselae database as
PDIN1, and was assigned a new sequence type (ST),
ST63. Within the PubMLST database, most of the P. damselae subsp. damselae isolates (Table 2) originated
from an unusual cetacean mortality event in Italy dur-
ing 2013 (11). Neighbor-joining phylogenetic analysis
suggested that the strain from Israel sequenced in
this study was not strongly related to any other avail-
able ST and showed closest resemblance to isolate
ST45 from a bottlenose dolphin from Italy (Appendix
Figure, https://wwwnc.cdc.gov/EID/article/29/1/
22-1345-App1.pdf). Identification of P. damselae subsp. damselae was
supported and confirmed by molecular, pheno-
typic, and genomic characterization. The 16S rRNA
sequence showed a similarity of 99.17% with oth-
er P. damselae subsp. damselae strains in GenBank. When tested for hemolysis, the isolate exhibited
a weak hemolytic phenotype, producing narrow 181 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 DISPATCHES Figure 2. Histologic analysis of lungs and spleen of a bottlenose dolphin (Tursiops truncatus) with Photobacterium damselae subspecies
damselae pneumonia, eastern Mediterranean Sea. A) Lung tissue showed a nodular structure covered by fibrous capsule (right bottom
of figure panel) composed of numerous cholesterol clefts and areas of reactive fibrosis. Hyaline cartilage was observed, interpreted
as bronchi and bronchioles. Inset, higher magnification showing an aggregate of cholesterol clefts and hyaline cartilage (arrow). B) Abundant fibrous lung tissue (lower right half) and cellular infiltrates were also observed. C) Different area of the lung parenchyma
characterized by increased cellular infiltration. D) Spleen expressed an apparent contraction of the parenchyma showing diffuse
cellularity, with only a few defined lymphoid follicles, as well as megakaryocytes (arrows) indicative of extramedullary hematopoiesis. Inset: higher magnification showing 2 adjacent megakaryocytes (arrow). Hematoxylin and eosin stained. Scale bars indicate 500 µm in
panel A and 200 µm in panels B–D. Figure 2. Histologic analysis of lungs and spleen of a bottlenose dolphin (Tursiops truncatus) with Photobacterium damselae subspecies
damselae pneumonia, eastern Mediterranean Sea. A) Lung tissue showed a nodular structure covered by fibrous capsule (right bottom
of figure panel) composed of numerous cholesterol clefts and areas of reactive fibrosis. Hyaline cartilage was observed, interpreted
as bronchi and bronchioles. P. damselae Pneumonia in Bottlenose Dolphin Inset, higher magnification showing an aggregate of cholesterol clefts and hyaline cartilage (arrow). B) Abundant fibrous lung tissue (lower right half) and cellular infiltrates were also observed. C) Different area of the lung parenchyma
characterized by increased cellular infiltration. D) Spleen expressed an apparent contraction of the parenchyma showing diffuse
cellularity, with only a few defined lymphoid follicles, as well as megakaryocytes (arrows) indicative of extramedullary hematopoiesis. Inset: higher magnification showing 2 adjacent megakaryocytes (arrow). Hematoxylin and eosin stained. Scale bars indicate 500 µm in
panel A and 200 µm in panels B–D. used in human and veterinary medicine in this re-
gion. Tests results for T. gondii, canine distemper vi-
rus, and Brucella spp. showed negative results, mak-
ing P. damselae subsp. damselae the only culturable
pathogen identified in the dolphin. Results of molecular detection for T. gondii, canine
distemper virus, and Brucella spp. were negative for all
tested samples. Examination of lung tissue (Figure 2,
panels A–C) showed a nodular structure covered by fi-
brous tissue composed of extensive cellular infiltration,
numerous cholesterol clefts, and areas of reactive fibrosis. A second section of the lung showed extensive tissue lysis
and concentric fibrosis of blood vessels. In part of the sec-
tion, a locally extensive cellular infiltration was observed. An area of necrosis was accompanied by a neutrophilic
inflammatory reaction and intralesional bacterial colo-
nies. Two additional tissue sections showed diffuse solid
fibrosis, multiple cholesterol clefts, and aggregations of
leukocytes. Histopathologic analysis indicated an appar-
ent contraction of the parenchyma with occasional lym-
phoid follicles and diffuse cellularity within the spleen
(Figure 2, panel D), which were suggestive of extramed-
ullary hematopoiesis. Morphologic features of both or-
gans included severe chronic suppurative pneumonia
and splenic lymphoid depletion, possibly resulting in
extramedullary hematopoiesis in the spleen. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 Conclusions We report detection of P. damselae subsp. damselae in
a bottlenose dolphin in the Mediterranean Sea. This
report adds to the increasing baseline data regarding
the health of these marine mammals and provides
molecular information for a pathogen capable of in-
fecting a large variety of animals in the marine envi-
ronment, as well as humans. This study was supported by the Southern Marine Science
and Engineering Guangdong Laboratory (Guangzhou),
Guangzhou, China (grant SMSEGL20SC02) and by the
Kahn Foundation. D.M., N.D., E.B., Y.Z., A.S., and K.A. contributed to field
collections, necropsy procedure, and sample processing;
Z.Z.S., A.R., I.N., D.T., N.W., P.I., and M.R.E. contributed to
data processing, pathologic interpretation, and writing of the
manuscript; and I.N., N.F., A.R., S.B., and M.F. performed
bacterial isolation and molecular characterization. All authors
participated in drafting the manuscript, contributed to
writing the article, and approved the submitted version. y
This strain caused severe chronic suppurative
pneumonia in the absence of the dly gene. This result
supports previous indications that this virulence fac-
tor is not essential for pathogenesis (12). g
The antibacterial drug sensitivity test showed
susceptibility of the isolate to drugs most frequently 182 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 P. damselae Pneumonia in Bottlenose Dolphin About the Author bottlenose dolphins (Tursiops truncatus); a first description
from the eastern Mediterranean Sea. Vet Parasitol. 2018;258:74–8. https://doi.org/10.1016/j.vetpar.2018.06.009 bottlenose dolphins (Tursiops truncatus); a first description
from the eastern Mediterranean Sea. Vet Parasitol. 2018;258:74–8. https://doi.org/10.1016/j.vetpar.2018.06.009 Dr. Morick is a veterinarian, researcher, and head of the
marine pathology laboratory at the Morris Kahn Marine
Research Station, Haifa, Israel. His primary research
interests are marine animals, pathogen emergence, disease
transmission, aquatic animals, marine biology, marine
ecology, and public health. 7. Elia G, Decaro N, Martella V, Cirone F, Lucente MS,
Lorusso E, et al. Detection of canine distemper virus in
dogs by real-time RT-PCR. J Virol Methods. 2006;136:171–6. https://doi.org/10.1016/j.jviromet.2006.05.004 p //
g/
/j j
8. Bardenstein S, Waner T, Etinger M, Even Tof B, Blum S,
Bellaiche M, et al. First diagnosis of Brucella canis infection in
dogs in Israel. Isr J Vet Med. 2021;76:12–8. 9. Sharir Y, Kerem D, Gol’din P, Spanier E. Small size in the
common bottlenose dolphin Tursiops truncatus in the eastern
Mediterranean: a possible case of Levantine nanism. Mar
Ecol Prog Ser. 2011;438:241–51. https://doi.org/10.3354/
meps09282 Research Station, University of Haifa, Haifa 3498838, Israel; email:
dmorick@univ.haifa.ac.il Address for correspondence: Danny Morick, Morris Kahn Marine Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023 Address for correspondence: Danny Morick, Morris Kahn Marine
Research Station University of Haifa Haifa 3498838 Israel; email: References 1. Bearzi G, Fortuna CM, Reeves RR. Ecology and conservation
of common bottlenose dolphins Tursiops truncatus in the
Mediterranean Sea. Mammal Rev. 2009;39:92–123. https://doi.org/10.1111/j.1365-2907.2008.00133.x 1. Bearzi G, Fortuna CM, Reeves RR. Ecology and conservation
of common bottlenose dolphins Tursiops truncatus in the
Mediterranean Sea. Mammal Rev. 2009;39:92–123. https://doi.org/10.1111/j.1365-2907.2008.00133.x 10. Alba P, Caprioli A, Cocumelli C, Ianzano A, Donati V,
Scholl F, et al. A new multilocus sequence typing scheme and
its application for the characterization of Photobacterium
damselae subsp. damselae associated with mortality in
cetaceans. Front Microbiol. 2016;7:1656. https://doi.org/
10.3389/fmicb.2016.01656 g
j
2. Rivas AJ, Lemos ML, Osorio CR. Photobacterium damselae
subsp. damselae, a bacterium pathogenic for marine animals
and humans. Front Microbiol. 2013;4:283. https://doi.org/
10.3389/fmicb.2013.00283 2. Rivas AJ, Lemos ML, Osorio CR. Photobacterium damselae
subsp. damselae, a bacterium pathogenic for marine animals
and humans. Front Microbiol. 2013;4:283. https://doi.org/
10.3389/fmicb.2013.00283 3. Osorio CR, Vences A, Matanza XM, Terceti MS. Photobacterium damselae subsp. damselae, a generalist
pathogen with unique virulence factors and high genetic
diversity. J Bacteriol. 2018;200:e00002–00018. https://doi.org/10.1128/JB.00002-18 11. Casalone C, Mazzariol S, Pautasso A, Di Guardo G,
Di Nocera F, Lucifora G, et al. Cetacean strandings in Italy:
an unusual mortality event along the Tyrrhenian Sea coast in
2013. Dis Aquat Organ. 2014;109:81–6. https://doi.org/
10.3354/dao02726 4. Rivas AJ, Balado M, Lemos ML, Osorio CR. The
Photobacterium damselae subsp. damselae hemolysins
damselysin and HlyA are encoded within a new virulence
plasmid. Infect Immun. 2011;79:4617–27. https://doi.org/
10.1128/IAI.05436-11i 4. Rivas AJ, Balado M, Lemos ML, Osorio CR. The
Photobacterium damselae subsp. damselae hemolysins
damselysin and HlyA are encoded within a new virulence
plasmid. Infect Immun. 2011;79:4617–27. https://doi.org/
10.1128/IAI.05436-11i 12. Osorio CR, Romalde JL, Barja JL, Toranzo AE. Presence of
phospholipase-D (dly) gene coding for damselysin
production is not a pre-requisite for pathogenicity in
Photobacterium damselae subsp. damselae. Microb Pathog. 2000;28:119–26. https://doi.org/10.1006/mpat.1999.0330 5. Geraci JR, Lounsbury VJ. Marine mammals ashore: a field
guide for strandings. 2nd ed. National Aquarium in
Baltimore. College Station (TX): Texas A&M University
Press; 2005. Research Station, University of Haifa, Haifa 3498838, Israel; email:
dmorick@univ.haifa.ac.il 6. Bigal E, Morick D, Scheinin AP, Salant H, Berkowitz A,
King R, et al. Detection of Toxoplasma gondii in three common 6. Bigal E, Morick D, Scheinin AP, Salant H, Berkowitz A,
King R, et al. Detection of Toxoplasma gondii in three common 183 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 29, No. 1, January 2023
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Challenges to Ego-Depletion Research Go beyond the Replication Crisis: A Need for Tackling the Conceptual Crisis
|
Frontiers in psychology
| 2,017
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cc-by
| 4,284
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OPINION
published: 18 April 2017
doi: 10.3389/fpsyg.2017.00568 OPINION Edited by:
Martin S. Hagger,
Curtin University, Australia
Reviewed by:
Michael Philipp,
Massey University, New Zealand
Evan C. Carter,
United States Army Research
Laboratory, USA
Pierpaolo Primoceri,
University of Zurich, Switzerland
*Correspondence:
John Lurquin
john.lurquin@colorado.edu
Akira Miyake
akira.miyake@colorado.edu Edited by:
Martin S. Hagger,
Curtin University, Australia Reviewed by:
Michael Philipp,
Massey University, New Zealand
Evan C. Carter,
United States Army Research
Laboratory, USA
Pierpaolo Primoceri,
University of Zurich, Switzerland Reviewed by:
Michael Philipp,
Massey University, New Zealand
Evan C. Carter,
United States Army Research
Laboratory, USA
Pierpaolo Primoceri,
University of Zurich, Switzerland THE CONCEPTUAL CRISIS SURROUNDING THE
EGO-DEPLETION EFFECT We propose that compellingly resolving the controversy surrounding the ego-depletion effect
requires concerted efforts to address three interrelated conceptual problems, which jointly make
it difficult to derive unequivocal and testable predictions for any ego-depletion study. Below, we
illustrate these problems by referring to the strength model of self-control (Baumeister et al.,
2007) because this influential model has provided the basis for most of the existing ego-depletion
research. We emphasize, however, that these problems are general enough to be also applicable to
other models (e.g., Inzlicht and Schmeichel, 2012) and, hence, that field-wide efforts are needed to
satisfactorily address them. Specialty section:
This article was submitted to
Personality and Social Psychology,
a section of the journal
Frontiers in Psychology Received: 10 February 2017
Accepted: 28 March 2017
Published: 18 April 2017 Citation: Lurquin JH and Miyake A (2017)
Challenges to Ego-Depletion Research
Go beyond the Replication Crisis: A
Need for Tackling the Conceptual
Crisis. Front. Psychol. 8:568. doi: 10.3389/fpsyg.2017.00568 John H. Lurquin* and Akira Miyake* Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA Keywords: ego-depletion, self-control, the strength model, resource theories, replication crisis One important line of self-control research concerns the phenomenon known as ego-depletion,
the negative effect of performing a self-control task (Task 1) on performance on a subsequent
self-control task (Task 2). Although a 2010 meta-analysis reported a moderate effect size (d = 0.62)
for this phenomenon (Hagger et al., 2010), its replicability has since come under scrutiny with
the publication of some replication failures (Xu et al., 2014; Lurquin et al., 2016), including a
high-profile study involving 23 laboratories (Hagger et al., 2016). Some researchers even suggest
that the ego-depletion effect might not be real and that the reported results primarily reflect
publication bias (Carter and McCullough, 2014). This replication crisis has prompted a call for
additional replication attempts involving large sample sizes and preregistration (Carter et al., 2015). Although such replication efforts are undoubtedly important, we submit that, unless some
fundamental conceptual (and related methodological) issues are more satisfactorily addressed,
attempts to evaluate the ego-depletion effect would unlikely be successful. In this article, we outline
what we call the conceptual crisis for the ego-depletion literature, explain how these limitations
undermine replication attempts, and suggest possible ways to alleviate these problems. We do so by
noting some parallel problems that have faced cognitive psychologists studying attention, working
memory (WM), and executive functions (EFs), in the hope that such insights might contribute to
theoretical and empirical development in ego-depletion research. One important line of self-control research concerns the phenomenon known as ego-depletion,
the negative effect of performing a self-control task (Task 1) on performance on a subsequent
self-control task (Task 2). Although a 2010 meta-analysis reported a moderate effect size (d = 0.62)
for this phenomenon (Hagger et al., 2010), its replicability has since come under scrutiny with
the publication of some replication failures (Xu et al., 2014; Lurquin et al., 2016), including a
high-profile study involving 23 laboratories (Hagger et al., 2016). Some researchers even suggest
that the ego-depletion effect might not be real and that the reported results primarily reflect
publication bias (Carter and McCullough, 2014). This replication crisis has prompted a call for
additional replication attempts involving large sample sizes and preregistration (Carter et al., 2015). l h
h
h
l
ff
d
b dl
b
h
l Problem The field lacks clearly articulated and generally agreed-upon operational definitions of self-
control that can guide ego-depletion research. Although some studies refer to inhibitory April 2017 | Volume 8 | Article 568 Frontiers in Psychology | www.frontiersin.org The Conceptual Crisis in Ego-Depletion Research Lurquin and Miyake (e.g., the video-viewing task) even lack objective measures of task
performance that could be used as indices of self-control. control of some sort as their operational definition (e.g.,
Muraven et al., 2006; Tice et al., 2007), the term “inhibition”
is typically used in a rather generic sense, without being
specific
as
to
different
types
of
inhibitory
processes
postulated in the EF literature (Nigg, 2000; Friedman and
Miyake,
2004). More
problematic,
some
studies
define
self-control too broadly as the ability to control thoughts,
emotions,
and
behavior
(Segerstrom
and
Nes,
2007)
or
any monitoring and modification of behavior (Vohs et al.,
2005). control of some sort as their operational definition (e.g.,
Muraven et al., 2006; Tice et al., 2007), the term “inhibition”
is typically used in a rather generic sense, without being
specific
as
to
different
types
of
inhibitory
processes
postulated in the EF literature (Nigg, 2000; Friedman and
Miyake,
2004). More
problematic,
some
studies
define
self-control too broadly as the ability to control thoughts,
emotions,
and
behavior
(Segerstrom
and
Nes,
2007)
or
any monitoring and modification of behavior (Vohs et al.,
2005). This lack of independent validation of self-control tasks
is problematic, because it makes it difficult to derive an
unambiguous prediction for any ego-depletion study. For
example, according to the strength model, the ego-depletion
effect should be observed only when Tasks 1 and 2 (a)
both implicate self-control and (b) draw from the same self-
control resources. It is unclear, however, whether various task
combinations used in ego-depletion research actually meet these
necessary conditions. The justifications used for selecting self-control tasks are
equally unsatisfactory: Many studies use circular logic to justify
task selection by noting that the task was used before and had
a depleting effect. Even when some independent justifications
are provided, the attributes used to justify a task vary greatly,
including being intellectually demanding (Fennis et al., 2009),
requiring effort (Boucher and Kofos, 2012), and simply being
difficult (Webb and Sheeran, 2003). Consequently, wide-ranging
tasks like taking standardized tests (e.g., Converse and Deshon,
2009) or even balancing on one leg (Tyler and Burns, 2008) count
as self-control tasks. 1If the glucose-as-self-control-resources hypothesis (Gailliot et al., 2007) had
received strong support, this would have resolved the circular-logic problem, but,
given the growing evidence against this hypothesis (Beedie and Lane, 2012; Dang,
2016; Vadillo et al., 2016), the field still lacks independent, noncircular evidence for
the domain-generality assumption. Ways Forward Each researcher should explicitly articulate an operational
definition of self-control used in his/her study and justify task
selection with regard to that operational definition. To facilitate
the progress, however, more needs to be done by the field
as a whole. Parallel conceptual problems that have faced EF
research—another elusive and multifaceted concept—may be
relevant here. Although it is still far from achieving a field-
wide consensus (Baggetta and Alexander, 2016), attempts to
systematically classify and operationally define different facets of
EFs (e.g., updating, shifting, and inhibition; Miyake et al., 2000)
have contributed to developing some initial consensus, which has
helped researchers judge whether a task implicates EF processes. Analogous attempts would be helpful for self-control research,
especially if such efforts can help systematically examine which
facets of self-control are linked to the ego-depletion phenomenon
(e.g., Fujita, 2011; Heller et al., 2017). We find it justifiable to initially develop laboratory self-control
tasks on the basis of the experimenter’s intuition (Baumeister,
2016). We expect, however, that subsequent research would
validate their appropriateness as self-control indicators and offer
independent evidence that these tasks indeed draw on the
same pool of domain-general self-control resources. Without
knowing whether a particular task combination used in a study
meets these conditions, it is impossible to predict whether
one should expect a significant ego-depletion effect in that
study. Problem Concerning (a), a negative consequence of this problem is
illustrated by recent exchanges (Baumeister and Vohs, 2016b;
Hagger and Chatzisarantis, 2016) regarding the appropriateness,
as a self-control task, of the specific e-crossing task used in
Hagger et al. (2016) multilab replication study. A focal issue
was the necessity of an initial habit-forming block to make
the e-crossing task sufficiently demanding, but, tellingly, this
exchange did not reference any independent (non-ego-depletion)
research validating different versions of the e-crossing task as
effective (or not-so-effective) indices of self-control. Without
such independent evidence, any replication failures would
be open for alternative explanations based on task-selection
problems. Given this confusing state, it is hardly surprising that
the same task (e.g., 3-digit by 3-digit multiplication) has
been used as both the self-control (depletion) task (Stillman
et al., 2009) and the control (nondepletion) task (Burkley,
2008). If one cannot unambiguously determine whether or
not a particular task implicates self-control, it is impossible to
determine whether one should expect a significant ego-depletion
effect. Concerning (b), we do not know of any independent evidence
for this crucial domain-generality assumption. Although there
has been rigorous theoretical debate about, and empirical
investigation into, the domain generality/specificity of attention
(e.g., Wickens, 1984) and WM (e.g., Kane et al., 2004),
little consideration has been given to this important issue
in ego-depletion research, despite some prior evidence for
domain/process-specific ego-depletion effects (Persson et al.,
2007; Healey et al., 2011). Moreover, this domain-generality
assumption is built on circular logic: Domain-general self-
control resources must be present because the ego-depletion
effect is observed. This criticism is reminiscent of those
raised against resource theories in cognitive psychology, most
notably Kahneman’s (1973) seminal capacity theory of attention,
which, like the strength model, postulated a single pool of
general-purpose attentional resources fueling various mental
activities.1 2If fatigue is the more critical dimension (Baumeister and Vohs, 2016b;
Drummond and Philipp, 2017), independent evidence for linking fatigue levels to
self-control resources is needed. Ways Forward y
We do not know of any formal attempts to mechanically
specify how self-control resources are consumed when two
tasks
are
performed
consecutively
in
the
sequential-task
paradigm. It seems necessary not only to better specify
the underlying resource-consumption functions (preferably via
mathematical or computational modeling) but also to be
more explicit about critical moderating variables (e.g., when
to conserve or consume resources). To gain insights into
the underlying resource-performance functions, it might also
be helpful to systematically (parametrically) manipulate task
durations or attentional demands for Task 1 (Lee et al., 2016),
which unfortunately has rarely been done in ego-depletion
research. It is also important to provide more objective measures of
task performance to quantify the self-control demands associated
with Task 1 performance. One such possibility is to use
pupillometry (Beatty, 1982) as an index of the degree of effort or
attentional demands associated with the task performance2 (e.g.,
Hopstaken et al., 2015; Rondeel et al., 2015). CONCLUSION The recent replication efforts have succeeded in promoting
preregistration, open data, and large sample sizes, all of which
improve the reproducibility of scientific work. To resolve the
issue of whether ego-depletion is a real phenomenon, however,
it is also crucial to address the severe conceptual problems
that impede the derivation and testing of specific, falsifiable
predictions. Although tackling these issues is not easy, we
believe that effectively addressing them is a necessary step to
resolve the current controversy surrounding the ego-depletion
effect in a manner that satisfies its proponents and skeptics
alike. This theoretical issue has been neglected in ego-depletion
research, despite some relevant historical precedent. In an
influential critique, Navon (1984) articulated various problems
with resource theories (e.g., Kahneman, 1973), including
the aforementioned circularity problem and the ambiguity
surrounding the hypothesized resource-performance functions. This critique led some theorists to abandon the resource concept
altogether (Neuman, 1987) and others to attempt to better specify
the nature of resources and their consumption functions in the
form of computational models (e.g., Just and Carpenter, 1992;
Lovett et al., 1999). Models of ego-depletion phenomena are in
need of such formalization. FUNDING Such theoretical development is urgently needed following
the recent updates made to the strength model (Baumeister
and Vohs, 2016a) that, in our view, make the model flexible
enough to fit any data and, hence, unfalsifiable. In particular, this
revised model incorporates the notion of the “central governor”
(adopted from Evans et al., 2016), whose role is to determine
whether to expend or conserve the available self-control Publication of this article was funded by the University of
Colorado Boulder Libraries Open Access Fund. Problem The existing models purported to explain the ego-depletion
effect are currently too underspecified to allow other researchers
to unambiguously derive testable (falsifiable) predictions. For
example, the strength model does not specify how the self-
control resources are consumed by Tasks 1 and 2 and when
the available remaining resources are low enough to start
impairing subsequent performance on Task 2. Such key resource-
consumption parameters must be more formally specified before
one can determine whether an experiment should produce the
ego-depletion effect. Problem Various tasks used in ego-depletion research—such as watching
a video while ignoring words appearing onscreen and writing
essays without using certain letters—have not been independently
validated as effective measures of self-control. Some such tasks
have not been used outside ego-depletion research, and some April 2017 | Volume 8 | Article 568 Frontiers in Psychology | www.frontiersin.org 2 The Conceptual Crisis in Ego-Depletion Research Lurquin and Miyake AUTHOR CONTRIBUTIONS All authors listed, have made substantial, direct and intellectual
contribution to the work, and approved it for publication. 3Indeed, Logie (2016), a long-time proponent of the central executive (Baddeley
and Logie, 1999), recently declared that the field is now ready to “retire” it. Ways Forward resources. This addition seems to us a step backwards,
considering that WM theories, which have long featured the
“central executive” (Baddeley and Hitch, 1974; Baddeley, 1996),
have been trying to replace this vague, homunculus-like construct
with something more precise3. Without better specifying how
this central governor determines whether and when to consume
or conserve self-control resources, one cannot unambiguously
determine whether one should observe a significant ego-
depletion effect (for a more detailed critique of the central
governor model, see Inzlicht and Marcora (2016). One way to alleviate these problems is to conduct carefully
designed correlational research (e.g., latent-variable analysis)
and/or experimental studies using the simultaneous dual-task
interference paradigm to establish that various commonly
used tasks in ego-depletion research share some underlying
commonality, namely self-control resources. Tests of ego-
depletion would be more effective when the specific combination
of tasks used has already been shown to demonstrate a clear
overlap between them. In this regard, relying more on cognitive
(attention, WM, and EF) tasks for which such evidence of overlap
already exists might be helpful. REFERENCES Toward a
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during updating, inhibition and switching as revealed by pupillometry. Front. Hum. Neurosci. 9:494. doi: 10.3389/fnhum.2015.00494 Gailliot, M. REFERENCES Hagger, M. S., and Chatzisarantis, N. L. (2016). Commentary: misguided effort
with elusive implications, and sifting signal from noise with replication science. Front. Psychol. 7:621. doi: 10.3389/fpsyg.2016.00621 Baddeley, A. (1996). Exploring the central executive. Q. J. Exp. Psychol. 49A, 5–28. doi: 10.1080/713755608 Hagger, M. S., Chatzisarantis, N. L., Alberts, H. A., Anggono, C. O., Batailler,
C. B., Birt, A. R., et al. (2016). A multilab preregistered replication of the
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652873 Baddeley, A. D., and Hitch, G. J. (1974). “Working memory,” in The Psychology
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and the strength model of self-control: a meta-analysis. Psychol. Bull. 136,
495–525. doi: 10.1037/a0019486 Baddeley, A. D., and Logie, R. H. (1999). “Working memory: the multiple-
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seas: winners, losers, and recommendations. J. Exp. Soc. Psychol. 66, 153–158. doi: 10.1016/j.jesp.2016.02.003 Hopstaken, J. F., van der Linden, D., Bakker, A. B., and Kompier, M. A. (2015). The
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574–575. doi: 10.1177/1745691616652878 Inzlicht, M., and Schmeichel, B. J. (2012). What is ego depletion? ACKNOWLEDGMENTS We thank Michael J. Kane for providing thoughtful feedback on
this manuscript. 3Indeed, Logie (2016), a long-time proponent of the central executive (Baddeley
and Logie, 1999), recently declared that the field is now ready to “retire” it. April 2017 | Volume 8 | Article 568 Frontiers in Psychology | www.frontiersin.org 3 The Conceptual Crisis in Ego-Depletion Research Lurquin and Miyake REFERENCES T., Baumeister, R. F., DeWall, C. N., Maner, J. K., Plant, E. A.,
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et al. (2014). Failure to replicate depletion of self-control. PLoS ONE 9:e109950. doi: 10.1371/journal.pone.0109950 Xu, X., Demos, K. E., Leahey, T. M., Hart, C. N., Trautvetter, J., Coward, P.,
et al. (2014). Failure to replicate depletion of self-control. PLoS ONE 9:e109950. doi: 10.1371/journal.pone.0109950 Tice, D. M., Baumeister, R. F., Shmueli, D., and Muraven, M. (2007). Restoring the
self: positive affect helps improve self-regulation following ego depletion. J. Exp. Soc. Psychol. 43, 379–384. doi: 10.1016/j.jesp.2006.05.007 Soc. Psychol. 43, 379–384. doi: 10.1016/j.jesp.2006.05.007 Tyler, J. M., and Burns, K. C. (2008). After depletion: the replenishment of the
self’s regulatory resources. Self Identity 7, 305–321. Frontiers in Psychology | www.frontiersin.org REFERENCES doi: 10.1080/15298860701
799997 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Vadillo, M. A., Gold, N., and Osman, M. (2016). The bitter truth about sugar and
willpower: the limited evidential value of the glucose model of ego depletion. Psychol. Sci. 27, 1207–1214. doi: 10.1177/0956797616654911 Copyright © 2017 Lurquin and Miyake. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) or licensor are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. y
Vohs, K. D., Baumeister, R. F., and Ciarocco, N. J. (2005). Self-regulation and self-
presentation: regulatory resource depletion impairs impression management
and effortful self-presentation depletes regulatory resources. J. Pers. Soc. Psychol. 88, 632–657. doi: 10.1037/0022-3514.88.4.632 April 2017 | Volume 8 | Article 568 Frontiers in Psychology | www.frontiersin.org 5
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Our Gaelic Class
|
All Ireland review
| 1,900
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public-domain
| 1,560
|
EXTRACT
FROM
THE
"DAILY
EXPRESS."
"THE SELECTION AND PURCHASE OF CHRISTMAS
PRESENTS." ADDRESS
At
the
very
beginning
of
the
present
year
this
journal
was
established. This
year
is
now
drawing
to a
close,
and
during
its course
the lovers
of our native
tongue
have
been
very
successful
in promoting
its
ancient
and
sacred
cause. May
they
be
sevenfold
more
successful
a
year
hence. And
now,
dear
readers
of
this
journal
and
members
of the Gaelic
Class,
I pray
that
God
may
grant
you every joy during this Holy Season of Christmas
; that
you may
have
a happy
new
year,
and
that
very
many
returns
of that
event may
await
you Year
affer year, for a longer period than we would
like to state,
we have known
ladies and gentlemen
to put off the purchase
of the
presents
they wished
to make
until within
a few d iys ot Christ
mas, with
the result that they were hurried and flurried,
and shops
crowded,
and nine
times
out of ten, articles,
were purchased
that
neither
pleased
the donors
nor were
they
seasonable
or useful
to
the recipients. To obviate
a recurrence
of this unpleasant
state of
affairs
we
beg
thus ?arty to arouse
the attention
of our readers,
both
ladies
and
gentleman,
to
the
great
advantages
that
are
offered to them
to buy really useful
and seasonable
articles
(that
will
be undoubtedly
acceptable
to either
sex)
at
about
half
the
regular
prices. We
refer
to the
executors*
sale
of rich furs and
mantles
now proceeding
at Messrs
Barnardo's
108, Grafton-street. On
reference
to our advertising
columns
it will be seen "the
last
weeks
of the salegare
announced. The collection
is not only large,
but bewildering
in variety. There
are
fur-lined
coats
for gentle
men's
use in the city or the
field, dainty
wraps
for ladies
of the
same soft,
warm
material
in all varieties,
and garments
in real
sealskin,
now
so dear, but which
having
early laid in a large stock,
the firm are able to offer at astonishingly
low prices. Children
are
equally
well
provided
for, and all articles
are marked
at nearly
50 per cent, below
ordinary
prices. prosperous
may
you,
*OiteA5^A,
an
address
; Aop
teigmn,
class
; p?op? topACt
very
beginning
(from
p?op,
true,
and copA?,
begin
ning);
bliAtmA,
of
a
year
(gen. All Ireland Review All Ireland Review Our Gaelic Class
Author(s): Patrick Kangley and C. B. Source: All Ireland Review, Vol. 1, No. 51 (Dec. 22, 1900), p. 7
Published by: All Ireland Review
Stable URL: http://www.jstor.org/stable/20545106 . Accessed: 22/06/2014 03:13 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www jstor org/page/info/about/policies/terms jsp Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
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All use subject to JSTOR Terms and Conditions ALL IRELAND REVIEW. people
; muinttfl
ti a
gAetntge
the
people
(who
were
working
in behalf)
of
the
Gaelic
; f eAti,
old,
ancient
;
c?if,
a cause
(fern.)
; nAorhCA,
holy,
sacred
; -An c-feAti
c?if
nAotintA
wo,
that
ancient
and
sacred
cause,
i.e.,
of
the Gaelic
; ye An most
frequently
goes
before
the
noun
forming
a compound
noun
; <3*o, that,
yonder
; ?ifVOfteim,
high-career
or sway
; "oo tAX>A\nz. in-Aijvof\?inn,
to
pro
mote
: 50 m
oa, may
it be
(optativa
of
?p) ; peAcc,
seven
;
f eA?c
oce?nn,
seven-times
better
; peA^fv
comparative
of
mAitc,
good
: eine?cAi'? (future
of
etfugtm),
will
suc
ceed
; leo,
with
them
; btiA*?Ain
?'n
I? itvotu,
a
year
from
to day,
i.e? a
year
hence
; teugt?ijt?,
readers
;
Aitcrni,
I pray,
entreat
; Ajt *?\A, of
(on) God;
AOibinn,
pleasant,
joyful;
aj;a?o}
at
or with
you
; An
Aimft|i
tiAorhcA
f o, this
holy
season
; tl0T>tA5
(gen. of H 0*01^15,
fern.)
, Christmas
; foriA,
lucky,
prosperous;
huatj,
nesr;
50 m-b-A
f onA dgAib
-An ?uia*? ?tiATiAin,
may
you
have
a
happy
new
year
; beip
An, overtake
; 50
m-betnni
aix
10mAt)
ac? (To be Continued.) (To be Continued.) Professor Mahaffy's
letter is interesting,
as raising
a question,
generally
ignored,
so
far
as
I know,
by
ethnologists,
but which has often presented
itself
to
me,
viz.,
the
prominence
of
the
tall,
dark,
powerful,
brachycephalic
type, to be found in " Celtic "
regions,
and not satisfactorily
accounted
for by any of the
customary
Basque
or Berber,
Euskanan
or Auverg
nal
Iberian
or Celtic
theories. p?t)ttAic
mAcceAnslAig,
Sdu^t?if
"o' Aop
t?iginn
tiA SAe^ilge,
(TRANSLATION) p?t)ttAic
mAcceAnslAig,
Sdu^t?if
"o' Aop
t?iginn
tiA SAe^ilge,
(TRANSLATION) ?t)ttAic
mAcceAnslAig,
Sdu^t?if
"o' Aop
t?iginn
tiA SAe^ilge,
(TRANSLATION) All Ireland Review oj^Aib,
may
a good
many
of
them
overtake
you
; beifut),
optative
of
beij\im
; ioiuat?,
many
; aca
here
means
of
them
; Ajvfuio,
on
you
; retin,
prosperity
;
?a
feun,
in prosperous
circumstances
; fc??jtto?fi,
direc
tor,
conductor,
manager. tann, who lived on both
sides of the great flood, and
roamed the depths when
the world was
submerged
;
there
Partholanus
and
his
il?-starred
race?the
chroniclers
know
them all;
there the children
of
Nemed
in
their
own
Golgotha,
their
stones
all
care
fully
lettered,
these
not
so ancient
as
the
rest,
only
three
thousand years before the birth
of
Christ;
there the Clan Fomor,
a giant race, and the Firbolgs
with
their
correlatives,
Fir-Domnan
and
Fir-Gaileen
the
Tuatha
De
Da?an,
whom
the
prudent
annalist
condemns
to
a
place
amongst
the
dead?a
divine
race
they
will
not
die?they
flee
afar,
preferring
their
phantasmal
life
; even
the
advent
of
the
Talk
end will
not
slay them,
though
their glory
suffers
eclipse before the new faith. The children of Milith
are
there with
their long ancestry reaching
to Egypt
and
Holy
Land?Heber,
Heremon,
Amergin,
Ir,
with
all their descendants,
each beneath his lettered
stone;
Tiernmas
and Moh Corb, Ollav Fohla,
their
lines descending
through many
centuries
; all put
away
and
decently
composed
for
ever. No
confusion
now,
no
dissolving
scenes
or
aught
that
shocks
and
dusturbs,
no
conflicting
events
and
incredible
re-ap
pearances. Chronology
is
respected. The
critical
and historical
intellect
has provided
that all things
shall be done rightly and in order, that the obits and
oirths
and battles
should be natural
and
imposing,
and
worthy
of
the
annals
of
an
ancient
people _C.
" A Chance
of a Lifetims." )
o nAib
p? f
p?t)ttAic
mAcceAnslAig EXTRACT
FROM
THE
"DAILY
EXPRESS."
"THE SELECTION AND PURCHASE OF CHRISTMAS
PRESENTS." of
bliAt>Ain,
fern.);
tAtAi|\,
presence
; 1 tAtAip,
present
; cui^eAt),
was-put
(past
passive
of ctn^im,
I put
; bun,
foundation
;
*oo c
An bun,
was-established
; ifupteAbAn,
a
journal
(mas.)
;
AS
T)tMii*oim
te,
drawing
towards
; TieineAt),
end,
close
;
tinn,
period
of
time
; te
n-A tmn,
during
its
course
;
'eipi$
50
rriAit
te,
it
succeeded
well
with
; muincip, ,
ordinary
prices. bove
refers
to the last weeks
of the Executors'
Sale at g
PATRICK KANGLEY. Conductor
of
the Gaelic
Class. Conductor
of
the Gaelic
Class. The articles
on "Pre-Historic
Ireland"
in
the
"
A. I. R."
are
very
interesting. May
I
suggest
that
the
writer,
though
by
no
means
dogmatic,
yet
scarcely
makes
us
understand
how
very
complicated
the ethnolical
question
is, and how contradictory
the
evidence
? worthy
people. And
thus, regarding
the whole
from a point
of
view
sufficiently
remote,
a certain
epic
completeness
and
harmony
characterizes
that
vast
panoramic
suc
cession
of
age? and
races. OUR GAELIC
CUSS. wteAgRA
T)' Aos
1 0151 nn ha
gAetnlge. wteAgRA
1.0151
gAetnlge. 1
DpOF-tOfA? riA
bllAtmA
A
C? 1 lAtAin
X>0
cmneA-? An c-ift?pteAbAn
T? ?P
t)un. U? An
btiAt>Ain
fo
as
Dtuivoim
te
TjeipeAt)
Anoip,
A^up
le
n-A
linn
*o'
?itug
50
rriAit te nunncifi
nA g^e-?it^e
An c-peAn
c?ip
nAomtA
?x>
"oo tADA^z
1 ti-Ai|\t)
|\?im. go
m-bA
p eAcc
bpe?ftp
ad'
eipeocAit)
teo
bliAt>Ain
?'n
tAitroi?. Ajjup
Anoip
a teugt?i?v? An
IjupteAbAin
po,
Agtip
a
rhtnnan
t)'AOf
t?ig?nn
riA 5Ae>?1^5e>
?itcim
A|\
"O?a 50
m-b. Aoibtn
tiA^Aib
An
Aimpip
tiAorhtA
po
nA
tlo*otA5j
50
m-bAfonA
A5Aib
An
?luA'?-btiA^?Ain,
A^up
50
m-beifiii?)
a?i
loniAt)
ac? ^ Aib
? f etm. nal,
While
recognising
the limits
imposed by space, I
hope
that
we
may
learn
more
of
the
old
romances
translated
for us,
as well
as counterspells
to the Great
Enchantment
and
also
a continuance
of
those
more
b?n?ficient
spells which
the votaries
of
the
Irish
muses
put
us under
at your
behest. B. BARNABDO'S, BARNABDO'S, BARNABDO'S,
108,
?EAFTON-STBEET,
DUBLIN. BARNABDO'S,
108,
?EAFTON-STBEET,
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English
| null |
ON THE CHANGES INDUCED IN BLOOD BY FEEDING: A STUDY IN CELLULAR PHYSIOLOGY.
|
Lancet
| 1,912
|
public-domain
| 9,619
|
BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) Secondly, to determine, if possible, the function of the
granules in both leucocytes and lymphocytes. Thirdly, to
see whether the increased alkalinity of the blood during
gastric digestion would show itself by an increased affinity
for free acid dyes, as in Mylius’s reaction. And fourthly, to
reinvestigate the problem whether a deposit of stored
material, such as glycogen or fat, occurs in the blood cells
or platelets after a liberal meal. his recovery. As regards the after-history of my patients I can only say
that I have seen them at periods varying from seven years to
.a few weeks after their operation, and, except in a very few
instances, they have been quite satisfied and have told me
-that the joint was as strong as ever. In part proof of this
I would refer you to the cases in which after the semilunar
.cartilage in one knee has been removed, the patients have
returned to have a torn cartilage removed from the other, or
in one or two cases the other cartilage of the same knee. ’They would not have done this if the first operation had not
been successful. Again, where I have operated in the case
of both amateur and professional football players, they have
all played again, and many of them are still playing. One
Monday morning, a week or two ago, I noticed in the column
of the Daily Telegraph devoted to the account of Association
matches played the previous Saturday, that a player in an
important Second League match, from whose left knee I
removed both semilunar cartilages at the same operation on
March 16th, 1910, had not only played well, but scored the
- only goal of the match. platelets
Our experiments were made on men and frogs. We
started our investigation on six medical students, who
volunteered to act as subjects, and the following procedure
was adopted. After eating the usual breakfast, which
included at least two eggs and one glass of milk, no food of
any kind and no liquid, except water, was ingested until noon
of the next day; thus the men fasted for about 30 hours. Immediately before the breakfast, and after breakfast at
two-hour intervals, each student made four blood films,
except from 12 o’clock midnight until 8 A.M. the following
morning. BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) (WITH COLOURED PLATE.) THE experiments detailed below were undertaken with the
view of ascertaining whether feeding would produce changes
in the blood which could be demonstrated microscopically,
apart from the increase in number of white blood corpuscles
which we know to occur. Lily Huie,l working in Edinburgh and Oxford under
Mann’s direction, was able to show marked changes in the
nuclei of the gland cells of the insectivorous plant Drosera
after feeding the same with egg albumin, albumoses,
peptones, &c. The rapidity with which the nuclear changes
could be produced depended on the amount of predigestion
of the protein food substances. Subsequently, H. G. Butter-
field, working with Mann in Oxford, succeeded in dernon-
strating that if newts are starved in the summer time for a
fortnight, and if each newt is fed with a worm of the size of
a wooden match, all the nuclei of the most diverse animal
tissues, excepting only the nuclei of the nerve cells, would
stain much more deeply in the fed newts than in the control
animals. After the operation a rise in temperature for the first two
nights to 990 or 1000 F. is commonly met with, but this need
cause no alarm. Again, for the first 48 hours a great deal
of pain may be complained of, necessitating one or two
hypodermic injections of morphia. Otherwise the con-
valescence of the patient is perfectly straightforward, and
;at the end of a week the subcuticular suture is removed. After this is done a light dry dressing is applied, and my
infirmary patients are discharged as a rule on the tenth
- day, being then able to carry out full movements of the joint. In the case of one patient convalescence was disturbed by an
:attack of acute gangrenous appendicitis, for which operation
was promptly performed 11 days after the cartilage had been
removed, with a successful result. Another was seized with
acute lobar pneumonia (pneumococcal), but made a good
recovery; while a third contracted a whitlow a few days
following his operation, but this did not in any way affect
his recovery. In the present instance blood was chosen for the subject
of inquiry to ascertain whether metabolism in the nucleated
blood cells during the ingestion of food is likewise accom-
panied by a definite increase in the nucleo-proteins. 1 Huie, Lily : Changes in the Cell Organs of Drosera Rotundifolia,
produced by Feeding with Egg Albumin, Quarterly Journal of
Microscopical Sciences, vol. xxxix., N.S. (1896-97).
Further Study of
Cytological Changes produced in Drosera, ibid., vol. xlii. (1899).
2 Consult Mann’s Physiological Histology, Clarendon Press, 1902. 1069
DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. 1069
DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING 1069
. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. 1069 case of the internal cartilage, from the inner border of the
patellar tendon, backwards for about two inches in the
line of the articulation. This incision, I consider, gives
the best exposure, and if the internal lateral ligament
be not cut across there is no interference with the
after stability of the joint. After the skin is divided
.a strong aponeurosis is exposed, and is also divided
in the length of the incision. The capsule with under-
lying synovial membrane is then opened, and the interior
-of the joint exposed. Then, in the great majority of
- cases, the
tear or split will be quickly recognised,
- especially where it involves the anterior part of the cartilage
or where there is marked displacement of the torn portion. If after careful scrutiny nothing pathological is discovered, I
- divide the anterior attachment of the semilunar cartilage,
seize it with strong-toothed forceps, and drag it forwards. If
there be a posterior split the detached portion will suddenly
snap forwards between the condyles and tuberosity. This
snapping forwards is what actually happens during an attack,
and if witnessed it can be readily appreciated what a shock it
will be to the patient. In every case I remove not only the
detached piece, but endeavour to ablate the portion still
retaining its normal attachment, and since doing this have
had no patient returning with recurrence of symptoms. After the cartilage is removed the capsule and synovial
membrane are sutured in one layer with catgut. The
aponeurosis is similarly dealt with, and the skin incision
is closed with a subcuticular suture of silkworm gut. An
antiseptic dressing is then applied, with a thick layer of
wood-wool wadding, and secured by a domette bandage
firmly applied. No splint is used, and the patient
is told to commence to move his knee as soon as he
can. injuries to the semilunar cartilages in text-books and else-
where, this was unavoidable. 1069
DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. I
trust my small
con-
tribution to the subject may be of some value, and would add,
in conclusion, that I have always thought these accidents,
which claim as their victims a goodly number of those
engaged in the important industry of coal mining, have not
received the attention they deserved. case of the internal cartilage, from the inner border of the
patellar tendon, backwards for about two inches in the
line of the articulation. This incision, I consider, gives
the best exposure, and if the internal lateral ligament
be not cut across there is no interference with the
after stability of the joint. After the skin is divided
.a strong aponeurosis is exposed, and is also divided
in the length of the incision. The capsule with under-
lying synovial membrane is then opened, and the interior
-of the joint exposed. Then, in the great majority of
- cases, the
tear or split will be quickly recognised,
- especially where it involves the anterior part of the cartilage
or where there is marked displacement of the torn portion. If after careful scrutiny nothing pathological is discovered, I
- divide the anterior attachment of the semilunar cartilage,
seize it with strong-toothed forceps, and drag it forwards. If
there be a posterior split the detached portion will suddenly
snap forwards between the condyles and tuberosity. This
snapping forwards is what actually happens during an attack,
and if witnessed it can be readily appreciated what a shock it
will be to the patient. In every case I remove not only the
detached piece, but endeavour to ablate the portion still
retaining its normal attachment, and since doing this have
had no patient returning with recurrence of symptoms. After the cartilage is removed the capsule and synovial
membrane are sutured in one layer with catgut. The
aponeurosis is similarly dealt with, and the skin incision
is closed with a subcuticular suture of silkworm gut. An
antiseptic dressing is then applied, with a thick layer of
wood-wool wadding, and secured by a domette bandage
firmly applied. No splint is used, and the patient
is told to commence to move his knee as soon as he
can. Newcastle-on-Tyne. 3 Since the completion of this paper a very important article on the
Romanowsky staining of blood cells, by S. G. Scott, T O. Thompson, and
J. L. Hydrick, has appeared in Folia Hæmatologica, No. 12, p. 302 (1911).
The data given in this paper will help us in further investigations.
4 See Mann’s Physiological Histology. White Cells. In making comparisons between "control" and "fed"
"
films due attention was paid to the fact that in fasting cells
the resistance to pressure is less, and that therefore they are
apt to be spread out more, in consequence of which they
will present a paler appearance when examined under the
microscope lymphocytes. In the Giemsa preparations there are also a number of
polymorphonuclear leucocytes containing large granules,
which stain red with a slight tinge of purple. Some of
them are very large and some very small, there being no
uniformity in size.6 These granules are unstained in both
the triacid and hæmatoxylin methyl blue-eosin preparations. They are most numerous in the tenth-hour films and are
very scarce in the control films. microscope. A. Lymphocytes in man.-Compare Figs. 1, d, e, f, g, with
Figs. 2, f, g, A, i, k, and Figs. 3, g, h, i, k, l, m, gi, with
Figs. 4, A, i, k, l,m, n, o. Figs. 1 and 3 represent the
appearance met with in fasting films, and Figs. 2 and 4
those seen during full digestion. Figs. 1 and 2 represent
films from one of the students, while Figs. 3 and 4 represent
those of Mann. g
i
very
These granules have not been previously described, and
probably are stained metachromatically by the methylene-
azur ; they either represent a stage in the formation of the
neutrophile granules, which is not likely, or they represent
a special intracellular enzyme, or, probably, the formation of
the sol-phase in the cytoplasm, with which we mean that
part of the cytoplasm in which finally the neutrophile
granules are imbedded. The cells in the films made during full digestion are
smaller than those in the control films. The decrease in size
of the cell seems to be due almost entirely to a narrowing of
the rim of cytoplasm surrounding the nucleus, the nucleus
itself being but little affected, if any. By all three stainin
methods the films made during the periods of digestion and
of fast showed marked differences in the staining intensity
of both the nucleus and the cytoplasm. ’
. n
granules
2. Eosinophiles. Compare Fig. 1, e, with Figs. 2, d, e,
and Figs. 3, e, f, with Figs. 4, j, g. The diminution in size
and number of the granules is quite as well marked as in
the neutrophiles. BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) he following procedures were adopted in staining :- g p
g
I. Fixed by Gulland’s method. Stained in Mann’s acid hæmatein-
30 min. Rinsed in distilled water. Stained in Mann’s methylblue-
eosin-15 min. Rinsed in distilled water. Blotted with toilet paper. Examined in oil. Examined
oil. II. Fixed by Gulland’s method. Stained in "Giemsa’s" stain-12
hours. Rinsed in distilled water. Blotted with toilet paper. Examined in oil. III. Fixed by heat on xylol bath for 1 hour. Stained in Ehrlich’s
triacid mixture-15 min. Rinsed in distilled water. Blotted with
toilet paper. Examined in oil. In order to give all the films uniform treatment they were
kept for 10 days, to equalise the time factor between the
films made at the beginning and at the end of the experi-
ment. The slides with the films to be fixed and to be stained
by a given method were then fastened together in slide-
holders, were placed into and removed from the fixing bath
and the stain at the same time, and were then carried
through the various rinsing baths in a like manner. In short,
all films received as nearly the same treatment as possible,
as regards both fixation and staining. fading
Leucocytes in man.-1. Neutrophiles.-Compare Figs. 1,
a, b, with Figs. 2, a, b, c, and Figs. 3, a, b, c, d, with
Figs. 4, a, b, c, d, e. The diminution in size during the
digestion period can also be seen in these cells, but is not as
marked as in the case of the lymphocytes. The neutrophile
granules are larger and more abundant in the control films
as shown by the triacid and ’’ Giemsa
" stains. 5
In the
films made during the height of digestion--i.e., about the
eighth hour-there is a distinct, though not very marked,
diminution both in the size and in the number of granules. The granules are more numerous at the periphery of the
cells, leaving a clear zone around the nucleus. Occasionally
a cell is seen in which the granules are arranged in chains
radiating from the nucleus like the spokes in a wheel. Cells
may often be seen in which the granules at the periphery
are confluent. The increase in the staining intensity of the
nuclei is so marked that one can tell at a glance whether
any given preparation was made during the starvation or the
digestion period. BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) In these films made during the digestion
period the nuclei stain much more deeply, the maximum being
reached at the eighth and tenth hours. At this time the
films stained with Giemsa’s stain showed diffuse purplish-red
areas in the nuclei of some of the neutrophiles and
lymphocytes regards
staining. The films were studied with the view of noting changes in
the plasma, in the red cells, and in the white cells. 5 The peculiar staining of the neutrophile granules by our Giemsa
stain is probably due to the fixation f the films by Gulland’s method.
6 Ibid. Plasma and -Red. Cells. The Mylius-Ehrlich test showed a definite increase in the
alkalinity of the plasma during gastric digestion. The
changes could first be detected in the films made four hours
after the meal and were present until the eighth hour, the
maximum being reached at about the sixth hour. being
Changes in the viscosity of the plasma and alterations in
surface tension of the red cells were carefully looked for, but
the difficulties encountered were so great that we think it
best not to make definite statements concerning them at this
time. 5 The peculiar staining of the neutrophile granules by our Giemsa
stain is probably due to the fixation f the films by Gulland’s method.
6 Ibid.
7 Consult Mann’s Physiological Histology, pp. 29-38 BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) This was more
marked in the six-hour films, and the maximum was reached
at the eighth and tenth hours. In these films nearly all
the lymphocytes were small, with their nuclei and cytoplasm
very deeply stained, the rim of the cytoplasm being very
narrow and difficult to distinguish from the nuclei. An
occasional lymphocyte could be found which was somewhat
larger and less deeply stained than the rest, but none was as
large and pale as the average cell in the control films. After
the twelfth hour the differences in the films became less
noticeable. in the films made during the digestion period. The cyto-
plasm, although pale, was more deeply stained than the
nucleus. The films made four hours after the meal were the
earliest to show a definite change. In these the average
width of the rim of cytoplasm was less than in the control
or fasting and two-hour films, and both the cytoplasm and
nucleus were stained somewhat more deeply. This was more
marked in the six-hour films, and the maximum was reached
at the eighth and tenth hours. In these films nearly all
the lymphocytes were small, with their nuclei and cytoplasm
very deeply stained, the rim of the cytoplasm being very
narrow and difficult to distinguish from the nuclei. An
occasional lymphocyte could be found which was somewhat
larger and less deeply stained than the rest, but none was as
large and pale as the average cell in the control films. After
the twelfth hour the differences in the films became less
noticeable. Most of the nuclei of the lymphocytes, stained by
° Giemsa’s " method, show particularly during the fasting
condition, round, sometimes oval, vacuoles with sharply
defined surfaces, which latter in mid-focus appear as rings. The boundaries of the vacuoles stain more deeply than does
the rest of the nucleus, and in the surface membrane,
bounding the vacuole, minute granules may frequently be
seen. There is usually one vacuole in each nucleus, but
occasionally two vacuoles are met with. The vacuoles are
best seen in lymphocytes having very pale nuclei, when they
appear as dark "rings." Stained by other methods than the
Giemsa stain prepared by us the vacuoles can be seen, but are
not distinct, and in "Giemsa" preparations, kept unmounted
for a couple of months, the vacuoles are also difficult to see
because of the fading of the surface membrane. ncreased. Ibid.
7 Consult Mann’s Physiological Histology, pp. 29-38 BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) After
the twelfth hour the differences in the films became less
noticeable. Most of the nuclei of the lymphocytes, stained by
° Giemsa’s " method, show particularly during the fasting
condition, round, sometimes oval, vacuoles with sharply
defined surfaces, which latter in mid-focus appear as rings. The boundaries of the vacuoles stain more deeply than does
the rest of the nucleus, and in the surface membrane,
bounding the vacuole, minute granules may frequently be
seen. There is usually one vacuole in each nucleus, but
occasionally two vacuoles are met with. The vacuoles are
best seen in lymphocytes having very pale nuclei, when they
appear as dark "rings." Stained by other methods than the
Giemsa stain prepared by us the vacuoles can be seen, but are
not distinct, and in "Giemsa" preparations, kept unmounted
for a couple of months, the vacuoles are also difficult to see The films were stained in a modified Giemsa stain,
Ehrlich’s triacid mixture, and Mann’s acid haematein
and Mann’s methylblue-eosin. The methylene-azur of
’’ Giemsa’s
" stain was prepared by us according to Borrel’s
silver oxide method or by short treatment with 2 per cent. NaOH, the latter being neutralised equimolecularly with
centinormal hydrochloric acid and then extracted with
chloroform. 3
The films were fixed either by heat or by
Gulland’s method. We found that Ehrlich’s triacid mixture
stained best after the films had been placed for one hour on
a xylol bath, kept at its boiling point. For the other
staining methods ( ’ " Giemsa’s and Mann’s) the films were
fixed for exactly one minute in Gulland’s fixative (90 per
cent. absolute ethyl alcohol, 10 per cent. formalin), rinsed
in absolute alcohol, and dried by blotting with toilet paper. It must be remembered 4 that formaldehyde converts tissues
into acid methylene-compounds, and for this reason the
affinity for acid dyes is diminished, while that for basic dyes
is increased. in the films made during the digestion period. The cyto-
plasm, although pale, was more deeply stained than the
nucleus. The films made four hours after the meal were the
earliest to show a definite change. In these the average
width of the rim of cytoplasm was less than in the control
or fasting and two-hour films, and both the cytoplasm and
nucleus were stained somewhat more deeply. BY GUSTAV MANN
AND
JOHN G. GAGE.
(From the Physiological Department of the Tulane University
of Louisiana.)
(WITH COLOURED PLATE.) The last blood films were made two, four, six, and
eight hours following the first meal after the fast. eight
following
The films were air-dried, wrapped in tissue paper, marked,
and turned in to us at the end of the period. The slides on
which the students made the blood films were cleaned by us
to remove all
traces of sodium silicate, because the
alkalinity of the latter would have interfered with the
Mylius-Ehrlich reaction.2 The slides were placed in Mann’s
cleaning fluid of chromic and sulphuric acids 2 for two to
four hours, rinsed in several changes of distilled water, and
finally rinsed again, one at a time, in distilled water, dried
with tissue paper, and wrapped in packets of four, care
being taken to avoid touching the surface of the slides. o
y goa
Where a man is engaged in manual labour I allow
him to return to work in 10 or 12 weeks after the
operation, but in many cases-and this, of course, applies
to sufferers who have sustained the accident at football
. (other than professional), and so are not entitled to
workmen’s compensation-they have returned to work
as early as six or seven weeks after, and with no bad
result. I am afraid my remarks and conclusions have been,
perhaps, based too much on personal experience, but
inasmuch as so little has been authoritatively written about DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. I in the films made during the digestion period. The cyto-
plasm, although pale, was more deeply stained than the
nucleus. The films made four hours after the meal were the
earliest to show a definite change. In these the average
width of the rim of cytoplasm was less than in the control
or fasting and two-hour films, and both the cytoplasm and
nucleus were stained somewhat more deeply. This was more
marked in the six-hour films, and the maximum was reached
at the eighth and tenth hours. In these films nearly all
the lymphocytes were small, with their nuclei and cytoplasm
very deeply stained, the rim of the cytoplasm being very
narrow and difficult to distinguish from the nuclei. An
occasional lymphocyte could be found which was somewhat
larger and less deeply stained than the rest, but none was as
large and pale as the average cell in the control films. 8 Attention must be drawn to an exception to this rule, for E. Wace
Carlier has shown that marked nuclear changes occur in the liver of
rats after feeding them on carbohydrates and fats (see E. Wace Carlier’s
paper Concerning the Secretion of Ferments by the Liver Cells
and some of the Changes observable in them during Digestion, La
Cellule, 1905, tome xxii., 2nd fascicule, p. 431). We must bear in mind,
however, that the liver acts as a storehouse for both these food
substances, and that its increased nuclear activity in all probability is
connected with the production of definite ferments for the conversion
of dextrose into glycogen, &c. White Cells. The nuclei show the same increase in
staining intensity during the digestion period as do the
nuclei of the neutrophiles and the lymphocytes, but on the cytoplasm. These changes were best shown after ° Giemsa’s " stain
In the "fasting" " films, stained by this method, the rim of
the cytoplasm around the nucleus was wider and paler tha 1071
DR. G. MANN
& DR. J. G. GAGE : CHANGES INDUCED IN BLOOD BY FEEDING. 1071
DR. G. MANN
& DR. J. G. GAGE : CHANGES INDUCED IN BLOOD BY FEEDING. 1071
R. G. MANN
& DR. J. G. GAGE : CHANGES INDUCED IN BLOOD BY FEEDING. hours after breakfast ; Chart I. shows absence of very pale
lymphocytes and leucocytes during the eighth hour. hours after breakfast ; Chart I. shows absence of very pale
lymphocytes and leucocytes during the eighth hour. hours after breakfast ; Chart I. shows absence of very pale
lymphocytes and leucocytes during the eighth hour. whole they are paler than the nuclei of the other white
cells. 3. Mast cells.-A sufficient number for study could not be
found. y p
y
y
g
g
Experiments with Sodium Caseinogenate containing 5 per cent. Sodium Glycerophosphate. The change in the blood pictures, as far as the depth of
staining of the nuclei is concerned, is shown in the accom-
panying chart (1), which was picked out at random from a
large number of similar charts. On comparing the very pale y
p
p
Since previous experiments have shown that nuclear
changes are produced under normal conditions only by
protein foods, and not by carbohydrates or fats,8 we decided
on choosing a very concentrated purin-free protein food,
such as caseinogen. As further pure caseinogen, prepared by
Hammarsten’s method, is very indigestible, while its alkali
salts are easily digested, we thought of giving the combina-
tion of sodium caseinogenate (95 per cent.) and glycero-
phosphate (5 per cent.), sold commercially under the name
of sanatogen, a trial. This preparation was recommended
some years ago to Mann after an attack of influenza, by
Dr. Meredith Young, who had employed it in typhoid. As
the beneficial results of this preparation were much doubted
by one of our foremost colleagues, who at that time was at
the Tulane University, we decided to put its food value to
experimental test. ’ p
g
p
CHART 1. White Cells. experimental
A hundred frogs, which had not been fed during the
winter months, were starved for one additional week and,
apart from six frogs used as controls, each one was given
about one gramme of sanatogen made into a thick paste
with water. The frogs were killed at 10-minute intervals
during the first hour, then at intervals of one hour for
30 hours, and at 6-hour intervals up to 96 hours. The idea
of giving such a relatively large amount of sanatogen to the
frogs was to see how the blood would behave in the presence
of an excessive amount of protein food. At the proper time
each frog was pithed, the thorax opened, and blood films
made by rubbing the excised heart over the slides. The
films were quickly dried under an electric fan. All the organs
were removed as quickly as possible and placed at once in
Mann’s picro-corrosive-formaldehyde fluid, sectioned, and
stained. The results of the study of these tissues will appear
in an early paper. - early paper. The slides on which the blood films were made were treated
in the manner above described for the human blood films, and
the films were treated in the same manner throughout as were
the human blood films. In addition to the experiments on
frogs, Mann experimented on himself. He starved for 20
hours, then took 15 grammes of sanatogen in a breakfast-
cupful of water. cupful
The most notable change in his own blood films after
feeding on sanatogen is the marked increase in nuclear
metabolism, which is very evident even after staining in
hæmatein-eosin. (See Figs. 3 and 4.) The changes in the
granules of the polymorphonuclear leucocytes and in the
eosinophiles were much less marked than in the specimens
supplied by the students, and the diminution in the size of
the white cells was so insignificant as to be hardly noticeable,
but the great change induced in the general appearance of
the nuclei of the pale lymphocytes of all sizes is shown by
comparing Figs. 3, g to n, with Figs. 4, A to o. White Cells. The great
differences in the ratio of the dark, the medium, and the pale
nuclei of the leucocytes and lymphocytes in the sanatogen
experiment made on Mann are shown in the following
table :- and pale with the medium dark and very dark polymorpho-
nuclear leucocytes, and similarly the pale with the dark
small lymphocytes, it will be seen that feeding produces a
marked increase in the dark corpuscles. By adding the very
pale, the pale, and the medium dark corpuscles together and
comparing them with the very dark ones, the following table
is obtained - is obtained -
CHART 2. The last column gives the figures 4½ hours after the
first meal following the 30-hour fast, and is especially
interesting, for while after 26 and 28 hours’ fast the dark
leucocytes are to the pale in the ratio of 0 : 11 and 0 : 26,
after having taken food 4; hours previously the ratio
becomes as 4’5 : 1. If it were not for the fact that some
corpuscles begin feeding later and therefore pass later into
the active dark state, the change would be even more
marked. It will be seen that the maximum change in the conversion
of pale into dark leucocytes and lymphocytes occurs 6 to 8 is obtained
CHART 2. The dark, to the medium, to the pale polymorphs after 20 hours’
fasting were as 1: 33 : 17. fasting
While 7 hours after taking the sanatogen the ratio was 12 : 6 : 1. Similarly the small lymphocytes after 18 hours’ fast were 1 : 4’6 : 1’4. After 7 hours’ sanatogen 25 : 42 : 1. Changes in the frog after feeding on sanatogen.- White
blood cells.-The changes in the white blood cells of the
frogs correspond so closely with those found in man that
they will not be described in detail. The decrease in size
and number of eosinophile granules was especially well
marked. (See description of Fig. 5, as also the " red
granules " in the polymorphs, Fig. White Cells. 5, i and k.) In the 2-,
4-, 6-, and 36-hour films an occasional white cell undergoing
mitotic division could be found, while the mitoses during The last column gives the figures 4½ hours after the
first meal following the 30-hour fast, and is especially
interesting, for while after 26 and 28 hours’ fast the dark
leucocytes are to the pale in the ratio of 0 : 11 and 0 : 26,
after having taken food 4; hours previously the ratio
becomes as 4’5 : 1. If it were not for the fact that some
corpuscles begin feeding later and therefore pass later into
the active dark state, the change would be even more
marked. It will be seen that the maximum change in the conversion
of pale into dark leucocytes and lymphocytes occurs 6 to 8 1072
DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. [C DR. G. MANN
& DR. J. G. GAGE: CHANGES INDUCED IN BLOOD BY FEEDING. [C the 18th hour in all lymphocytes were very numerous
and frequent in the leucocytes. During the early period of
digestion, especially after 6 hours, amitotic division (see
Fig
6) was common. the 18th hour in all lymphocytes were very numerous
and frequent in the leucocytes. During the early period of
digestion, especially after 6 hours, amitotic division (see
Fig
6) was common. depends on an increase in the nucleo-protein content, as
proved microchemically by the iron reaction, it follows that,
the greater affinity for basic dyes shown by the blood cor-
puscles of animals, while in the act of absorbing food, must
come under the same heading as the changes observed in
other nucleated cells. We have thus definite evidence that
the metabolism of all nucleated cells is inseparable from the. increase in the nucleo-protein radical in that cell, and we
have therefore before us the problem of the manufacture of
purin substances from non-purin radicals. Fig. 6)
Red blood cells.-The first noticeable feature is
an
increase
in
size
of
these
cells,
together with an
increased affinity for both acid and basic dyes, during
the digestion period. Both the nuclei and the cyto-
plasm appear somewhat swollen and stain more deeply. This becomes first evident in the cytoplasm, and as
early as the sixth and eighth hour. White Cells. Marked nuclear
changes, however, are not noticeable until the twelfth hour,
at which time the nuclei are distinctly larger and more
reticular than in the control. There is little further
change until the 21st to the 23rd hour. At this time
the nuclei are still larger and the chromatin still less dense,
although staining more deeply, and the cytoplasm somewhat
paler than in the preceding hours. The maximum is reached
at the 24th hour. In the 24-hour films are large numbers of
cells undergoing division, as all stages can be found. (See
Fig. 6.) While the majority of the cells show evidence of
nuclear activity, more or less marked, there are still some
in (or maybe have reverted to) the resting condition. After
the 24th hour these changes decrease, and by the 36th hour
practically all the cells are in the resting condition, and
remain so until the 54th hour. At this time the cells show
nuclear activity and present an appearance similar to the
films made at the 22nd and 23rd hours. The changes are
still evident in the 60- and 66-hour films, but are less marked. while the resting condition is reached by the 72nd hour,
The 8-hour films again show nuclear activity, although less
marked than in the 54-hour films. It would, therefore,
appear that the feeding of the red cells has a certain
periodicity of from 24 to 36 hours. purin
non purin
It is more difficult to account for the diminution in the
size of the neutrophile corpuscles. We suggest the explana-
tion that the irritability and the contractility of the white
corpuscles are increased in feeding, in consequence of which
fed cells are more likely to become spherical and less likely
to be spread out by the pressure of the glass slide used in
making a film ; and further, that the increased osmotic
pressure in the blood plasma during the absorption of food
may cause a certain amount of exosmosis, and hence
shrinkage of the white cells apart from the diminution
caused by the partial disappearance of the zymogen
granules. granules. The granules in the leucocytes may be supposed to be-
either stored food material or zymogen granules, as Ehrlich
first pointed out in his "Farbenanalytischen Untersuchungen. All the figures show a magnification of 1500 linear diameters, except
Figs. 3 and 4, which are magnified 1000 diameters. All figures were-
drawn with the Abbe drawing apparatus, and each granule is in its.
correct place. place.
Figs. 1 and 2 represent human blood stained by "Giemsa" stain
prepared by us.
They show the alterations blood undergoes after
taking an ordinary breakfast. Fig. 1 shows the control or fasting period
immediately before breakfast after 10 hours’ fast, and Fig. 2 the
changes induced in 8 hours after the breakfast. DESCRIPTION OF THE FIGURES. periodicity
Sanatogen further stimulates blood cells to undergo nuclear
division, which during the early period is mostly amitotic
(see Fig. 6 and the accompanying figures, 1-9), while
later, at
the 24th hour, mitotic division figures are
present (Fig. 7). The division figures are well brought out
by the hæmatein and methylblue-eosin stain. They were seen
with more difficulty in the Giemsa preparations and in
the triacid films, while the mitotic division of the white cells
was best brought out by the triacid mixture. Therefore it is
evident that sanatogen acts as a strong stimulus as far as
the recuperative powers of the blood are concerned. How it
behaves towards other tissues, as already pointed out, will
be shown later. Whether this stimulative action depends
upon the administration of
a rich protein food-viz.,
caseinogen-or whether upon glycerophoric acid, or on the
fortunate combination of these two radicals, is a question
which we soon hope to be able to answer definitely. control cells. Fig. 2, e, represents the diminished affinity for eosin produced by
feeding, and Fig. 2, d, the typical increase in the nucleo-protein
content and probably a stage in the restoration of the eosinophile
granules changes
ou s a te
t e b ea
ast.
Figs. 3 and 4 are stained by Mann’s heematein-eosin. Fig. 3 shows
the effect of 20 hours’ fast, and Fig. 4 represents the change induced
7 hours after taking 15 grammes of sanatogen Fig. 2, e, represents the diminished affinity for eosin produced by
feeding, and Fig. 2, d, the typical increase in the nucleo-protein
content and probably a stage in the restoration of the eosinophile
granules. corpuscles
frog.
description
Figs. 1, a and b, are two typical neutrophile leucocytes. In both, ire,
addition to the neutrophile granules, a few scattered red granules are-
seen. Fig 1, c, is a typical eosinophilous cell. Fig. 1, d, is an exception-
ally large lymphocyte, and; Fig. 1, e, f, and g, three small lymphocytes,.
of which e shows a very distinct vacuole. granules.
Figs. 2, f and h are large, i an intermediate, and g and k small
lymphocytes. very
Fig. 2, a, b, c, are three neutrophile leucocytes ; in a the typical
red
granules are seen, and this figure shows the average amount of
granules. In Fig. 2, c,
the place of the neutrophile granules seems to.
have been taken by vacuoles, but in reality the granules are less.
refractive than in the control cells. ou s a te taking 5 grammes o sanatogen.
Fig. 5 represents the white corpuscles of frogs’ blood, and Figs. 6 and
7 the red corpuscles of the frog.
For detailed description see below. lymphocytes.
Figs. 3, a, b, c, d, are neutrophile leucocytes, and the granules are-
only represented in a. Fig. 3, e, f, are two eosinophiles ; the granules.
in e are distinctly smaller than in f. The disparity in the size of the
granules is sometimes even greater. lymphocytes.
Figs. 4, a, b, c, d, e, are neutrophile leucocytes, with the granules.
only shown in a, b, c. The granules are less distinct and not so purple
as in Fig
3, a. Fig. 3,
Figs. 4, h, i, k, I, are large, and m, n, o are small lymphocytes ;.
;.
p shows size of red blood corpuscle. DESCRIPTION OF THE FIGURES. granules
greater.
Figs. 3, k, I, m, are large, n an intermediate, and g, h,
i small
lymphocytes. S2urcma.rf. p
corpuscle. Fig. 5, a, is a typical " fasting
" eosinophile ; Fig. 5, b, an eosinophile
six hours after feeding with sanatogen; Fig. 5, c, an eosinophile in the
process of reforming the eosinophile granules 16 hours after feeding
with sanatogen. Fig. 5, d, shows an eosinophile surrounded by
numerous bacteria. Notwithstanding the very severe septicaemia
leading to the destruction of the cell body, the eosinophile granules are
but little affected. Fig. 5, e, is a typical resting or fasting lymphocyte with the baso-
phile cytoplasm aggregated in two places to form pseudopodia-like
processes. Fig. 5, /, is a lymphocyte 12 hours after feeding, and g a
similar one 16 hours after feeding with sanatogen. Fig. 5, h, is a small
fasting neutrophile leucocyte, without any red granules ; while i and
k show numerous purplish-red granules 18 and 24 hours after feeding
with sanatogen. produce
great changes
The second explanation is based on the fact that the
nucleated corpuscles in the blood must get nourishment from
the blood plasma and must metabolise as long as they are
alive. One would further expect the nucleated corpuscles to
be feeding at such times when the blood plasma contains
the most food material-that is, while food is being absorbed
from the alimentary canal. As Mann and his pupils have
previously established, for both plants and animals, the fact
that absorption of protein food materials always produces an
increase in the stainability of the nucleus, and as the latter
,
’
9
l
b
. r sanatogen. Fig. 6 is a red blood corpuscle of the frog six hours after feeding with
sanatogen, and is undergoing amitotic division. The cell membrane is. seen distinctly above the constricted part of the nucleus. distinctly
part
Fig. 7 is a red blood corpuscle of the frog undergoing mitosis, stained
with hæmatein and methylblue-eosin. distinctly
part
Fig. 7 is a red blood corpuscle of the frog undergoing mitosis, stained
with hæmatein and methylblue-eosin. g;
methylblue eosin. Fig. 8 is a red blood corpuscle of the frog in Fig. corpuscle
frog
resting
Fig. 9 is a red blood corpuscle of the frog at the seventh hour of
digestion, showing an increase in the stainability of both the cytoplasm
and of the nucleus. g
p
g
g
Fig. S2urcma.rf. The principal points for consideration are : during the
digestion period there is a marked increase in the staining
intensity of all nuclei ; the rim of cytoplasm in the lympho-
cytes becomes narrower; the granules in the leucocytes
decrease both in size and in number; and the leucocytes
may also show a diminution in size. ’
’
: granules. Figs. 2, f and h are large, i an intermediate, and g and k small
lymphocytes. lymphocytes. Figs. 3, a, b, c, d, are neutrophile leucocytes, and the granules are-
only represented in a. Fig. 3, e, f, are two eosinophiles ; the granules. in e are distinctly smaller than in f. The disparity in the size of the
granules is sometimes even greater granules is sometimes even greater. Figs. 3, k, I, m, are large, n an intermediate, and g, h,
i small
lymphocytes. y
As far as the deeper staining of the nuclei is concerned
two explanations suggest themselves. We may assume, since
there is an actual increase in the number of white cells after
a meal, that these new cells in the circulation, being younger,
are therefore smaller, and that for the same reason they
stain more deeply. Analogously younger leucocytes might
contain fewer and smaller granules. This explanation of
the change in the microscopic appearance of the blood does
not seem to us to be the correct one, as we would have to
assume either a vast increase in the number of new cells or
a correspondingly large destruction of those white cells,
which were circulating in the blood before food was taken,
to produce the great changes we have found to occur. :
-
’
;
,
;
. . ! lymphocytes. Figs. 4, a, b, c, d, e, are neutrophile leucocytes, with the granules. only shown in a, b, c. The granules are less distinct and not so purple
as in Fig. 3, a. as
Fig. 3,
Figs. 4, h, i, k, I, are large, and m, n, o are small lymphocytes ;. ;. p shows size of red blood corpuscle. as
Fig. 3,
Figs. 4, h, i, k, I, are large, and m, n, o are small lymphocytes ;. ;. p shows size of red blood corpuscle. Fig. 3,
Figs. 4, h, i, k, I, are large, and m, n, o are small lymphocytes ;. ;. p shows size of red blood corpuscle. White Cells. "
If food material, they should be used up during starvation,
while if zymogen granules they should not diminish during
starvation, since Mann has observed that zymogen granules. in the salivary glands and along the alimentary canal and
the Nissl’s granules in the nerve cells are not diminished in A NOTE ON A CASE OF DOUBLE
CATARACT: morning
equally satisfactory. But on the third morning a change had taken place. The
lids were red and slightly swollen, and there was muco-pus
on the dresing ; the eye had obviously become septic. I was
in the country, and was on the lawn of the house I was at for
the time when a motor car appeared with a Salvation Army
man in it. He brought a note from Dr. Milne, but what was
still more eloquent than any description the dressing taken
from the eye that morning : it was smeared with muco-pus. I left in the motor- at once and arrived at Hadley Wood,
where the patient was residing, 70 miles off, in under three and
a half hours. Dr. Milne gave an anaesthetic, and I thoroughly
examined the eye ; the incision was firmly healed, the pupil
circular and central, but the iris was cloudy and there was
muco-pus hanging about the conjunctiva. I thoroughly
cleansed the eye with a solution of formalin 1 in 2000-1
could not get any peroxide-I reapplied the bandage, and
the patient went back to bed. On the following morning
there was no more pus, the cornea was clear, and fingers
could be seen. I hoped that the progress of suppuration had
been stopped. The day after matters had changed ver
much for the worse, the incision had given way and was
blocked by a bulging, pus-infiltrated iris. There followed
the usual course of a panophthalmitis. Later in the same
day I saw the patient again in consultation with Mr. Treacher
Collins and we had to point out that there was no chance of
saving the eye. After that it was treated with hot com-
presses and the inflammation gradually quieted down, and
when I last saw General Booth on June 28th the eye was
shrinking but giving no trouble. THE worldwide interest in General Booth’s great career
and far-reaching work was, if possible, deepened by the
general knowledge that towards the end of his life he
carried on that work with the profound disabilities incident
upon cataract. I consider it almost a public duty to record
the clinical history of his case. y
General Booth first consulted me on Dec. 2nd, 1908. S2urcma.rf. 16
with an ordinary pupil; there was some improvement when
it was dilated, so I prescribed a solution of atropine, which
was used from time to time up to a few days before the
extraction, which did not take place until more than two
years later. At this time (March, 1910) the patient com-
plained of giddy attacks, and at times of lapses of memory. In April his urine was albuminous, but the albumin had
disappeared at the end of June. ,
by taking up bacte
a,
Occasionally leucocytes will have. to combat bacterial
invasion, but how little, even in cases of severe septicaemia,
the granules of ecsinophilous cells may be affected is shown
’in Fig. 5, d, where an eosinophile leucocyte is surrounded by
numerous bacilli without any apparent change in the granules,
although the cytoplasm has become disintegrated. disappeared
By Nov. 8th, 1910, the vision had become worse, and he
could only make out J. 19. I advised him to dispense with
an operation as long as he possibly could. disappeared
By Nov. 8th, 1910, the vision had become worse, and he
could only make out J. 19. I advised him to dispense with
an operation as long as he possibly could. although
cytoplasm
disintegrated. Finally, the blood pictures of each individual are quite
characteristic, as will be seen by comparing the white cells
in Figs. 1 and 2, representing those of a young student, with
Figs.,3 and 4, representing those of the elder of us, both
showing characteristic nuclear differences. Figs. 1 and 2 are
magnified 1500 times, while Figs. 3 and 4 are only magnified
1000 diameters. operation
long
possibly
I saw him in April, June, and October, 1911. The vision
was becoming slowly worse, and at this last date he could
barely make out J.20 with the pupil fully dilated. The lens
was almost entirely opaque and very dark coloured. operation
long
possibly
I saw him in April, June, and October, 1911. The vision
was becoming slowly worse, and at this last date he could
barely make out J.20 with the pupil fully dilated. The lens
was almost entirely opaque and very dark coloured. entirely opaque
very
On Feb. 22nd, 1912, he again consulted me. His sight
had now become so bad that he could barely lind his way
about. S2urcma.rf. I advised an operation, and at the same time pointed
out to him that failure meant total blindness, but that the
risk was very small. He decided to have the operation per-
formed, but wished to postpone it for about two months, and
eventually May 23rd was fixed. On that day, assisted by
Mr. Herbert L. Eason and Dr. E. W. Milne, I performed the
operation under cocaine-a simple extraction without iridec-
tomy. The operation was most satisfactory, there was no
difficulty of any kind, the lens escaped easily, no soft matter
remained behind, and the iris returned to its place. There
was no bleeding into the anterior chamber, and the patient
could see my fingers plainly. The eye was bandaged and
the patient was put to bed. On the following morning the
condition could not have been better. I did not remove the
bandage, but lifted up the dressing. He partly opened the
eye and found that he could see with it. On the following
morning the condition was equally satisfactory. The main general fact, determined histologically, that
cellular metabolism in both plants and animals is invariably
accompanied by an increase in nuclear metabolism, as
evidenced by the increase in nucleo-protein material, is being
investigated macrochemically by us at present, and our results
will be communicated shortly. S2urcma.rf. There were muco-purulent discharge and lymph
in the pupil; the cicatrix was bulging and the iris was
pushed into it; the eye was stony hard and very painful,
and there was bare perception of light. There was also
moderate conjunctivitis of the left eye. I sent him to a
nursing home, and a vaccine prepared from the discharge
from the right eye was injected within 12 hours, but it did
no good. On August 21st I eviscerated the right eye. Recovery from the operation was rather slow but uninter-
rupted. I ordered him an artificial eye, which he wore with
comfort. they
zymogen granules. As the white blood corpuscles are constantly changing
their positions relative to the plasma immediately surround
ing them, it is most likely that the granules of the leucocyte
:are used by each individual cell, or, to put it differently, that
they are intracellular zymogen granules utilised by the cell
themselves in rendering the available food material suitable
- for their own needs, instead of being used extracellularly as
in the salivary glands. At the same time the comparatively
small amount of diminution in the size and number of the
granules makes it very likely that these granules subserve
some main function other than that of acting on the already
’digested food materials in the plasma. We suggest that they
digest body cells which have died, as will happen, for
example, to the nerve cells in the distal part of a severed
nerve, or during the atrophy which the mammary and
lymphatic glands undergo after lactation, or in cases of
.atrophy owing to pressure of neighbouring organs as in
’cancer, or by taking up bacteria,
&c. On March 10th, 1910, the vision of the left eye was J. 16
with an ordinary pupil; there was some improvement when
it was dilated, so I prescribed a solution of atropine, which
was used from time to time up to a few days before the
extraction, which did not take place until more than two
years later. At this time (March, 1910) the patient com-
plained of giddy attacks, and at times of lapses of memory. In April his urine was albuminous, but the albumin had
disappeared at the end of June. On March 10th, 1910, the vision of the left eye was J. S2urcma.rf. 9 is a red blood corpuscle of the frog at the seventh hour of
digestion, showing an increase in the stainability of both the cytoplasm
and of the nucleus. Fig. 10 is a red blood corpuscle of the frog at the height of digestion.. showing the increased nuclear metabolism. SupþlelJ/ellt to "THE LANCET," To illustrate the article on The Changes in Blood by Feeding,
by Dr. Gustav Mann and Dr. John G. Gage. To illustrate the article on The Changes in Blood by Feeding,
by Dr. Gustav Mann and Dr. John G. Gage. 1073
R. CHARLES HIGGENS : A NOTE ON A CASE OF DOUBLE CATARACT. 1073 the rabbit by even a 22 days’ fast. Since the granules in th
leucocytes are not diminished during a fast, but are to some
- extent during the period of digestion, we may conclude tha
they are of the nature of zymogen granules. e
t
-
s
s
he would not use it nor his goggles, as from time to time they
prevented the people seeing him and him from seeing them. He said that he had come to me straight from Newport,
Monmouthshire. He told me that 48 hours before his right
eye became suddenly painful and that the sight was lost in a
few hours. There were muco-purulent discharge and lymph
in the pupil; the cicatrix was bulging and the iris was
pushed into it; the eye was stony hard and very painful,
and there was bare perception of light. There was also
moderate conjunctivitis of the left eye. I sent him to a
nursing home, and a vaccine prepared from the discharge
from the right eye was injected within 12 hours, but it did
no good. On August 21st I eviscerated the right eye. Recovery from the operation was rather slow but uninter-
rupted. I ordered him an artificial eye, which he wore with
comfort. e
t
-
s
s
he would not use it nor his goggles, as from time to time they
prevented the people seeing him and him from seeing them. He said that he had come to me straight from Newport,
Monmouthshire. He told me that 48 hours before his right
eye became suddenly painful and that the sight was lost in a
few hours. A NOTE ON A CASE OF DOUBLE
CATARACT:
THE CASE OF GENERAL BOOTH.
BY CHARLES HIGGENS, F.R.C.S. ENG.
SENIOR CONSULTING OPHTHALMIC SURGEON, GUY’S HOSPITAL. A NOTE ON A CASE OF DOUBLE
CATARACT:
THE CASE OF GENERAL BOOTH. BY CHARLES HIGGENS, F.R.C.S. ENG. SENIOR CONSULTING OPHTHALMIC SURGEON, GUY’S HOSPITAL. A NOTE ON A CASE OF DOUBLE
CATARACT: I
found the lens of the right eye almost entirely opaque ; he
could see large objects, such as a hand held in front of it,
but could read none of the test types. There was central
opacity of the left lens, the vision was 6/60 (= 1/10 of
normal) aided by a - 6 lens. On Dec. 16th I removed the
cataract from the right eye by simple extraction without
iridectomy, and uninterrupted recovery took place. iridectomy,
uninterrupted recovery
place. A month later-i. e., on Jan. 15th, 1909-the pupil was not
- quite central, being drawn slightly upwards towards the scar
of the operation wound ; there was no prolapse of iris and the
pupil was active. Vision 6/24 c. + 12 D. J. 8 c. z-15 D. There was some opaque capsule. On April 1st, 1909, the
vision was 6/18 badly c. + 13, J. 2 c. + 15. On April 8th
the capsule was needled, and on the 15th vision was 6/12 c. + 13 and J. 1 c. z- 15. There was some entropion of the
lower lid, which, however, righted itself. lid, which, however, righted
I have no note of the case after this until August 18th,
1909. The patient was going about doing his usual work and
reading and writing with ease. On the morning of this day
Tie appeared in my consulting room at about 4 A.M. He had
been on a motor tour rushing about in heat and dust, and,
contrary to my directions, without at times any protection
whatever for his eyes. His car possessed a wind screen, but lid, which, however, righted
I have no note of the case after this until August 18th,
1909. The patient was going about doing his usual work and
reading and writing with ease. On the morning of this day
Tie appeared in my consulting room at about 4 A.M. He had
been on a motor tour rushing about in heat and dust, and,
contrary to my directions, without at times any protection
whatever for his eyes. His car possessed a wind screen, but shrinking
giving
.ZM<M*&.—This
case may be classed amongst what Sir
James Paget (I think it was) described as ’’ The Disasters of
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https://openalex.org/W4299507474
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https://cdr.lib.unc.edu/downloads/w0892g92x
|
English
| null |
Multilevel Deficiency of White Matter Connectivity Networks in Alzheimer’s Disease: A Diffusion MRI Study with DTI and HARDI Models
|
Carolina Digital Repository (University of North Carolina at Chapel Hill)
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cc-by
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Hindawi Publishing Corporation
Neural Plasticity
Volume 2016, Article ID 2947136, 14 pages
http://dx.doi.org/10.1155/2016/2947136 Hindawi Publishing Corporation
Neural Plasticity
Volume 2016, Article ID 2947136, 14 pages
http://dx.doi.org/10.1155/2016/2947136 Tao Wang,1,2,3 Feng Shi,2 Yan Jin,2 Pew-Thian Yap,2 Chong-Yaw Wee,2
Jianye Zhang,4 Cece Yang,1,3 Xia Li,1,3 Shifu Xiao,1,3 and Dinggang Shen2,5 g
A Lab, Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 3Alzheimer’s Disease and Related Disorders Center, Shanghai Jiao Tong University, Shanghai, China
4Department of Radiology, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China
5Department of Brain and Cognitive Engineering, Korea University, Seoul, Republic of Korea Alzheimer’s Disease and Related Disorders Center, Shanghai Jiao Tong University, Shanghai, China 4Department of Radiology, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China
5Department of Brain and Cognitive Engineering, Korea University, Seoul, Republic of Korea orrespondence should be addressed to Shifu Xiao; xiaoshifu@msn.com and Dinggang Shen; dgshen@med.unc.ed Received 23 June 2015; Accepted 22 November 2015 Received 23 June 2015; Accepted 22 November 2015 Academic Editor: Clive R. Bramham Copyright © 2016 Tao Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Alzheimer’s disease (AD) is the most common form of dementia in elderly people. It is an irreversible and progressive brain disease. In this paper, we utilized diffusion-weighted imaging (DWI) to detect abnormal topological organization of white matter (WM)
structural networks. We compared the differences between WM connectivity characteristics at global, regional, and local levels
in 26 patients with probable AD and 16 normal control (NC) elderly subjects, using connectivity networks constructed with the
diffusion tensor imaging (DTI) model and the high angular resolution diffusion imaging (HARDI) model, respectively. At the
global level, we found that the WM structural networks of both AD and NC groups had a small-world topology; however, the AD
group showed a significant decrease in both global and local efficiency, but an increase in clustering coefficient and the average
shortest path length. We further found that the AD patients had significantly decreased nodal efficiency at the regional level, as
well as weaker connections in multiple local cortical and subcortical regions, such as precuneus, temporal lobe, hippocampus, and
thalamus. The HARDI model was found to be more advantageous than the DTI model, as it was more sensitive to the deficiencies
in AD at all of the three levels. 2. Methods 2.1. Participant Recruitment. This study involved 26 patients
who were diagnosed with probable AD at the Alzheimer’s
Disease and Related Disorders Center (ADRDC) in the
tertiary hospital of Shanghai Mental Health Center (SMHC)
at Shanghai Jiao Tong University School of Medicine. 16
cognitively healthy elderly subjects from the community of
Shanghai Chang Ning district were included as the normal
control (NC) group. Subjects were enrolled via self-referral or
referral from families or physicians. The study was conducted
from May 2011 to May 2012 at ADRDC. The SMHC Institu-
tion’s Ethical Committee approved the consent form and the
study protocol. The study was carried out in accordance with
the Declaration of Helsinki. Informed consent was obtained
from all subjects and/or their legal guardians. y
[
]
WM abnormalities in AD were reported in previous
studies. Liu et al. [15] performed voxelwise TBSS to compare
fractional anisotropy (FA) between the AD patients and the
healthy controls. Multiple WM tracts, such as parahippocam-
pal WM, cingulum, uncinate fasciculus, inferior and supe-
rior longitudinal fasciculus, and corpus callosum, showed
decreased FA in the AD group. Jin et al. [16] used the
tract-based clustering method to relate fornix degeneration
to cognitive decline in AD with various diffusion-derived
measures. Mean diffusivity (MD) was shown to be more sen-
sitive to the group difference among AD and normal controls
than FA did. Li et al. [17] proposed a spectral diffusional
connectivity framework to explore the connectivity deficit in
AD. The framework was based on studying eigenvalues of the
Laplacian matrix of the diffusion tensor field at the voxel level. The peaks of the diffusional connectivity spectra were shifted
in the AD group compared to the normal controls that did
not shift. Daianu et al. [18] found widespread breakdown in
AD in the 68-ROI based connectivity networks with multiple
connectivity metrics on the “𝑘-core” structure. The ages of the AD subjects enrolled ranged from 50s to
90s. Prior to enrollment, patients provided their medical his-
tory and were given physical and neurological examinations,
laboratory tests, and both T1-weighted and fluid-attenuated
inversion recovery (FLAIR) MRI scans. 2. Methods Enrollment criteria
included (1) the National Institute of Neurological and Com-
municative Disorders and Stroke/Alzheimer’s Disease and
Related Disorder Association (NINCDS/ADRDA) criteria
for probable AD [27]; (2) the Diagnostic and Statistical
Manual for Mental Disorders, 4th edition (DSM-IV) criteria
for the Alzheimer’s dementia; (3) a Hachinski Ischemia Score
less than 4; (4) the systolic blood pressure between 95 and
160 and the diastolic blood pressure between 60 and 95; (5)
identification of a responsible and consistent caregiver; (6)
absence of diabetes, renal impairment, significant systemic
conditions, psychiatric disorders, seizures, or traumatic brain
injuries that could compromise their cognitive or brain
functions; (7) significant brain abnormalities on the patient’s
T1-weighted MRI; (8) clinical score requirements. In the
Chinese version of the Mini-Mental Status Exam (MMSE)
[28], there are three cut-off thresholds for AD diagnosis
according to education levels: (1) AD subjects who had not
been educated exhibited MMSE scores <18; (2) those with
elementary school education exhibited MMSE scores <21; (3)
those with higher than middle school education exhibited
MMSE scores <25. The Clinical Dementia Rating (CDR) scale
[29] was equal or more than 1. Importantly, WM tracts can be used to form the connec-
tivity networks that give comprehensive pictures of interac-
tions between different brain regions. A WM connectivity
network can be described mathematically as a graph consist-
ing of (1) a collection of nodes, representing the ROIs and
(2) a set of edges between nodes, describing the connections
(e.g., fiber counts) between ROI pairs. The characteristics of a
connectivity network can be described using metrics at three
hierarchical levels: global, regional, and local.hl The stability of connectivity networks is influenced by
multiple factors, including field strength [19, 20], scanners
[21], imaging acquisition parameters [22], and tractography
parameters [23]. Zhan et al. [24] compared several trac-
tography and feature extraction methods in relation to AD
diagnostic accuracy. Among these factors, the choice of
diffusion models is found to be the most influential. The most
commonly used approach, namely, diffusion tensor imaging
(DTI), is based on the Gaussian assumption of water diffu-
sion. This approach works well in regions with unidirectional
fiber bundles, but this model may fail in regions with
fiber crossings, which may introduce tractography errors in
these regions. To address this issue, advanced models for high
angular resolution diffusion imaging (HARDI) were pro-
posed to estimate orientation distribution functions (ODF)
[25, 26] at each voxel. 1. Introduction On the other hand, magnetic resonance imaging (MRI)
has been widely recognized as a noninvasive means for
neurodiagnosis and disease staging. Previous studies using
T1-weighted structural MRI revealed AD-induced gray mat-
ter (GM) atrophy in multiple brain regions, including the
hippocampal and entorhinal cortices [4, 5], the temporal and
cingulate gyri, the precunei, the insular cortices, the caudate
nuclei, the frontal cortices [6], the sensorimotor cortices, the
occipital poles, the cerebellum, and the medial thalami [7].f Alzheimer’s disease (AD) is the most common form of
dementia in elderly people and is characterized by chronic
cortical atrophy and neurodegeneration, resulting in behav-
ioral changes, loss of memory and language function, and
general cognitive decline [1]. It is an irreversible and progres-
sive brain disease and usually diagnosed in people older than
65. Nearly 36 million people worldwide are affected by AD,
with 5.2 million alone just in the United States [2].l On the other hand, diffusion-weighted magnetic reso-
nance imaging (DWI) [8] can recover the local profile of
water diffusion in tissue, yielding information on white mat-
ter (WM) integrity and connectivity that is not available from
standard structural MRI. Specifically, tractography methods Tau and amyloid beta (A𝛽) in cerebrospinal fluid are
considered to be reliable biomarkers of AD [3]. However,
the invasiveness, cost, and availability associated with the
measurement of these quantities are significant drawbacks. 2 Neural Plasticity at multiple levels on AD, to our knowledge, has not been
attempted previously. [9, 10] can be used to fit continuous streamlines through
directional diffusion data at each voxel for reconstructing
WM fiber tracts. With the obtained tractography, WM
integrity can be analyzed with both region-of-interest- (ROI-)
based analysis, for example, tract-based spatial statistics
(TBSS) [11] and fiber clustering [12, 13], and parcellation-
based connectome analysis [14]. Neural Plasticity tractography was manually visualized and checked in Par-
aView (Kitware, http://www.paraview.org/).h Table 1: The demography and clinical scores of the subjects in the
study. The 𝑝values are based on the two-sample 𝑡-tests except the
gender. The gender ratio was examined by the Chi-squared test. The Automated Anatomical Labeling (AAL) template [33]
is a widely used high-resolution T1-weighted brain parcella-
tion based on a single adult subject, which includes 90 cortical
and subcortical brain regions for the cerebrum. The names
and abbreviations of these 90 ROIs are listed in Table 2. First,
we nonlinearly registered the AAL template to each subject’s
segmented T1-weighted image using HAMMER [34]. Then,
the T1-weighted image was rigidly aligned to the FA image. The original 90 ROIs of the AAL template were transferred
to each individual’s DWI space using the deformation fields
and the affine transformation matrix generated during the
registration step. These ROIs were used as nodes in the
connectivity network for each subject. NC (𝑛= 16)
AD (𝑛= 26)
𝑝value
Age (years)
70.1 ± 7.5
69.5 ± 7.1
0.81
Male/female
11/5
8/18
0.03
Education (years)
10.6 ± 3.2
10.4 ± 3.9
0.91
CDR
0.0 ± 0.0
2.0 ± 0.7
<0.001
MMSE
25.3 ± 3.6
15.2 ± 6.5
<0.001 out in our analysis. As expected, the group difference was
observed in the MMSE (𝑝< 0.001) and CDR scores (𝑝<
0.001). out in our analysis. As expected, the group difference was
observed in the MMSE (𝑝< 0.001) and CDR scores (𝑝<
0.001). Two ROIs were considered anatomically connected, if
there were fibers traversing them. In the network, the edge,
connecting the nodes representing these two ROIs, was
defined as the number of fibers connecting them. Two ROIs
were considered connected if there were no less than four
fibers between them, which has been proven effective to
reduce false-positive connections [35–37]. As a result, the
WM connectivity network, represented by a symmetric 90 ×
90 matrix, was constructed for each subject. The network was
weighted and undirected. 2.2. MR Image Acquisition. MRI images were scanned with a
Siemens MAGNETOM VERIO 3 T scanner at SMHC. Neural Plasticity T1-
weighted images were obtained with 128 sagittal slices using
the 3D magnetization prepared rapid acquisition gradient
echo (MPRAGE) sequence with the following parameters: TR
= 2,530 ms, TE = 3.39 ms, flip angle = 7∘, and spatial resolution
= 1 × 1 × 1.3 mm3, and the acquisition time was 8 minutes 7
seconds. The DWI images were acquired with 75 axial slices
by using an echo planar imaging (EPI) sequence that covered
the whole brain. The acquisition parameters were as follows:
TR = 10,000 ms, TE = 91 ms, and spatial resolution = 2 ×
2 × 2 mm3. A total volume of 62 directions was acquired,
where two volumes were without diffusion gradient (𝑏=
0) and the rest 60 volumes were with diffusion gradient of
𝑏= 1,000 s/mm2. The acquisition time was 5 minutes and 42
seconds. 2.5. Multilevel Network Measures. Three hierarchical levels
(global, regional, and local) of complex network measures
were used to compare the measures of connectivity networks
constructed in Section 2.4 between the AD group and the NC
group. The measures were calculated with the GRETNA tool-
box (https://www.nitrc.org/projects/gretna/). For a detailed
review of those measures, please see [38]. 2.3. Image Preprocessing. T1-weighted images were first
resampled to be 1 mm isotropic, intensity inhomogeneity
corrected [30], and skull stripped to remove nonbrain tissues
[31]. The resulting images were then tissue segmented to
separate GM, WM, and cerebrospinal fluid (CSF) with FSL
FAST (http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/). DWI images
were skull stripped and then corrected for eddy-current
induced distortion with FSL. FA and MD images were then
extracted from DWI data after diffusion tensor fitting. 2.5.1. Global Measures. Global and local network efficiencies
are used to describe global and local characteristics of parallel
information transfer in a network. Global network efficiency
quantifies the exchange of information across the entire brain: 𝐸glob =
1
𝑁(𝑁−1)
𝑁
∑
𝑖=1
𝑁
∑
𝑗=1,𝑗̸=𝑖
1
𝐿𝑖𝑗
,
(1) (1) 2.4. Network Construction. Whole-brain tractography was
performed with both DTI and HARDI models. For DTI, the
diffusion tensors were fitted to the data using a weighted-
least-squares estimation algorithm and the eigenvector of
the largest eigenvalue was taken as the principal diffusion
direction [32]. Seed points were chosen as voxels with FA >
0.3. The maximum turning angle was set to 45∘and tracking
was stopped when FA < 0.2. 2. Methods By detecting the peaks (i.e., local
maxima) of the ODF, one can then infer the number of
directions contained in each voxel. The NC group was cognitively functioning healthy indi-
viduals. The NC group did not have any history of cognitive
decline, neurological disorders, or uncontrolled systemic
medical disorders. Their CDR scales were equal to 0. All
subjects in the study were required to have fewer than two
lacunar ischemia strokes (of diameter <1 cm) in the brain, as
revealed by FLAIR. The demography and clinical scores for the AD group and
the NC group are listed in Table 1. No significant differences
between the two groups were observed in age or education. The difference was observed in gender. However, the effects of
gender, age, education level, and brain size would be regressed In this study, we investigate the global, regional, and local
changes of whole-brain connectivity networks in AD patients
in comparison to healthy elderly subjects. DTI and HARDI
models are used to construct two different sets of connectivity
networks for comparison. Such a systematic network analysis 3 Neural Plasticity Neural Plasticity The allowed fiber length
was at minimum, 20 mm, and at maximum, 300 mm. For
HARDI, ODFs were estimated with dictionary-based spher-
ical deconvolution [26]. A maximum of three peaks were
detected from the ODF at each voxel [26]. Four seeds were
randomly sampled within each seed voxel. The criteria of
fiber tracking were the same for both methods. The resulting where 𝐿𝑖𝑗is the shortest absolute path length between node
𝑖and node 𝑗. 𝑁is the total number of nodes. Similarly, local
network efficiency of node 𝑖is defined as 𝐸𝑖
loc =
1
𝑁𝐺𝑖(𝑁𝐺𝑖−1)
𝑁𝐺𝑖
∑
𝑗=1
𝑁𝐺𝑖
∑
𝑘=1,𝑘̸=𝑗
1
𝐿𝑗𝑘
,
(2) (2) where 𝐺𝑖is a subgraph comprising nodes directly connected
to node 𝑖, and 𝑁𝐺𝑖is the node number of the subgraph 𝐺𝑖. Therefore, the average local network efficiency for the whole
brain is 𝐸loc = (1/𝑁) ∑𝑁
𝑖=1 𝐸𝑖
loc. 4 Neural Plasticity Table 2: Names and abbreviations of the 90 ROIs defined in the AAL template. Table 2: Names and abbreviations of the 90 ROIs defined in the AAL template. ∗The odd and even indices indicate the regions in the left and right hemispheres, respectively. Neural Plasticity The
nodal efficiency 𝐸𝑖is defined as The global clustering coefficient gives an overall indica-
tion of clustering in a network. It is the average of absolute
local clustering coefficients of all nodes: 𝐸𝑖=
1
𝑁−1
𝑁
∑
𝑗=1,𝑗̸=𝑖
1
𝐿𝑖𝑗
,
(5) 𝐶= 1
𝑁
𝑁
∑
𝑖=1
𝐶𝑖,
𝐶𝑖=
𝐸𝑖
𝐾𝑖(𝐾𝑖−1) /2,
(3) (5) (3) where 𝐿𝑖𝑗is the shortest path length between node 𝑖and
node 𝑗. Therefore, 𝐸𝑖measures the overall information flow
between a given node 𝑖and the rest of the nodes in the
network. The node 𝑖is defined as a hub if 𝐸𝑖is at least 1
standard deviation (SD) above the average nodal efficiency
of the network. where 𝐶𝑖is the local clustering coefficient for node 𝑖, 𝐸𝑖
is the number of edges in the subgraph 𝐺𝑖of node 𝑖, and
𝐾𝑖denotes the number of nodes in 𝐺𝑖. In other words, 𝐶𝑖
is the proportion of edges between the nodes within the
neighborhood of node 𝑖divided by the number of edges that
could possibly exist between them. In addition, the average
shortest path length of the network is defined as 2.5.3. Local Measures. The network edges, that is, the fiber
counts between each pair of ROIs, were directly used to
describe the local characteristics of a network. 2.6. Statistical Analysis. The nonparametric permutation test
was used to evaluate statistical differences of brain network
properties between the AD and NC groups. First, linear
regression was performed on all the network measures at
global, regional, and local levels (described in Section 2.5),
respectively, with age, gender, level of education, and intracra-
nial volume (ICV) as covariates. Then, after removing those
factors on the measures, the regressed measures were per-
muted 5,000 times to assess the statistical significance of the
differences [36]. The significance level was set as 𝑝< 0.05,
with false discovery rate (FDR) [43] for multiple comparison
correction. To compare the performance between the DTI
and HARDI methods, the same analysis was performed to the
networks constructed by each method, respectively. 𝐿=
1
𝑁(𝑁−1)
𝑁
∑
𝑖=1
𝑁
∑
𝑗=1,𝑗̸=𝑖
𝐿𝑖𝑗. (4) (4) The human brain exhibits the “small-world” property charac-
terized by dense local clustering between neighboring nodes
and high global network efficiency with short path lengths
due to the existence of relatively few long-range connections
[39–41]. Neural Plasticity Index∗
Region
Abbreviation
1, 2
Precentral gyrus
PreCG
3, 4
Superior frontal gyrus (dorsal)
SFGdor
5, 6
Orbitofrontal cortex (superior)
ORBsupb
7, 8
Middle frontal gyrus
MFG
9, 10
Orbitofrontal cortex (middle)
ORBmid
11, 12
Inferior frontal gyrus (opercular)
IFGoperc
13, 14
Inferior frontal gyrus (triangular)
IFGtriang
15, 16
Orbitofrontal cortex (inferior)
ORBinf
17, 18
Rolandic operculum
ROL
19, 20
Supplementary motor area
SMA
21, 22
Olfactory
OLF
23, 24
Superior frontal gyrus (medial)
SFGmed
25, 26
Orbitofrontal cortex (medial)
ORBmed
27, 28
Rectus gyrus
REC
29, 30
Insula
INS
31, 32
Anterior cingulate gyrus
ACG
33, 34
Middle cingulate gyrus
MCG
35, 36
Posterior cingulate gyrus
PCG
37, 38
Hippocampus
HIP
39, 40
Parahippocampal gyrus
PHG
41, 42
Amygdala
AMYG
43, 44
Calcarine
CAL
45, 46
Cuneus
CUN
47, 48
Lingual gyrus
LING
49, 50
Superior occipital gyrus
SOG
51, 52
Middle occipital gyrus
MOG
53, 54
Inferior occipital gyrus
IOG
55, 56
Fusiform gyrus
FFG
57, 58
Postcentral gyrus
PoCG
59, 60
Superior parietal gyrus
SPG
61, 62
Inferior parietal lobule
IPL
63, 64
Supramarginal gyrus
SMG
65, 66
Angular gyrus
ANG
67, 68
Precuneus
PCUN
69, 70
Paracentral lobule
PCL
71, 72
Caudate
CAU
73, 74
Putamen
PUT
75, 76
Pallidum
PAL
77, 78
Thalamus
THA
79, 80
Heschl gyrus
HES
81, 82
Superior temporal gyrus
STG
83, 84
Temporal pole (superior)
TPOsup
85, 86
Middle temporal gyrus
MTG
87, 88
Temporal pole (middle)
TPOmid
89, 90
Inferior temporal
ITG
∗The odd and even indices indicate the regions in the left and right hemispheres, respectively. 5 Neural Plasticity Crossing
DTI
(a)
Crossing
HARDI
(b)
Figure 1: Directional glyphs at the intersection of the left corticospinal tract and the corpus callosum given by (a) the DTI method and
the HARDI method. Crossing
HARDI
(b) (b) Figure 1: Directional glyphs at the intersection of the left corticospinal tract and the corpus callosum given by (a) the DTI method and (b)
the HARDI method Figure 1: Directional glyphs at the intersection of the left corticospinal tract and the corpus callosum given by (a) the DTI method and (b)
the HARDI method. 2.5.2. Regional Measures. The nodal efficiency was computed
to represent the regional characteristics of a network. The
nodal efficiency 𝐸𝑖is defined as 2.5.2. Regional Measures. The nodal efficiency was computed
to represent the regional characteristics of a network. Neural Plasticity Mathematically, it can be represented by the ratio of
the normalized global clustering coefficient 𝛾= 𝐶real/𝐶rand
to the normalized shortest path length 𝜆= 𝐿real/𝐿rand, where
𝐶rand and 𝐿rand are the global clustering coefficient and the
normalized shortest path length of a random network. A
random network was simulated by iteratively rewiring 50%
pairs of random edges of the existing brain network for 1,000
times. Then, small-worldness can be measured as 𝜎= 𝛾/𝜆
[42] and it should be greater than 1 if the graph demonstrates
the small-world property. 3. Results In both cases, the AD group, when compared
to the NC group, showed decreases in global efficiency and
local efficiency but increases in the normalized shortest path
length (𝜆) and the normalized clustering coefficient (𝛾). Also,
all results given by the HARDI method were statistically
significant (𝑝< 0.05), while most results by the DTI method
were not, except global efficiency. Table 3 lists the values of
these measures for the AD and NC groups by both the DTI
and HARDI methods. The AD networks actually showed higher small-worldness
than the NC networks did, in both DTI and HARDI cases
(𝜎AD > 𝜎NC). In both cases, the AD group, when compared
to the NC group, showed decreases in global efficiency and
local efficiency but increases in the normalized shortest path
length (𝜆) and the normalized clustering coefficient (𝛾). Also,
all results given by the HARDI method were statistically
significant (𝑝< 0.05), while most results by the DTI method
were not, except global efficiency. Table 3 lists the values of
these measures for the AD and NC groups by both the DTI
and HARDI methods. method can handle fiber crossings in the intersection
between the left corticospinal tract and the corpus callosum.h method can handle fiber crossings in the intersection
between the left corticospinal tract and the corpus callosum. The DTI method, on the other hand, was not able to do so. Figure 2 shows the tractography results based on a seed ROI
near the brain stem. The HARDI method was able to produce
significantly more fibers than the DTI method. The DTI method, on the other hand, was not able to do so. Figure 2 shows the tractography results based on a seed ROI
near the brain stem. The HARDI method was able to produce
significantly more fibers than the DTI method. gi
yi
Figure 3 shows the 90 × 90 connectivity matrices (≥4
fiber connections) with both DTI and HARDI methods
from a randomly selected subject in our dataset. The binary
difference between the two matrices is also shown, where
the entries with +1 denote connections detected by HARDI
but not DTI, and −1 for connections detected by DTI
but not HARDI. For this selected subject, the meaningful
connections (≥4 fiber connections) account for 38% and 52%
out of the total connections for DTI and HARDI, respectively. 3. Results 3.1. DTI versus HARDI. We compared the DTI and HARDI
networks, in terms of their ability, to distinguish the AD
group from the NC group. Figure 1 shows that HARDI 6 Neural Plasticity ROI
DTI
(a)
ROI
HARDI
(b)
Figure 2: Tractography results based on a seed ROI at the brain stem with (a) the DTI method and (b) the HARDI method. ROI
DTI
(a) ROI
HARDI
(b) DTI
(a) (b) (a) Figure 2: Tractography results based on a seed ROI at the brain stem with (a) the DTI method and (b) the HARDI method. 0
500
100
1500
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
DTI
0
500
100
1500
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
HARDI
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
HARDI-DTI
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1
Figure 3: The 90 × 90 connectivity matrices built with the DTI method and the HARDI method, respectively. The right panel shows the
binary difference between the left two matrices for a selected subject, where the entries with +1 denote connections detected by HARDI but
not DTI, and −1 for connections detected by DTI but not HARDI. 0
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
HARDI-DTI
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1 0
0
500
100
1500
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
HARDI 0
5
1
1
90
80
70
60
50
40
30
20
10
20 30 40 50 60 70 80 90
10
DTI DTI Figure 3: The 90 × 90 connectivity matrices built with the DTI method and the HARDI method, respectively. The right panel shows the
binary difference between the left two matrices for a selected subject, where the entries with +1 denote connections detected by HARDI but
not DTI, and −1 for connections detected by DTI but not HARDI. The AD networks actually showed higher small-worldness
than the NC networks did, in both DTI and HARDI cases
(𝜎AD > 𝜎NC). 3. Results In addition, 4 ROIs, such
as the left insula (INS.L), the right caudate nucleus (CAU.R),
and the bilateral pallida (PAL), were identified as the hubs in
the AD group but not in the NC group. 8 ROIs, such as the
right medial superior frontal gyrus (SFGmed.R), the bilateral
posterior cingulate gyri (PCG), the right calcarine cortex
(CAL.R), the right cuneus (CUN.R), the bilateral superior
occipital gyri (SOG), and the left middle occipital gyrus
(MOG.L), were identified as the hubs in the NC group but not
in the AD group. For the DTI case, most of the hubs identified
in the HARDI case were also detected. The AD group had the
exact 16 hubs as in the HARDI case, while the NC group only
had 19 hubs. The right calcarine cortex (CAL.R) and the left
middle occipital gyrus (MOG.L) were only identified in the
HARDI case for the NC group, while the left medial superior
frontal gyrus (SFGmed.L) was only identified in the DTI case. The hub distributions in the AD and NC groups are shown in
Figure 4 for both methods. In both DTI and HARDI cases, when compared to the
NC group, the AD group showed reduced nodal efficiency
(𝑝< 0.05, FDR corrected) in the bilateral superior occipital
gyri (SOG), the right middle occipital gyrus (MOG.R), the
right rectus gyrus (REC.R), the left posterior cingulate gyrus
(PCG.L), the right parahippocampal gyrus (PHG.R), the
right middle temporal pole (TPOmid.R), the right inferior
occipital gyrus (IOG.R), the right fusiform gyrus (FFG.R),
the right precuneus (PCUN.R), and the bilateral cunei
(CUN). Besides all of the regions shown above, the right
posterior cingulate gyrus (PCG.R), the right calcarine cortex
(CAL.R), and the left precuneus (PCUN.L) showed the
significantly reduced nodal efficiency only in the HARDI
case, while the left middle temporal pole (TPOmid.L) showed
the reduced efficiency only in the DTI. The comparison
between the ROIs that had the reduced efficiency in the two
groups for the DTI and HARDI cases is shown in Figure 5. 3. Results Particularly, the thicker the line in
Figure 7, the greater the difference in the connection between
the two groups. The identified differences in connections
spread over the entire brain. A large portion of these dif-
ferences was located in the limbic system and subcortical
regions. It is obvious that the HARDI model was able to
detect noticeably more pairs of different connections between
the groups (30 pairs in HARDI versus 20 pairs in DTI). For example, the connections through the left supplementary
motor area (SMA.L), the right lingual gyrus (LING.R), the
left superior parietal gyrus (SPG.L), the bilateral thalami
(THA), the left middle temporal gyrus (MTG.L), and the left
hippocampus (HIP.L) were only shown in the HARDI case. connection. After performing the permutation test [36] on
each connection, the axial and the sagittal views of those
significantly different connections (𝑝
<
0.05) between
the AD group and the NC group, with the DTI and
HARDI method, are illustrated in Figure 6. Additionally, the
connectogram, a circular representation tool called Circos
(http://www.cpan.org/ports/) [44], was used to demonstrate
those connections with the two models in Figure 7. In
both figures, the stronger connections (higher fiber counts
between a pair of ROIs) in the AD group are shown in blue,
and the weaker connections (lower fiber counts between a
pair of ROIs) are in red. Particularly, the thicker the line in
Figure 7, the greater the difference in the connection between
the two groups. The identified differences in connections
spread over the entire brain. A large portion of these dif-
ferences was located in the limbic system and subcortical
regions. It is obvious that the HARDI model was able to
detect noticeably more pairs of different connections between
the groups (30 pairs in HARDI versus 20 pairs in DTI). For example, the connections through the left supplementary
motor area (SMA.L), the right lingual gyrus (LING.R), the
left superior parietal gyrus (SPG.L), the bilateral thalami
(THA), the left middle temporal gyrus (MTG.L), and the left
hippocampus (HIP.L) were only shown in the HARDI case. dorsolateral (SFGdor), the bilateral supplementary motor
areas (SMA), the bilateral median cingulate gyri (MCG), the
bilateral precunei (PCUN), the bilateral putamina (PUT),
and the bilateral thalami (THA). 3. Results From the difference map in the right panel of Figure 3, it is
obvious that more connections can be detected with HARDI
compared to DTI. 3.3. Regional Characteristics. An ROI is defined as a network
hub, if its nodal efficiency is 1 SD greater than the mean nodal
efficiency of the network. For the HARDI case, we identified
20 hub nodes in the NC group, including 6 association
cortical regions, 13 paralimbic cortical regions, and 1 primary
cortical region. Only 16 hub nodes were identified in the
AD group, including 5 association regions and 11 paralimbic
regions. In both groups, 12 ROIs were identified as hubs
in common, including the bilateral superior frontal gyri, 3.2. Global Characteristics. Both the NC and the AD groups
showed small-world organization (𝜎> 1) in their networks. 7 Neural Plasticity Neural Plasticity Table 3: The comparison of the global connectivity characteristics between the AD and the NC groups with the DTI and HARDI models,
respectively. 𝐸glob
𝐸loc
𝛾
𝜆
𝜎
DTI
AD
524 ± 93
674 ± 134
1.46 ± 0.18
1.20 ± 0.06
1.22 ± 0.11
NC
617 ± 114
748 ± 121
1.39 ± 0.12
1.16 ± 0.04
1.20 ± 0.07
Difference
−93
−74
0.07
0.03
0.02
𝑝value
0.03
0.12
0.12
0.07
0.20
HARDI
AD
426 ± 100
578 ± 126
1.66 ± 0.24
1.27 ± 0.08
1.31 ± 0.16
NC
543 ± 134
674 ± 144
1.53 ± 0.19
1.22 ± 0.06
1.25 ± 0.11
Difference
−116
−95
0.14
0.05
0.06
𝑝value
0.01
0.05
0.03
0.03
0.04 Table 3: The comparison of the global connectivity characteristics between the AD and the NC groups with the DTI and HARDI models,
respectively. connection. After performing the permutation test [36] on
each connection, the axial and the sagittal views of those
significantly different connections (𝑝
<
0.05) between
the AD group and the NC group, with the DTI and
HARDI method, are illustrated in Figure 6. Additionally, the
connectogram, a circular representation tool called Circos
(http://www.cpan.org/ports/) [44], was used to demonstrate
those connections with the two models in Figure 7. In
both figures, the stronger connections (higher fiber counts
between a pair of ROIs) in the AD group are shown in blue,
and the weaker connections (lower fiber counts between a
pair of ROIs) are in red. 3.4. Local Characteristics. We utilized the fiber counts
between a pair of ROIs to measure the strength of their 4. Discussion This study investigates the impact of AD on the topological
characteristics of the WM connectivity network at three
hierarchical levels, global, regional, and local level, through
tractography data reconstructed using DTI and HARDI
methods, respectively. The main findings are as follows: (1)
the global and average local network efficiency are reduced
in AD, with increased shortest path length; (2) the number
of regional hubs and nodal efficiency decreases in AD; (3)
the local connections weaken in AD; (4) the HARDI method
has an advantage over the DTI method in identifying more
abnormal network characteristics at all the three levels. At the global level, consistent with the previous studies
[41, 45, 46], our results indicate that the WM connectivity
networks of both AD and NC have the small-world topology. 3.4. Local Characteristics. We utilized the fiber counts
between a pair of ROIs to measure the strength of their Neural Plasticity 8 PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R CAU.R
PAL.R
PUT.R
PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R
CAU.R
PAL.R
PUT.R
PCUN.L
SOG.L
SMA.L
MCG.L
PCG.L
THA.L
PUT.L
SFGdor.L
SFGmed.L
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
SOG.R
CUN.R
AD
NC
DTI
SMA.L
SFGdor.L
PUT.LTHA.L
MCG.L PCUN.L
PCG.L
SOG.L
MOG.L
SOG.R
CUN.R
CAL.R
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
AD
NC
HARDI
L
R
L
R
Hubs in the AD group and the NC group given by the DTI method and the HARDI method, respectively. Each spher
of an ROI. Refer to Table 2 for the label of each ROI. PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R
CAU.R
PAL.R
PUT.R
AD
L
R L R AD PCUN.L
SOG.L
SMA.L
MCG.L
PCG.L
THA.L
PUT.L
SFGdor.L
SFGmed.L
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
SOG.R
CUN.R
NC
DTI NC
DTI PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R CAU.R
PAL.R
PUT.R
SMA.L
SFGdor.L
PUT.LTHA.L
MCG.L PCUN.L
PCG.L
SOG.L
MOG.L
SOG.R
CUN.R
CAL.R
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
AD
NC
HARDI
L
R
Figure 4: Hubs in the AD group and the NC group given by the DTI method and the HARDI method, respectively. Each sphere represents
the center of an ROI. Refer to Table 2 for the label of each ROI. 4. Discussion Therefore, it is
likely that, in people with AD, information may flash in a Neural Plasticity 9 TPOmid.L
PCG.L
SOG.L
CUN.L
SOG.R
CUN.R
PCUN.R
IOG.R
FFG.R
PHG.R
REC.R
TPOmid.R
MOG.R
L
R
DTI
PCUN.L
PCG.L
SOG.L
CUN.L
SOG.R
CAL.R
PCUN.R
PCG.R
CUN.R
IOG.R FFG.R PHG.R
REC.R
TPOmid.R
MOG.R
HARDI
Figure 5: ROIs that have the reduced nodal efficiency in the AD group compared to the NC group (𝑝< 0.05), given by the DTI and HARDI
methods, respectively. Each sphere represents the center of an ROI and its size is proportional to the nodal efficiency. Refer to Table 2 for the
label of each ROI. TPOmid.L
PCG.L
SOG.L
CUN.L
SOG.R
CUN.R
PCUN.R
IOG.R
FFG.R
PHG.R
REC.R
TPOmid.R
MOG.R
L
R
DTI R DTI DTI
PCUN.L
PCG.L
SOG.L
CUN.L
SOG.R
CAL.R
PCUN.R
PCG.R
CUN.R
IOG.R FFG.R PHG.R
REC.R
TPOmid.R
MOG.R
HARDI HARDI Figure 5: ROIs that have the reduced nodal efficiency in the AD group compared to the NC group (𝑝< 0.05), given by the DTI and HARDI
methods, respectively. Each sphere represents the center of an ROI and its size is proportional to the nodal efficiency. Refer to Table 2 for the
label of each ROI. temporal pole, middle (TPOmid), and the parahippocampal
gyrus (PHG) are involved in semantic memory processing
and recognition [56] and show atrophy and neuronal loss
in AD [57, 58]. Notably, the decreased efficiency in the
temporal lobes was observed to be mainly located in the right
hemisphere. Together, the reduced nodal efficiency suggests
that possible WM degeneration in these brain regions may
negatively affect information transmission and functional
integration in AD patients.i certain brain region but fail to transmit to other regions effec-
tively to form stable memories. The normalized clustering
coefficient is a ratio of local information transfer capability
in a network to that of a random network. In AD, its increase
reflects the reinforcement of information transfer capability. Likewise, previous studies have also found a greater clustering
coefficient and a longer absolute path length in AD, which
may indicate that the organization of the cortical network is
least optimal in AD [49].i g
At the local level, weaker connection (lower fiber counts)
happens predominantly in the area of the bilateral precunei
(PCUN), the right cuneus (CUN.R), the left middle temporal
gyrus (MTG.L), and left hippocampal gyrus (HIP.L). 4. Discussion PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R CAU.R
PAL.R
PUT.R
SMA.L
SFGdor.L
PUT.LTHA.L
MCG.L PCUN.L
PCG.L
SOG.L
MOG.L
SOG.R
CUN.R
CAL.R
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
AD
NC
HARDI
L
R
Figure 4: Hubs in the AD group and the NC group given by the DTI method and the HARDI method, respectively. Each s
h
f
ROI R f
T bl 2 f
h l b l f
h ROI PCUN.L
SMA.L
SFGdor.L
MCG.L
THA.L
INS.L
PUT.L
PAL.L
PCUN.R
SMA.R
SFGdor.R
MCG.R
THA.R CAU.R
PAL.R
PUT.R
AD
L
R R AD SMA.L
SFGdor.L
PUT.LTHA.L
MCG.L PCUN.L
PCG.L
SOG.L
MOG.L
SOG.R
CUN.R
CAL.R
SMA.R
PCUN.R
SFGdor.R
SFGmed.R
MCG.R
PCG.R
PUT.R
THA.R
NC
HARDI NC
HARDI Figure 4: Hubs in the AD group and the NC group given by the DTI method and the HARDI method, respectively. Each sphere represents
the center of an ROI. Refer to Table 2 for the label of each ROI. Although the AD networks show a slightly elevated small-
world attribute, most global measures are lower in AD, com-
pared to those in NC. AD patients show significant decreases
in global efficiency and average local efficiency, but increases
in normalized shortest path length. Global efficiency and
average local efficiency are known to reflect the overall ability
of information transfer between different nodes in a network. They are comprehensive indices for the capability of parallel
information processing. The reductions in those measures
can be attributed to the degeneration of WM, which indicates
that connections between cortical regions are abnormal. The
less strength of connections between cortical regions is due to the damaged WM integrity, resulting in longer pathways
that connect different regions in the brain. The breakdown
in the optimal brain balance between the local specialization
and the global integration causes information processing
to malfunction in AD. Similarly to previous studies [47],
we have also found that the normalized weighted shortest
path length increases in the AD group. Shortest path length
ensures interregional effective communication, or prompt
transfer of information between regions, which constitutes
the basis of cognitive processes [48]. The WM damage can
lead to an increase in shortest path length. 4. Discussion These
areas, which are mostly associated with linguistic integration,
emotion, and semantic memory [56, 59], are affected in AD
patients [57, 58]. It is worth noting that precuneus, cuneus,
and temporal lobe also show reduced nodal efficiency at the
regional level. In addition to the typically well-known affected
regions in AD, the right amygdala (AMYG.R) and the right
middle frontal gyrus (MFG.R) show weaker connections
as well. These regions are the structures mostly involved
in emotional processing, perceptions, psychological states,
and behavioral responses [60]. Weaker connections can
also be identified at the right thalamus (THA.R), which is
known to have a significantly reduced volume in AD [61]. Interestingly, a few regions show increased local connection
in the AD group than in the NC group, for example, the
connection between the left insula (INS.L) and left inferior
parietal lobule (IPL.L). It is possible that this may result from
the compensation to weak connections in the neighboring
regions. At the regional level, several hubs identified in NC are
not shown in AD, such as precuneus (PCUN) and posterior
cingulate gyrus (PCG). These two regions also demonstrate
reduced connectivity in functional magnetic resonance imag-
ing (fMRI) studies in patients with amnesic mild cognitive
impairment (aMCI), a stage with high risk in developing
AD [50], which may suggest that these two regions maintain
pivotal roles in both structural and functional default mode
networks in AD [51].hfi The AD networks also show decreased nodal efficiency
in many cortical regions, mainly located in the bilateral
cunei (CUN), the right precuneus (PCUN.R), the bilateral
posterior cingulate gyri (PCG), the right temporal pole,
middle (TPOmid.R), and the right parahippocampal gyrus
(PHG.R). The cuneus (CUN), the precuneus (PCUN), and
the posterior cingulate gyrus (PCG) are thought to be
involved in the episodic memory information transmission
and malfunction in AD [52]. Although the degeneration of
the posterior cingulate gyrus was originally interpreted as
not being a direct consequence of degeneration in the medial
temporal lobe, recent studies have revealed that this area has
atrophy and metabolic abnormalities in incipient AD [52–
54]. In a study that examined the cingulum tract in AD, both
the anterior and posterior regions were affected [55]. The
posterior cingulate region is a key “hub” affected in AD. The Overall, the HARDI method outperforms the DTI
method in terms of differentiating AD and NC at all three
levels. HARDI Figure 6: The axial and the sagittal views of the significantly different connections (𝑝< 0.05) based on the fiber counts between the AD
group and the NC group given by the DTI and HARDI methods, respectively. The stronger connections (higher fiber counts between a pair
of ROIs) in the AD group are shown in blue, while the weaker connections (lower fiber counts between a pair of ROIs) are in red. Refer to
Table 2 for the label of each ROI. Figure 6: The axial and the sagittal views of the significantly different connections (𝑝< 0.05) based on the fiber counts between the AD
group and the NC group given by the DTI and HARDI methods, respectively. The stronger connections (higher fiber counts between a pair
of ROIs) in the AD group are shown in blue, while the weaker connections (lower fiber counts between a pair of ROIs) are in red. Refer to
Table 2 for the label of each ROI. statistical power in distinguishing the groups for all the
measures, according to the 𝑝values in Table 3. The group
differences of all the measures are statistically significant in
the HARDI case, while most of them are not statistically
significant in the DTI case. At the regional level, the HARDI
method detects more regions with reduced nodal efficiency. These include the bilateral posterior cingulate gyri (PCG) and
the bilateral precunei (PCUN), while the results of the DTI
method only show the unilateral deficiency of these regions. The results from the HARDI method are more consistent with
the pathology of AD, as the bilateral posterior cingulate gyri
and precunei are both associated with memory processing
and affected in AD [62, 63]. At the local level, the two methods
show the greatest difference. The HARDI method is able to
identify 50% more of the weaker connections in AD than
the DTI method (30 pairs versus 20 pairs). This may be because the HARDI method is able to find the correct tract
directions at the fiber crossing regions and can find more
connections in the NC group. Specifically, the left superior
parietal gyrus (SPG.L), the right thalamus (THA.R), and
the left middle temporal gyrus (MTG.L), especially the left
hippocampus (HIP.L) and the left cuneus (CUN.L), are only
found using the HARDI method. During the early onset of
AD, the superior parietal gyrus and the middle temporal
gyrus undergo neuronal loss [64]. 4. Discussion At the global level, the HARDI method has more 10 Neural Plasticity REC.L
INS.L
CAU.L
IPL.L
PCG.L
PCUN.L
SOG.L
ORBsup.R
TPOmid.R
REC.R
CAU.R
PHG.R HES.R
ITG.R
FFG.R
PCG.R
PCUN.R
SOG.R
MFG.R
ORBinf.R
INS.R
OLF.R
AMYG.R
THA.R
LING.R
CAL.R
CUN.R
IFGtriang.L
SAM.L
MCG.L
THA.L
HIP.L
CUN.L
MOG.L
MTG.L
SPG.L
SAM.L
MFG.R
MCG.L
IFGtriang.L
SPG.L
ORBsup.R
TPOmid.R
AMYG.R
ORBinf.R
OLF.R
REC
INS
CAU
THA
PHG.R ITG.R
FFG.R
HIP.L
MTG.L
MOG.L
LING.R
PCG
CAL.R
IPL.L
PCUN
SOG
CUN
ORBmed.L
REC.L
INS.L CAU.L
ROL.L
IPL.L
MCG.L
PCG.L
PCUN.L
SOG.L
ORBsup.R
TPOmid.R
REC.R
CAU.R
PHG.R HES.R
ITG.R
ANG.R
FFG.R
PCG.R
PCUN.R
MOG.R
CUN.R
SOG.R
SOG
ORBmed.L
ORBsup.R
REC
TPOmid.R
PHG.R ITG.R
FFG.R
MCG.L
CAU
INS.L
ROL.L
HES.R
PCUN
IPL.L
PCG
ANG.R
CUN.R
MOG.R
Axial view
Sagittal view
DTI
HARDI
Figure 6: The axial and the sagittal views of the significantly different connections (𝑝< 0.05) based on the fiber counts between
group and the NC group given by the DTI and HARDI methods, respectively. The stronger connections (higher fiber counts betwe
of ROIs) in the AD group are shown in blue, while the weaker connections (lower fiber counts between a pair of ROIs) are in red
Table 2 for the label of each ROI. ORBmed.L
REC.L
INS.L CAU.L
ROL.L
IPL.L
MCG.L
PCG.L
PCUN.L
SOG.L
ORBsup.R
TPOmid.R
REC.R
CAU.R
PHG.R HES.R
ITG.R
ANG.R
FFG.R
PCG.R
PCUN.R
MOG.R
CUN.R
SOG.R
Axial view SOG
ORBmed.L
ORBsup.R
REC
TPOmid.R
PHG.R ITG.R
FFG.R
MCG.L
CAU
INS.L
ROL.L
HES.R
PCUN
IPL.L
PCG
ANG.R
CUN.R
MOG.R Sagittal view REC.L
INS.L
CAU.L
IPL.L
PCG.L
PCUN.L
SOG.L
ORBsup.R
TPOmid.R
REC.R
CAU.R
PHG.R HES.R
ITG.R
FFG.R
PCG.R
PCUN.R
SOG.R
MFG.R
ORBinf.R
INS.R
OLF.R
AMYG.R
THA.R
LING.R
CAL.R
CUN.R
IFGtriang.L
SAM.L
MCG.L
THA.L
HIP.L
CUN.L
MOG.L
MTG.L
SPG.L
SAM.L
MFG.R
MCG.L
IFGtriang.L
SPG.L
ORBsup.R
TPOmid.R
AMYG.R
ORBinf.R
OLF.R
REC
INS
CAU
THA
PHG.R ITG.R
FFG.R
HIP.L
MTG.L
MOG.L
LING.R
PCG
CAL.R
IPL.L
PCUN
SOG
CUN
Axial view
Sagittal view
DTI Axial view REC.L
INS.L
CAU.L
IPL.L
PCG.L
PCUN.L
SOG.L
ORBsup.R
TPOmid.R
REC.R
CAU.R
PHG.R HES.R
ITG.R
FFG.R
PCG.R
PCUN.R
SOG.R
MFG.R
ORBinf.R
INS.R
OLF.R
AMYG.R
THA.R
LING.R
CAL.R
CUN.R
IFGtriang.L
SAM.L
MCG.L
THA.L
HIP.L
CUN.L
MOG.L
MTG.L
SPG.L
SAM.L
MFG.R
MCG.L
IFGtriang.L
SPG.L
ORBsup.R
TPOmid.R
AMYG.R
ORBinf.R
OLF.R
REC
INS
CAU
THA
PHG.R ITG.R
FFG.R
HIP.L
MTG.L
MOG.L
LING.R
PCG
CAL.R
IPL.L
PCUN
SOG
CUN
Axial view
Sagittal view HARDI PreCG(1)
SFGdor(3)
ORBsupb(5)
MFG(7)
ORBmid(9)
IFGoperc(11)
IFGtriang(13)
ORBinf(15)
ROL(17)
SMA(19)
OLF(21)
SFGmed(23)
ORBmed(25)
REC(27)
INS(29)
ACG(31)
MCG(33)
PCG(35)
HIP(37)
PHG(39)
AMYG(41)
CAL(43)
CUN(45)
LING(47)
SOG(49)
MOG(51)
IOG(53)
FFG(55)
PoCG(57)
SPG(59)
IPL(61)
SMG(63)
ANG(65)
PCUN(67)
PCL(69)
CAU(71)
PUT(73)
PAL(75)
THA(77)
HES(79)
STG(81)
TPOsup(83)
MTG(85)
TPOmid(87)
ITG(89)
PreCG(2)
SFGdor(4)
ORBsupb(6)
MFG(8)
ORBmid(10)
IFGoperc(12)
IFGtriang(14)
ORBinf(16)
ROL(18)
SMA(20)
OLF(22)
SFGmed(24)
ORBmed(26)
REC(28)
INS(30)
ACG(32)
MCG(34)
PCG(36)
HIP(38)
PHG(40)
AMYG(42)
CAL(44)
CUN(46)
LING(48)
SOG(50)
MOG(52)
IOG(54)
FFG(56)
PoCG(58)
SPG(60)
IPL(62)
SMG(64)
ANG(66)
PCUN(68)
PCL(70)
CAU(72)
PUT(74)
PAL(76)
THA(78)
HES(80)
STG(82)
TPOsup(84)
MTG(86)
TPOmid(88)
ITG(90)
FRO
INS
TEM
PAR
OCC
LIM
SBC
FRO
INS
TEM
PAR
OCC
LIM
SBC
DTI
(a) PreCG(1)
SFGdor(3)
ORBsupb(5)
MFG(7)
ORBmid(9)
IFGoperc(11)
IFGtriang(13)
ORBinf(15)
ROL(17)
SMA(19)
OLF(21)
SFGmed(23)
ORBmed(25)
REC(27)
INS(29)
ACG(31)
MCG(33)
PCG(35)
HIP(37)
PHG(39)
AMYG(41)
CAL(43)
CUN(45)
LING(47)
SOG(49)
MOG(51)
IOG(53)
FFG(55)
PoCG(57)
SPG(59)
IPL(61)
SMG(63)
ANG(65)
PCUN(67)
PCL(69)
CAU(71)
PUT(73)
PAL(75)
THA(77)
HES(79)
STG(81)
TPOsup(83)
MTG(85)
TPOmid(87)
ITG(89)
PreCG(2)
SFGdor(4)
ORBsupb(6)
MFG(8)
ORBmid(10)
IFGoperc(12)
IFGtriang(14)
ORBinf(16)
ROL(18)
SMA(20)
OLF(22)
SFGmed(24)
ORBmed(26)
REC(28)
INS(30)
ACG(32)
MCG(34)
PCG(36)
HIP(38)
PHG(40)
AMYG(42)
CAL(44)
CUN(46)
LING(48)
SOG(50)
MOG(52)
IOG(54)
FFG(56)
PoCG(58)
SPG(60)
IPL(62)
SMG(64)
ANG(66)
PCUN(68)
PCL(70)
CAU(72)
PUT(74)
PAL(76)
THA(78)
HES(80)
STG(82)
TPOsup(84)
MTG(86)
TPOmid(88)
ITG(90)
FRO
INS
TEM
PAR
OCC
LIM
SBC
FRO
INS
TEM
PAR
OCC
LIM
SBC
HARDI
(b) (a) (b) Figure 7: Connectograms showing significantly different connections (𝑝< 0.05) based on the fiber counts between the AD group and the
NC group, given (a) the DTI method and (b) the HARDI method. The thickness of each line indicates the extent of the difference between
the corresponding connections in the two groups. The stronger connections (higher fiber counts between a pair of ROIs) in the AD group
are shown in blue, while the weaker connections (lower fiber counts between a pair of ROIs) are in red. Refer to Table 2 for the label of each
ROI. standard for regional parcellation makes the definition of ROI
not very precise, especially on the boundary. Registration
error may also play a role in this issue. Therefore, it may affect
the accuracy in the analysis of connectivity networks [71,
72]. Thirdly, the underlying biological relationship between
the network properties and the AD progression is currently
unclear. Studying the intermediate stage, for example, MCI,
may be beneficial for further understanding of the relation-
ship [73, 74]. HARDI In the future work, we will include participants
from this stage to perform a more comprehensive study on
this topic. and network efficiency in young healthy individuals, while
Zhan et al. [67] developed a machine learning framework to
classify different stages of AD with fiber counts as features. However, sometimes, fiber count may not be a suitable feature
in connectivity studies. For instance, in [68, 69], the networks
constructed with the mean FA, MD, and fiber length provided
better performance in identifying high-risk autistic infants
than fiber count. Therefore, we will consider incorporating
other network measures in our future work since they may
provide additional insights into connectivity breakdown,
especially for the case that the fiber count based networks
cannot reveal the progression of AD.h The conventional statistical analysis on network prop-
erties is often performed in a univariate manner, that is,
pairwise comparison between groups. This might overlook
the interaction among sets of connections in group differ-
ence. On the other hand, instead of doing simple pairwise
comparison, a classification framework is able to consider all
individual connection features, as well as their relationships,
for selecting the most discriminative features for classifica-
tion [68–70]. Ensemble learning algorithm, such as random
forest, is one of this type of classification algorithms that can
be applied to identify discriminative connectivity patterns in
a multivariate manner for AD or MCI classification. This will
be our future work. HARDI Besides the neocortical
atrophy, subcortical structures, such as the thalamus, also
suffer atrophy and may contribute to cognitive decline and
emotion disorder in AD [65]. Fiber count is one of the most commonly used measures
in evaluating connectivity characteristics. For example, Den-
nis et al. [66] computed graph theory metrics based on the
fiber count to track changes in both structural connectivity 11 Neural Plasticity Neural Plasticity
11
PreCG(1)
SFGdor(3)
ORBsupb(5)
MFG(7)
ORBmid(9)
IFGoperc(11)
IFGtriang(13)
ORBinf(15)
ROL(17)
SMA(19)
OLF(21)
SFGmed(23)
ORBmed(25)
REC(27)
INS(29)
ACG(31)
MCG(33)
PCG(35)
HIP(37)
PHG(39)
AMYG(41)
CAL(43)
CUN(45)
LING(47)
SOG(49)
MOG(51)
IOG(53)
FFG(55)
PoCG(57)
SPG(59)
IPL(61)
SMG(63)
ANG(65)
PCUN(67)
PCL(69)
CAU(71)
PUT(73)
PAL(75)
THA(77)
HES(79)
STG(81)
TPOsup(83)
MTG(85)
TPOmid(87)
ITG(89)
PreCG(2)
SFGdor(4)
ORBsupb(6)
MFG(8)
ORBmid(10)
IFGoperc(12)
IFGtriang(14)
ORBinf(16)
ROL(18)
SMA(20)
OLF(22)
SFGmed(24)
ORBmed(26)
REC(28)
INS(30)
ACG(32)
MCG(34)
PCG(36)
HIP(38)
PHG(40)
AMYG(42)
CAL(44)
CUN(46)
LING(48)
SOG(50)
MOG(52)
IOG(54)
FFG(56)
PoCG(58)
SPG(60)
IPL(62)
SMG(64)
ANG(66)
PCUN(68)
PCL(70)
CAU(72)
PUT(74)
PAL(76)
THA(78)
HES(80)
STG(82)
TPOsup(84)
MTG(86)
TPOmid(88)
ITG(90)
FRO
INS
TEM
PAR
OCC
LIM
SBC
FRO
INS
TEM
PAR
OCC
LIM
SBC
DTI
(a)
PreCG(1)
SFGdor(3)
ORBsupb(5)
MFG(7)
ORBmid(9)
IFGoperc(11)
IFGtriang(13)
ORBinf(15)
ROL(17)
SMA(19)
OLF(21)
SFGmed(23)
ORBmed(25)
REC(27)
INS(29)
ACG(31)
MCG(33)
PCG(35)
HIP(37)
PHG(39)
AMYG(41)
CAL(43)
CUN(45)
LING(47)
SOG(49)
MOG(51)
IOG(53)
FFG(55)
PoCG(57)
SPG(59)
IPL(61)
SMG(63)
ANG(65)
PCUN(67)
PCL(69)
CAU(71)
PUT(73)
PAL(75)
THA(77)
HES(79)
STG(81)
TPOsup(83)
MTG(85)
TPOmid(87)
ITG(89)
PreCG(2)
SFGdor(4)
ORBsupb(6)
MFG(8)
ORBmid(10)
IFGoperc(12)
IFGtriang(14)
ORBinf(16)
ROL(18)
SMA(20)
OLF(22)
SFGmed(24)
ORBmed(26)
REC(28)
INS(30)
ACG(32)
MCG(34)
PCG(36)
HIP(38)
PHG(40)
AMYG(42)
CAL(44)
CUN(46)
LING(48)
SOG(50)
MOG(52)
IOG(54)
FFG(56)
PoCG(58)
SPG(60)
IPL(62)
SMG(64)
ANG(66)
PCUN(68)
PCL(70)
CAU(72)
PUT(74)
PAL(76)
THA(78)
HES(80)
STG(82)
TPOsup(84)
MTG(86)
TPOmid(88)
ITG(90)
FRO
INS
TEM
PAR
OCC
LIM
SBC
FRO
INS
TEM
PAR
OCC
LIM
SBC
HARDI
(b)
Figure 7: Connectograms showing significantly different connections (𝑝< 0.05) based on the fiber counts between the AD group and the
NC group, given (a) the DTI method and (b) the HARDI method. The thickness of each line indicates the extent of the difference between
the corresponding connections in the two groups. The stronger connections (higher fiber counts between a pair of ROIs) in the AD group
are shown in blue, while the weaker connections (lower fiber counts between a pair of ROIs) are in red. Refer to Table 2 for the label of each
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constraints,” Human Brain Mapping, vol. Acknowledgments This work was partially supported by the National Natural
Science Foundation of China (81201030, 61210001, 81571298,
and 61473190), the China Ministry of Science and Technol-
ogy (2009BAI77B03), the National Key Clinical Disciplines
at Shanghai Mental Health Center (OMA-MH, 2011-873),
the Shanghai Science and Technology Committee Grants
(134119a2600, 14411965000), the Shanghai Jiao Tong Univer-
sity Technological Innovation Special Fund (YG2014MS39),
the SHSMU-ION Research Center for Brain Disorders, and
the Shanghai Jiao Tong University K. C. Wong Medical
Fellowship Fund. This work was also supported in part by
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many global measures, such as global efficiency, average
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A method for assessing implementation success of a peer-led suicide prevention program
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Implementation science
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cc-by
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Wyman et al. Implementation Science 2015, 10(Suppl 1):A42
http://www.implementationscience.com/content/10/S1/A42 Wyman et al. Implementation Science 2015, 10(Suppl 1):A42
http://www.implementationscience.com/content/10/S1/A42 Implementation
Science Open Access Objective reaching their classmates with the prevention concepts. In analytic models examining predictors of school-level
exposure, a higher proportion of student population
trained as PLs and greater retention of PLs predicted
higher population exposure to the prevention program,
congruent with diffusion of innovations theory, whereas
frequency of meetings did not. To summarize a first stage of research on implementa-
tion of a peer leader suicide prevention program by test-
ing the utility of a method for tracking and reporting
each school’s success in retaining and preparing Peer
Leaders. From 7th Annual Conference on the Science of Dissemination and Implementation in Health
North Bethesda, MD, USA. 8-9 December 2014 From 7th Annual Conference on the Science of Dissemination and Implementation in Health
North Bethesda, MD, USA. 8-9 December 2014 Contribution to the field Peer leader programs that prepare opinion leaders to
spread healthy practices through their social networks
reduce high-risk sex behaviors and show promise in pre-
venting adolescent substance use and suicidal behavior. However, knowledge of implementation processes is very
limited. To address this limitation, we drew on the Stages
of Implementation Completion (SIC) framework to mea-
sure a key phase of peer leader implementation. Identified an efficient method (derived from the SIC) for
assessing a school’s success in preparing/retaining peer
leaders. This approach shows promise in providing
schools actionable data to increase impact of peer-led
programs. Methods 40 high schools were randomly assigned to either immedi-
ate Sources of Strength (n = 20) or waitlist (n = 20). The
schools were underserved by mental health services and
over-represented by youth at high risk for suicide (e.g.,
American Indians). In the 20 implementing schools, 656
students (18-71 per school) received Peer Leader (PL)
training. Adult mentors facilitated PL meetings to rein-
force program concepts and help PLs plan and execute
activities to spread healthy coping practices. Using a
framework derived from the Stages of Implementation
Completion (SIC), school reports of PL meeting dates/
attendance were codified as indices of school success in
retaining and preparing PLs. Surveys with 5,712 students
showed wide school-level variation in success of PLs in Authors’ details
1School & Community-Based Prevention Program, Department of Psychiatry,
University of Rochester School of Medicine, Rochester, NY 14642, USA. 2Center for Prevention Implementation Methodology (Ce-PIM), Northwestern
University, Feinberg School of Medicine, Chicago, IL 60611, USA. 3Oregon
Social Learning Center, Eugene, OR 97401, USA. 4Institute for Prevention
Research, Department of Preventive Medicine, Keck School of Medicine, USC,
Los Angeles, CA 90034, USA. * Correspondence: peter_wyman@urmc.rochester.edu
1School & Community-Based Prevention Program, Department of Psychiatry,
University of Rochester School of Medicine, Rochester, NY 14642, USA
Full list of author information is available at the end of the article © 2015 Wyman et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated. Authors’ details
1 Published: 20 August 2015 doi:10.1186/1748-5908-10-S1-A42
Cite this article as: Wyman et al.: A method for assessing
implementation success of a peer-led suicide prevention program. Implementation Science 2015 10(Suppl 1):A42. doi:10.1186/1748-5908-10-S1-A42
Cite this article as: Wyman et al.: A method for assessing
implementation success of a peer-led suicide prevention program. Implementation Science 2015 10(Suppl 1):A42. © 2015 Wyman et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Trends of Shoreline Position: An Approach to Future Prediction for Balasore Shoreline, Odisha, India
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Open journal of marine science
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cc-by
| 6,825
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Trends of Shoreline Position: An Approach
to Future Prediction for Balasore Shoreline,
Odisha, India Nilay Kanti Barman1, Soumendu Chatterjee2, Ansar Khan1
1Department of Geography and Environment Management, Vidyasagar University, Midnapore, India
2Department of Geography, Presidency University, Kolkata, India
Email: nilay@csws.in, scgeovu@yahoo.co.in, khanansar@gmail.com
Received 23 September 2014; revised 2 November 2014; accepted 21 November 2014 Copyright © 2015 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ How to cite this paper: Barman, N.K., Chatterjee, S. and Khan, A. (2015) Trends of Shoreline Position: An Approach to Fu-
ture Prediction for Balasore Shoreline, Odisha, India. Open Journal of Marine Science, 5, 13-25.
http://dx doi org/10 4236/ojms 2015 51002 Open Journal of Marine Science, 2015, 5, 13-25
Published Online January 2015 in SciRes. http://www.scirp.org/journal/ojms
http://dx.doi.org/10.4236/ojms.2015.51002 Open Journal of Marine Science, 2015, 5, 13-25
Published Online January 2015 in SciRes. http://www.scirp.org/journal/ojms
http://dx.doi.org/10.4236/ojms.2015.51002 Abstract The present study aims to analyze the shift in shoreline due to coastal processes and formulate
available for best estimate of future shoreline positions based on precedent shorelines. Informa-
tion on rates and trends of shoreline change can be used to improve the understanding of the un-
derlying causes and potential effects of coastal erosion which can support informed coastal man-
agement decisions. In this paper, researchers go over the changes in the recent positions of the
shoreline of the Balasore coast for the 38 years from 1975 through 2013. The study area includes
the Balasore coastal region from Rasalpur to Udaypur together with Chandipur, Choumukh, Chan-
drabali as well as Bichitrapur. Transects wise shoreline data base were developed for approxi-
mately 67 kilometers of shoreline and erosional/accretional scenario has also been analysed by
delineating the shoreline from Landsat imageries of 1975, 1980, 1990, 1995, 2000, 2005, 2010
and 2013. A simple Linear Regression Model and End Point Rate (EPR) have been adopted to take
out the rate of change of shoreline and its future positions, based on empirical observations at 67
transects along the Balasore coast. It is found that the north eastern part of Balasore coast in the
vicinity of Subarnarekha estuary and Chandrabali beach undergo high rates of shore line shift. The
shoreline data were integrated for long- (about 17 years) and short-term (about 7 years) shift
rates analysis to comprehend the shoreline change and prediction. For the prediction of future
shoreline, the model has been validated with the present shoreline position (2013). The rate of
shoreline movement calculated from the fixed base line to shoreline position of 1975, 1980, 1990,
1995, 2000, 2005 and 2010 and based on this, the estimated shoreline of 2013 was calculated. The
estimated shoreline was compared with the actual shoreline delineated from satellite imagery of
2013. The model error or positional shift at each sample point is observed. The positional error
varies from −4.82 m to 212.41 m. It has been found that model prediction error is higher in the left
hand side of river Subarnarekha. The overall error for the entire predicted shoreline was found to
be 41.88 m by Root Mean Square Error (RMSE). In addition, it was tested by means difference be- How to cite this paper: Barman, N.K., Chatterjee, S. and Khan, A. 1. Introduction Shoreline shifting is the uncontrollable result of coastal erosion/accretion, the consequence of near shore cur-
rents. With reverence to the sediment supply, a certain portion of coastline may have excess, be in balance, or
have a discrepancy in its sediment budget. Enormous drop or boost in the sediment supply, in a little span of
time or a stretched period, creates a shortage/excess in the sediment budget which causes shoreline shifting. Multi-year shoreline shifting mapping seems to be an important task for coastal monitoring and appraisal. Recently, satellite remote sensing data in combination with Geographic Information System (GIS) is being used
in shoreline extraction and mapping [1]-[3]. Several studies have already been done on shoreline change and
prediction, such as empirical analysis relating to hard stabilization structures to beach dynamics [4] [5] analysis
of natural beach loss and gain [6] identification of relative changes among coastal units [7] and process response
of a shoreline [8]. Shifting in shoreline and rate of shoreline shifting have also been calculated by various studies. The general method regarding this comprises with field observations such as tide and wave properties measure-
ments, revealing and assessment of shoreline from toposheets, aerial photographs and multi date satellite image-
ries. Several models have been proposed for prediction of shoreline from comparison with historical data such as
End Point Rate (EPR) model [8] Average of Rates (AOR), Linear Regression (LR) and Jackknife (JK) [9]. The
EPR model is based on the supposition that observed past rate of change is the best approximation available for
predicting future while LR model is based on robust linear prediction method which detects short-range changes
in the long-standing trend. This process recognizes the linear and high-order polynomial model which best fits
the data according to that Minimum Description Length (MDL) condition that determines the nature and regu-
larities in observed data. The length of the data by which the data itself can be described is used for measuring
such regularities [10]. Prediction of future position of coastlines, from multi-temporal satellite images, using
these models are reliant on several influencing factors like accurateness of shoreline recognition (accuracy of
satellite data and method used), time duration of shoreline data attainment, number of data points taken into
consideration during the measurement of shoreline position and temporal variability of shoreline etc. [11] [12]. Linear Regression Model, End Point Rate, Root Mean Square Error, Shoreline Change, Shoreline
Prediction Linear Regression Model, End Point Rate, Root Mean Square Error, Shoreline Change, Shoreline
Prediction Abstract (2015) Trends of Shoreline Position: An Approach to Fu-
ture Prediction for Balasore Shoreline, Odisha, India. Open Journal of Marine Science, 5, 13-25. http://dx.doi.org/10.4236/ojms.2015.51002 N. K. Barman et al. tween actual and predicted shoreline positions using “t” test and it has been found that predicted
shore line is not significantly different from actual shoreline position at (t132 = 0.278) p < 0.01. tween actual and predicted shoreline positions using “t” test and it has been found that predicted
shore line is not significantly different from actual shoreline position at (t132 = 0.278) p < 0.01. Keywords 1. Introduction The study
area is mainly covered by the Holocene alluviums along with the recent Subarnarekha river’s deposition, which
has a natural gradient to the east and south east direction with a high water holding capacity of brackish water
environment. The pH of the soil is varies from 6.5 - 8.0 (pre monsoon) and 6.2 - 8.2 (post monsoon). The mon-
soonal phase is much more significant in this area. The temperature experienced in a range of minimum of 9˚C
in winter to a maximum of 38˚C in summer. Relative humidity is prevailing here in between 90% - 96% at most
of the months. At the time of the low atmospheric pressure during summer and monsoon period wind dominant-
ly blows from the offshore areas. There is no such forest cover but, some brackish environment floral species
like Sesuvium Portolacrustum, Ipomia Bioloba and some herbs like Lantena camera, Akanthesia, Calatropis
gigantia are found in the study area with some planted trees like Casuarina, Eucalyptus and Acacia auriculifor-
mis and also some indigenous floral species like Coconut, Banana, Bamboo and Mango. Figure 1. Location map of the study area with three blocks i.e. Balasore, Baliapal and Bhograi. Figure 1. Location map of the study area with three blocks i.e. Balasore, Baliapal and Bhograi. Figure 1. Location map of the study area with three blocks i.e. Balasore, Baliapal and Bhograi. state Odisha. As a result of increasing coastal hazards mainly tropical cyclone and other allied coastal hazards,
oceanic processes like transgression and regression and also due to affecting on physical aspects of the envi-
ronment by different types of human activities (deforestation, unscientific agricultural practices, excavators
works) shore line becomes shifted here much more frequently. Being a deltaic low lying coastal stretch this area
is monotonously flat alluvium surface of ground level varying between 2.5 m to 3.5 m above MSL. The study
area is mainly covered by the Holocene alluviums along with the recent Subarnarekha river’s deposition, which
has a natural gradient to the east and south east direction with a high water holding capacity of brackish water
environment. The pH of the soil is varies from 6.5 - 8.0 (pre monsoon) and 6.2 - 8.2 (post monsoon). The mon-
soonal phase is much more significant in this area. The temperature experienced in a range of minimum of 9˚C
in winter to a maximum of 38˚C in summer. 1. Introduction Spatial modeling in respect to the competent chronological illustration of the dynamic coastal setting is an in-
credibly demanding and challenging research. An extensive range of appliance can be made potential with the
exact inference of chronological shoreline shifting rates. The prediction model regarding future shoreline shift-
ing and its positions are very much necessary to efficiently resolve the shifting in the shoreline and to permit us
to maintain the structural and financial losses in the coastal region. So, it is essential to generate a digital data-
base of chronological shoreline position, with the help of standardized remote sensing and GIS technology as
well as to generate a data record that can be used to make possible the present and future analyses of shoreline
shifting. The present study involves an endeavor to appraise an investigative model for predicting the future
shoreline position in order to monitor the shoreline shift along the coast in Balasore district of Orissa, India. 2. Study Area The study area includes an unconsolidated coastal sector of Subarnarekha delta, extends from Rasalpur to Udai-
pur. The study area covers an area of about 142519.76 hectors Stretching between 21˚20'25''N 85˚50'45''E and
21˚39'55''N 87˚12'37''E in Figure 1. From the administrative point of view, these areas cover three blocks along
with seven police station of coastal part of Balasore district. On the other hand this area is also geomorphologi-
cally productive, reach in habitat diversity and environmentally hazards prone under the events of tropical cyc-
lones, tidal waves, climate change and sea level rise. The study area is surrounded in the north by Mayurbanja
district, in the south by Bay of Bengal in the east by West Bengal state and in the west by Bhadrak district of 14 N. K. Barman et al. Figure 1. Location map of the study area with three blocks i.e. Balasore, Baliapal and Bhograi. state Odisha. As a result of increasing coastal hazards mainly tropical cyclone and other allied coastal hazards,
oceanic processes like transgression and regression and also due to affecting on physical aspects of the envi-
ronment by different types of human activities (deforestation, unscientific agricultural practices, excavators
works) shore line becomes shifted here much more frequently. Being a deltaic low lying coastal stretch this area
is monotonously flat alluvium surface of ground level varying between 2.5 m to 3.5 m above MSL. 3.1.1. Data Processing 3.1.1. Data Processing
Eight satellite imageries of the years 1975, 1980, 1990, 1995, 2000, 2005, 2010 and 2013 have been taken into
consideration. Landsat MSS and TM-5 data sets have been acquired from USGS. Landsat MSS image of 1975
has been resampled to 30m to match the spatial resolution of Landsat TM-5 images of 1980, 1990, 1995, 2000,
2005, 2010 and 2013. All the data sets are projected in UTM projection with zone no 45 and WGS 84 datum. A
reference line has been considered as base line and image of 1975, 1980, 1990, 1995, 2000, 2005, 2010 and 2013
have been co-registered using first order polynomial model with base data with 0.5 pixel Root Mean Square Error
(RMSE) accuracy. 1. Introduction Relative humidity is prevailing here in between 90% - 96% at most
of the months. At the time of the low atmospheric pressure during summer and monsoon period wind dominant-
ly blows from the offshore areas. There is no such forest cover but, some brackish environment floral species
like Sesuvium Portolacrustum, Ipomia Bioloba and some herbs like Lantena camera, Akanthesia, Calatropis
gigantia are found in the study area with some planted trees like Casuarina, Eucalyptus and Acacia auriculifor-
mis and also some indigenous floral species like Coconut, Banana, Bamboo and Mango. 3. Modeling Shoreline Shifting 3.1. Data Base Three satellite data were taken into contemplation spanning over 38-year time period (1975-2013). Multi resolu-
tion satellite data such as Landsat MSS and Landsat TM-5 are used in this study because of the unavailability of 15 N. K. Barman et al. same resolution imagery over the chosen period. The details of the satellite imagery, acquisition details and reso-
lutions are given in Table 1. same resolution imagery over the chosen period. The details of the satellite imagery, acquisition details and reso-
lutions are given in Table 1. 3.1.3. EPR Model for Shoreline Prediction The shorelines experience both attrition and accumulation over an extensive range of spatio-temporal scales. The prediction precision of shoreline situation depends on the capturing of the chronological processes and gives
the most excellent information concerning the future shoreline positions. In shoreline analysis research, extra-
polation of a constant rate of change is the most commonly used method to predict the shoreline [14]. Shoreline
shifting rates are recurrently applied to sum up the historical shoreline shifting and their expectations prediction. A number of methods have been used for prediction of shoreline position as a function of time, rate of erosion
and deposition or sea-level rise such as non-linear mathematical models e.g. higher order polynomial, exponen-
tial model, cyclic series models [15]. Among them, the most easy and practical ones are the End Point Rate
(EPR) (Figure 3) and the Linear Regression (LR) models. In the present study, the EPR model has been imple-
mented to predict the future position of shoreline of Balasore coastal tract. The model is based on the assumption that the observed periodical rate of change of shoreline position is the
best estimate for prediction of the future shoreline [8] and no prior knowledge regarding the sediment transport
or wave interference is required because the cumulative effect of all the underlying processes are assumed to be
captured in the position history [15]. The position of the potential shoreline for a given data is investigated using the rate of shoreline shifting
(slope), time space between observed and predicted shoreline and model intercept which can be expressed as (1) 1
2
t
t
t
Y
X
u
β
β
=
+
±
(1) 1
2
t
t
t
Y
X
u
β
β
=
+
± The subscript t is used to index the observations of a sample. The total number of observations, also called
the sample size, will be denoted by n . Thus, for a sample of size n , the subscript t runs from 1 to n . Each
observation comprises an observation on a dependent variable, written as
tY for observation t , and an obser-
vation on a single explanatory variable, or independent variable, written as
t
X . The mathematical structure of
the model which is necessary for model calibration is described in the following manner. 3.1.2. Shoreline Delineation Automatic shoreline delineation is a complex process due to the presence of water saturated zone at the land-
water boundary [12] [13]. In order to delineate the actual shoreline position, two methods have been imple-
mented-image classification and Normalized Vegetation Indexing (NDVI) (Figure 2). The bimodal natures of
histograms of MSS and TM images have been found to be two different peaks for land and water. According to
histogram observation, images have been classified using the ISODATA classification (unsupervised) technique 16
Figure 2. Shoreline delineation by unsupervised classification and normalized vegetation index (NDVI). Figure 2. Shoreline delineation by unsupervised classification and normalized vegetation index (NDVI). 16 16 N. K. Barman et al. Table 1. Details of multi resolution satellite date of acquisition and resolution. Satellite and Sensors
Date of Acquisition
Path/Row
Band Used
Spatial Resolution
LANDSAT MSS
1975/02/12
139/45
Visible and NIR
60 × 60 m
LANDSAT TM
1980/12/15
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
1990/08/19
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
1995/09/13
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
2000/06/19
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
2005/03/21
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
2010/09/15
139/45
Visible and NIR
30 × 30 m
LANDSAT TM
2013/05/21
139/45
Visible and NIR
30 × 30 m Table 1. Details of multi resolution satellite date of acquisition and resolution. and two classes (land, water) have been taken to differentiate the land and water interface. In the vegetation in-
dexing processes, Normalized Difference Vegetation Index (NDVI) has been used to separate the land water
margin. The pixels signifying the shoreline have been converted into vector layer to get the real shoreline posi-
tion. The shoreline (2013), which obtained by the applying of above methods has been matched up to with the
40 ground control points (GCPs). and two classes (land, water) have been taken to differentiate the land and water interface. In the vegetation in-
dexing processes, Normalized Difference Vegetation Index (NDVI) has been used to separate the land water
margin. The pixels signifying the shoreline have been converted into vector layer to get the real shoreline posi-
tion. The shoreline (2013), which obtained by the applying of above methods has been matched up to with the
40 ground control points (GCPs). (2) Intercept of End Point Rate (
)
EPR
β (2) Intercept of End Point Rate (
)
EPR
β PR intercept can be calculated as if only forward shoreline progradation has to be found EPR intercept can be calculated as if only forward shoreline progradation has to be found (
)
(
)
earliest
recent
earliest
recent
EPR
earliest
recent
earliest
earliest
recent
earliest
recent
y
y
y
y
y
x
x
x
x
x
x
β
−
−
=
−
=
−
−
(4) (4) Shoreline position (
)
2
Y
and the elapsed time (
1
2
X
X
− Shoreline position (
)
2
Y
and the elapsed time (
)
1
2
X
X
−
. Since the end point line can extend beyond the most recent point ( )t , [2] can be rewritten to use that position Since the end point line can extend beyond the most recent point ( )t , [2] can be rewritten to use that position Since the end point line can extend beyond the most recent point ( )t , [2] can be rewritten to use that position
(
)
2
Y
and the elapsed time (
)
1
2
X
X
− (
)
2
Y
and the elapsed time (
)
1
2
X
X
− (
)
earliest
recent
EPR
predicted
recent
recent
earliest
recent
ˆ
t
y
y
Y
m
x
x
y
x
x
−
=
=
−
−
(5) (5) Hence, the shorelines extracted from 1975, 1980, 1990, 1995, 2000, 2005, 2010 and 2013 images which were
segmented at 1 km interval and the location of the midpoint of each segments are sampled for the entire 67 km
Balasore shoreline. The UTM coordinates ( x and y ) for each sample point were used to calculate
EPR
m
for
both x and y locations of each point. Initially, the model was calibrated based on 1975, 1980, 1990, 1995,
2000, 2005 and 2010 shoreline sample and the rate of movement (
)
EPR
m
was calculated to predict the shore-
line of 2013. (1) Rate of Shoreline Movement (
)
EPR
m (1) Rate of Shoreline Movement (
)
EPR
m The rate of shoreline movement for a given set of samples,
EPR
m
can be calculated as earliest
recent
EPR
earliest
recent
y
y
m
x
x
−
=
−
(3) earliest
recent
EPR
earliest
recent
y
y
m
x
x
−
=
−
(3) (3) 3.1.4. Position of Predicted Shoreline The EPR model employs the shoreline taken out from the two end points of satellite imageries, the earliest ( )
1Y
and the recent positions (
)
2
Y
. If researchers use Y to indicate predicted shoreline positions, X for time in-
terval/date, β for model intercept and
EPR
m
for the rate of shoreline change, and then the first equation [1]
can be written as EPR
EPR
ˆY
m
X
β
=
+
(2) (2) EPR
EPR
ˆY
m
X
β
=
+ 17 N. K. Barman et al. Figure 3. Techniques of End Point Rate (EPR) calculation by arc distance and time between earliest and most re-
cent shoreline position (After USGS, 2009). Figure 3. Techniques of End Point Rate (EPR) calculation by arc distance and time between earliest and most re-
cent shoreline position (After USGS, 2009). Figure 3. Techniques of End Point Rate (EPR) calculation by arc distance and time between earliest and most re-
cent shoreline position (After USGS, 2009). (2) Intercept of End Point Rate (
)
EPR
β Then the model was again calibrated with 2005 and 2010 shoreline sample points, based on the
reference line, shoreline change rates has been derived and the future shoreline position of the study area was
predicted for both short term (7 years) and long term (17 years). The positional shift in the model (estimated shoreline of 2013) was validated with respect to actual image
(extracted shoreline of 2013). The estimated shoreline was also validated with the 40 Ground Control Points
(GCPs) collected from the field during the satellite overpass time and during high tide. The validation (location
error in model estimated shoreline) was carried out in terms of RMSE [6]. The location errors at each sample
point can be plotted as error vectors, which have the constituents in x and y directions and the length of the
average vector are measured. RMSE gives a measure of accuracy which exhibits, how far, on average; the ob-
served values are from the assumed true value. The equations are listed below as (
)
(
)
2
2
1
model
actual
model
actual
1
RMSE
n
i
n
x
x
y
y
−
=
=
−
+
−
∑
(6) (6) where
model
x
and
model
y
are the model generated and
actual
x
and
actual
y
are the actual x and y coordi- where
model
x
and
model
y
are the model generated and
actual
x
and
actual
y
are the actual x and y coordi- 18 18 N. K. Barman et al. N. K. Barman et al. nates of the shoreline sample points. To minimize the error, an investigational step has been adopted in this
study. Positional shift in each sample points has been calculated by contrasting the actual and estimated shore-
line of 2013. The position of future shoreline prediction was tested by applying the error estimated at each sam-
ple points. nates of the shoreline sample points. To minimize the error, an investigational step has been adopted in this
study. Positional shift in each sample points has been calculated by contrasting the actual and estimated shore-
line of 2013. The position of future shoreline prediction was tested by applying the error estimated at each sam-
ple points. 4. Validation of EPR Model The End Point Rate (EPR) model has been adopted for estimating the position of future shoreline. But prior to
the prediction of future shoreline, the model has been validated with the current circumstances. To calculate the
rate of shoreline shifting, the shoreline position in 2005 and 2010 was applied and based on this; the estimated
shoreline of 2013 was calculated. The estimated shoreline was compared with the actual shoreline demarcated
from satellite imagery of 2013 (Figure 4). The positional error varies from 4.82 m to 212.41 m. It has been
found that model prediction error is very high at the mouth of Subarnarekha river. The overall error for the en-
tire predicted shoreline was found to be 41.18 m (RMSE). 4.1. Long-Term Rates (2013-2030) Long-term rates of shoreline change, in meters per year, were calculated at each transect by finding the slope of
the best-fit line through all shoreline positions from the earliest (1975) to the most recent (1980, 1990, 1995,
2000, 2005, 2010 and 2013) (Figure 5). Long-term rates also were calculated without shorelines from the years
of 1975 to 1980, 1990, 1995, 2000, 2005, 2010 and 2013 to examine the potential impact of including or ex-
cluding these data on the measured rates of change. When calculating linear regression rates, at least three ob-
tainable shoreline survey years were necessary at each transect. The linear regression method of calculating 19
Figure 4. Actual shore line position (2013) and predicted shoreline position (2030)
along 67 km shoreline with 1 km linear space transect. Figure 5. Transect wise distribution of coefficient of determination values as irregularity. Figure 4. Actual shore line position (2013) and predicted shoreline position (2030)
along 67 km shoreline with 1 km linear space transect. 19
Figure 5. Transect wise distribution of coefficient of determination values as irregularity. Figure 5. Transect wise distribution of coefficient of determination values as irregularity. N. K. Barman et al. shoreline change rates assumes a linear trend of change between the earliest and most recent shoreline dates. In
areas where a linear trend does not exist and shoreline positions have not progressed uniformly in one direction
through time, it is expected that the resulting linear fit to the data will be poorer, and the linear regression rate
will have a higher reported uncertainty. The metadata for the long-term transect shape files provide descriptions
of the four attribute fields associated with the linear regression rate calculations. Additional information can be
found in the Massachusetts Shoreline Change Mapping and Analysis Project, 2013 update [16] or Section 7 of
the DSAS user guide [17]. 4.2. Short-Term Rates (2013-2020) 4.2. Short-Term Rates (2013-2020) Short-term rates of change were calculated at each transect for the more recent 38 years of shoreline data (be-
ginning in between 1975-1980 and ending with data from 1990-2013) using the linear regression method (Fig-
ure 3). In addition, short-term end-point rates were calculated at any transect that had only two shorelines
available within this time period. The end-point rate is calculated by taking the difference in shoreline position
between the two dates and dividing that by the duration of time between surveys to report a rate in meters per
year. The end-point rate simply represents the net change between the surveys (Figure 6), annualized to facili-
tate comparisons with long-term linear regression rates. The short-term transect metadata files provide descrip-
tions of the attribute field associated with the end-point rate calculation. 5. Shoreline Shifting over Time 5.1. Shoreline Dynamics during the Period of 1975-2013 5.1. Shoreline Dynamics during the Period of 1975-2013 The 67 km long Balasore shoreline has been delineated from the satellite imageries of different years (1975,
1980, 1990, 1995, 2000, 2005, 2010 and 2013) using the vegetation cover (NDVI) and image classification
technique (Figure 2). The delineate shoreline of 2010 was validated using 40 GCPs and the positional shift shows that classification
method does better marking out of shoreline in contrast to NDVI. The positional shift is also articulated in terms
of Root Mean Square Error (RMSE), which are 2.55 m and 4.62 m for the classification and NDVI techniques
respectively. It has been also noted that the shift in the classification-detected shoreline varied from 1.4 to 3.75 m while in
NDVI the shift varied from 2.0 to 9.31 m. Based on this apprise, classification technique for delineating shore-
line has been adopted for all other applications. It has also been found that the coast line of Balasore district is
experiencing deposition. The position of shoreline at different years (Figure 7) shows the shifting of coast-line
seaward. It also shows the erosional scenario of Balasore coast for the period from 1975 to 2010 indicating sig-
nificant erosion in the northern part of the coast line (left bank portion of Subarnarekha river estuary) and also in
thewestern and eastern parts of Burahbalang and Dugdugi estuary. It may be due to the hydrodynamics com-
plexity of these areas. Whereas the mouth of the Subarnarekha river is gradually prograding towards the sea and
rest part of the study area (southern part) is considerably stable in nature. Figure 6. Transect wise End Point Rate (m/year) along 67 km shore line of Balasore coastal zone. Figure 6. Transect wise End Point Rate (m/year) along 67 km shore line of Balasore coastal zone. 20 20 N. K. Barman et al. Figure 7. Shoreline position at different years from fixed base along same crenulations of shore line. Figure 7. Shoreline position at different years from fixed base along same crenulations of shore line. 5.2. Future Shoreline Prediction Using EPR Model and Error Adjustment Using the EPR model (Figure 3), the shoreline of the study area has been predicted for short term (2017) and
long term (2030). In this prediction, shoreline shifting rate (Figure 8) has been calculated from chronological
observations and disastrous impacts like tsunami and storm have not been taken into consideration. Figure 9
shows the past, present and future positions of shorelines. 5.1. Shoreline Dynamics during the Period of 1975-2013 Using the EPR model (Figure 3), the shoreline of the study area has been predicted for short term (2017) and
long term (2030). In this prediction, shoreline shifting rate (Figure 8) has been calculated from chronological
observations and disastrous impacts like tsunami and storm have not been taken into consideration. Figure 9
shows the past, present and future positions of shorelines. The predicted shoreline indicates that the maximum erosion will take place in the northern part and no signifi-
cant change has occurred in rest of the study area. The same observation was found in the change scenario of
shoreline from 1972 to 2010, in Figure 10 which suggests that the cumulative effect of various processes like
sediment transport system, littoral process, which is captured in the observed rate of change is relevant to predict
the future shoreline. An error adjustment procedure of EPR model has been proposed in this study. The positional shifts in x and
y direction were calculated by comparing the actual and estimated shoreline of 2010. The calculated shift was
applied or adjusted to predict the future shoreline of 2017 and 2030. The shift adjusted predicted shoreline is
shown in Figure 11. The obtained results of the present study suggest that the utilization of remote sensing data in addition with
the GIS technology and statistical technique are very appropriate for extraction of shoreline and its shifting cal-
culation. Simple ISODATA binary classification technique is applicable for delineation of shoreline. The spatial
modeling along with temporal representation of dynamic coastline of Balasore district signifies that the coastline
of some places is suffering erosion and it has shifted inside. The magnitude of erosion is higher in the northern part of the coastline in the left bank area of Subarnarekha
river estuary and also in the estuarine part of river Dugdugi and Burahbalang, which is seen from the imagery of
1972 to 2010 and the model predicted shoreline also depicts the same. The southern part of the shoreline near
Rasalpur, Joydevkasba is considerably stable, indicated by the same model. The validation of EPR model with 21 N. K. Barman et al. Figure 8. Transect wise rate of shore line change (m/year) along 67 km shore line of Balasore
coastal zone. Figure 9. Comparing the trend of shore lines in regards of earliest and forward predicted as 2020 and 2030. image delineated shoreline was found to be useful. 5.1. Shoreline Dynamics during the Period of 1975-2013 The cross validation shows that the model can predict co
sistent guesstimate of the shoreline position with satisfactory accuracy. The trend of the future shoreline shifti
is matched with the present situation. A method for adjustment of model error is suggested in this work whi
can be validated to realize its efficiency. 6. Conclusion
Shorelines are constantly moving in reaction to winds waves tides sediment supply alterations in relative s image delineated shoreline was found to be useful. The cross validation shows that the model can predict con-
sistent guesstimate of the shoreline position with satisfactory accuracy. The trend of the future shoreline shifting
is matched with the present situation. A method for adjustment of model error is suggested in this work which
can be validated to realize its efficiency. 6. Conclusion Shorelines are constantly moving in reaction to winds, waves, tides, sediment supply, alterations in relative sea
level and anthropogenic conducts. Shoreline changes are not invariable through time and recurrently switch
from erosion to accretion and vice versa. Cyclic and non-cyclic processes alter the position of the shoreline over
an assortment of timescales, from each day and seasonal reworking of winds and waves to transform in sea 22 N. K. Barman
Figure 10. Erosional scenario of Balasore shoreline from 1972 to 2010. The red colour depicts erosion, which is
maximum in the north east part of the beach. Figure 11. Areal and linear view of shoreline position regards to recent shore line position (2013). Long term and
short time predicted shore line position with elapsed time 17 years and 7 years respectively. N. K. Barman et al. Figure 10. Erosional scenario of Balasore shoreline from 1972 to 2010. The red colour depicts erosion, which is
maximum in the north east part of the beach. gure 10. Erosional scenario of Balasore shoreline from 1972 to 2010. The red colour depicts erosion, which is
ximum in the north east part of the beach. Figure 10. Erosional scenario of Balasore shoreline from 1972 to 2010. The red colour depicts erosion, w
maximum in the north east part of the beach. Figure 11. Areal and linear view of shoreline position regards to recent shore line position (2013). Long term and
short time predicted shore line position with elapsed time 17 years and 7 years respectively. Figure 11. Areal and linear view of shoreline position regards to recent shore line position (2013). Long term and
short time predicted shore line position with elapsed time 17 years and 7 years respectively. gure 11. Areal and linear view of shoreline position regards to recent shore line position (2013). Long term and
ort time predicted shore line position with elapsed time 17 years and 7 years respectively. 23 23 N. K. Barman et al. level over a century to thousands of years. The shoreline “rate of change” statistic thus reproduces a collective
summary of the processes that distorted the shoreline for the time period analyzed. level over a century to thousands of years. The shoreline “rate of change” statistic thus reproduces a collective
summary of the processes that distorted the shoreline for the time period analyzed. 6. Conclusion Shoreline shifting analysis of the Balasore coast was carried out using chronological shoreline arrangements
spanning 38 years from 1975 to 2013. The Balasore coast was alienated into 5 regions for the purposes of this
present study. Long-term and short-term linear regression rates of shoreline shifting were considered for a total
of 67 transects covering 67 kilometers of shoreline. End-point rates were calculated in locations where there
were inadequate data to use the linear regression method. The uppermost statistically considerable long-term and
short term rates of shifting were experienced at Chandrabali coastal sector (Left bank side of river Subarnarek-
ha). The present work also facilitates us to comprehend the trends in shoreline behavior due to the influence of
natural forces or anthropogenic alterations and how those trends are reproduced in shoreline shifting data. Shoreline shifting trends recognized in this study include (a) unidirectional long term shoreline movement
(Chandrabali), considered by unremitting erosion or accretion, which can take place in both high-energy open
ocean settings or low-energy settings; (b) fluctuating shoreline shifting (Choumukh), which are highly movable
shorelines that have undergone both erosion and accretion on a long-term basis; (c) constrained shorelines in
which shifting of the shoreline indicator is restricted by natural or anthropogenic features (Chandipur); and (d)
anthropogenic shoreline rearrangement such as the infilling of embayments for development purposes (Subar-
napur). The shoreline shifting in the present coast experiences a greater range of variability only due to the frequently
presence of tropical cyclone and allied coastal hazards. The significant fluctuations of short term and long term
shoreline position (positional error 4.82 m - 212.41 m) in a particular transect is also the results of cumulative
effects of different coastal hazards of this geomorphic part. Acknowledgements The authors are awfully indebted to Prof. John Pethick, World Bank Expert on coastal vulnerability, UK, for his
precious comments and suggestions for improvement on the manuscript. We are also thankful to anonymous re-
viewers for their thoughtful suggestions to perk up this manuscript significantly. Finally, we thank our survey
and research team for their generous cooperation in this study. References [1]
Lee, J. and Jurkevich, I. (1990) Coastline Detection and Tracing in SAR Images. IEEE Transactions in Geosciences
and Remote Sensing, 28, 662-668. http://dx.doi.org/10.1109/TGRS.1990.572976 [1]
Lee, J. and Jurkevich, I. (1990) Coastline Detection and Tracing in SAR Images. IEEE Transactions in Geosciences
and Remote Sensing, 28, 662-668. http://dx.doi.org/10.1109/TGRS.1990.572976 [2]
White, K. and El Asmar, H. (1999) Monitoring Changing Position of Coastlines Using Thematic Mapper Imagery, an
Example from the Nile Delta. Geomorphology, 29, 93-105. http://dx.doi.org/10.1016/S0169-555X(99)00008-2 [2]
White, K. and El Asmar, H. (1999) Monitoring Changing Position of Coastlines Using Thematic Mapper Imagery, an
Example from the Nile Delta. Geomorphology, 29, 93-105. http://dx.doi.org/10.1016/S0169-555X(99)00008-2 [3]
Bertacchini, E. and Capra, A. (2010) Map Updating and Coastline Control with Very High Resolution Satellite Images:
Application to Molise and Puglia Coasts (Italy). Italian Journal of Remote Sensing, 42, 103-115. [3]
Bertacchini, E. and Capra, A. (2010) Map Updating and Coastline Control with Very High Resolution Satellite Images:
Application to Molise and Puglia Coasts (Italy). Italian Journal of Remote Sensing, 42, 103-115. [4]
Wright, H. and Pilkey, Jr., (1989) The Effect of Hard Stabilization upon Dry Beach Width. Proceed
Zone ‘89, American Society of Civil Engineers. 676-790. [5]
Basco, D. (1991) Boundary Conditions and Long Term Shoreline Change Rates for the Southern Virgin
tline. Shore and Beach, 59, 8-13. [6]
Paine, J. and Morton, R. (1989) Shoreline and Vegetation Line Movement, Texas Gulf Coast 1974 to 1982. The Uni-
versity of Texas at Austin, Bureau of Economic and Geology and Geological Circular, 89-1. 50. [7]
Siddiqui, M. and Maajid, S. (2004) Monitoring of Geomorphological Changes for Planning Reclamation Work in
Coastal Area of Karachi, Pakistan. Advances in Space Research, 33, 1200-1205. [7]
Siddiqui, M. and Maajid, S. (2004) Monitoring of Geomorphological Changes for Planning Reclamation Work in
Coastal Area of Karachi, Pakistan. Advances in Space Research, 33, 1200-1205. http://dx.doi.org/10.1016/S0273-1177(03)00373-9 8]
Fenster, M., Dolan, R. and Elder, J. (1993) A New Method for Predicting Shoreline Positions from His
Journal of Coastal Research, 9, 147-171. 9]
Dolan, R., Fenster, M. and Holmes, S. (1991) Temporal Analysis of Shoreline Recession and Accretion
Coastal Research, 7, 723-744. [10] Rissanen, J. (1978) Modeling by Shortest Data Description. Automatica, 14, 465-471. http://dx.doi.org/10.1016/0005-1098(78)90005-5 [11] Douglas, B. and Crowell, M. (2000) Long-Term Shoreline Position Prediction and Error Propagation. Journal of 24 24 N. K. Barman et al. Coastal Research, 16, 145-152. Coastal Research, 16, 145-152. S. and Bhattacharya, A. (2009) Shoreline Change Analysis and Its Application to Prediction: A Remote Sensin
tatistics Based Approach. Marine Geology, 257, 11-23. http://dx.doi.org/10.1016/j.margeo.2008.10.006 [13] Ryu, J., Won, J. and Min, K. (2002) Waterline Extraction from Landsat TM Data in a Tidal Flat: A Case Study in Gos-
mo Bay, Korea. Remote Sensing of Environment, 83, 442-456. http://dx.doi.org/10.1016/S0034-4257(02)00059-7 [14] Eliot, J. and Clarke, D. (1989) Temporal and Spatial Bias in the Estimation of Shoreline Rate-of-Change Statistics from
Beach Survey Information. Coastal Management, 17, 129-156. http://dx.doi.org/10.1080/08920758909362081 [14] Eliot, J. and Clarke, D. (1989) Temporal and Spatial Bias in the Estimation of Shoreline Rate-of-Change Statistics from
Beach Survey Information. Coastal Management, 17, 129-156. [15] Li, R., Liu, J. and Felus, Y. (2001) Spatial Modelling and Analysis for Shoreline Change and Coastal Erosion Moni-
toring. Marine Geodesy, 24, 1-12. http://dx.doi.org/10.1080/01490410121502 [16] Thieler, E.R., O’Connell, J.F. and Schupp, C.A. (2001) The Massachusetts Shoreline Change Project—1800s to 1994. USGS Administrative Report to the Massachusetts Office of Coastal Zone Management, Boston, 26 p. [17] Himmelstoss, E.A. (2009) DSAS 4.0—Installation Instructions and User Guide. In: Thieler, E.R., Himmelstoss, E.A.,
Zichichi, J.L. and Ergul, A., Eds., The Digital Shoreline Analysis System (DSAS) Version 4.0—An ArcGIS Extension
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The Limbic Degradation of Aging Brain: A Quantitative Analysis with Diffusion Tensor Imaging
|
The scientific world journal/TheScientificWorldjournal
| 2,014
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cc-by
| 5,127
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Academic Editor: Bernhard Schaller Academic Editor: Bernhard Schaller Copyright © 2014 Hediye Pınar Gunbey et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Introduction. The limbic system primarily responsible for our emotional life and memories is known to undergo degradation with
aging and diffusion tensor imaging (DTI) is capable of revealing the white matter integrity. The aim of this study is to investigate age-
related changes of quantitative diffusivity parameters and fiber characteristics on limbic system in healthy volunteers. Methods. 31
healthy subjects aged 25–70 years were examined at 1,5 TMR. Quantitative fiber tracking was performed of fornix, cingulum, and
the parahippocampal gyrus. The fractional anisotropy (FA) and apparent diffusion coefficient (ADC) measurements of bilateral
hippocampus, amygdala, fornix, cingulum, and parahippocampal gyrus were obtained as related components. Results. The FA
values of left hippocampus, bilateral parahippocampal gyrus, and fornix showed negative correlations with aging. The ADC values
of right amygdala and left cingulum interestingly showed negative relation and the left hippocampus represented positive relation
with age. The cingulum showed no correlation. The significant relative changes per decade of age were found in the cingulum and
parahippocampal gyrus FA measurements. Conclusion. Our approach shows that aging affects hippocampus, parahippocampus,
and fornix significantly but not cingulum. These findings reveal age-related changes of limbic system in normal population that
may contribute to future DTI studies. Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 196513, 7 pages
http://dx.doi.org/10.1155/2014/196513 Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 196513, 7 pages
http://dx.doi.org/10.1155/2014/196513 Hindawi Publishing Corporation
e Scientific World Journal
Volume 2014, Article ID 196513, 7 pages
http://dx.doi.org/10.1155/2014/196513 Hediye PJnar Gunbey,1 Karabekir Ercan,2 AyGe Serap FJndJkoglu,3
H. Taner Bulut,4 Mustafa Karaoglanoglu,2 and Halil Arslan2
1 Radiology Department, Ondokuz Mayıs University, Kurupelit, 55139 Samsun, Turkey
2 Radiology Department, Ataturk Research and Education Hospital, Ankara, Turkey
3 Radiology Department, Medipol University Hospital, Istanbul, Turkey
4Radiology Department, Adıyaman University, Adıyaman, Turkey Correspondence should be addressed to Hediye Pınar Gunbey; hpgunbey@hotmail.com Received 5 February 2014; Revised 2 March 2014; Accepted 3 March 2014; Published 13 April 2014 1. Introduction Age
Number of subjects
Mean age ± SD
Gender FM/M
≥18, <30
5
22.1 ± 4.0
3/2
≥30, <40
5
32.5 ± 3.0
3/2
≥40, <50
7
45.0 ± 2.6
3/4
≥50, <60
7
53.8 ± 2.3
3/4
≥60, <70
7
65.3 ± 3.7
2/5 investigated the integrity of whole components, including the
hippocampus, amygdala, parahippocampal gyrus, cingulum,
and fornix together with diffusion tensor imaging (DTI).i g
DTI data were acquired using a single-shot spin-echo
echo planar image (SE-EPI) sequence. The diffusion sensi-
tizing gradients were applied simultaneously along sixteen
noncollinear directions (𝑏= 1000 s/mm2) as well as an acqui-
sition without diffusion weighting (𝑏= 0). The other acqui-
sition parameters were TR = 8108 ms, TE = 75 ms, NSA =
3, flip angle = 90∘, FOV = 224 mm, matrix
=
256 ×
256 pixels, 2 mm axial slices, and no slice gap. Three aver-
ages were applied for sufficient signal-to-noise ratio (SNR). Eddy current artifacts were minimized by the intrasequence
registration tool utilized in the postprocessing. Fiber tracking
was performed for 3D segmentation of fornix and cingulum
as two major white matter tracts of the limbic system, and
additionally for the parahippocampal gyrus (Figure 1). The
fiber assignment by continuous tracking (FACT) algorithm,
which starts tracking by every single voxel and goes over
all the voxels of the image volume, was used for recon-
struction of these fiber tracts [21]. The fiber tracking was
performed with manually defined region of interest (ROI)
placed according to color-coded maps based on the guide-
lines of Concha et al. [12]. Fiber tracking was terminated
with a fractional anisotropy (FA) threshold of 0.20 and
when the angle between two principal Eigen vectors was
greater than 70∘. Quantitative analysis was obtained from
the statistical evaluation of parameters of pixels occupied
by the reconstructed fibers. The diffusivity parameters FA
and apparent diffusion coefficient (ADC) measurements were
calculated for each selected fiber bundle and also for the
hippocampus, amygdala, and parahippocampal gyrus as the
gray matter components. gf
g
g (
)
DTI is a noninvasive specific neuroimaging technique
that enables measurement of restricted water diffusion in
brain tissue. It is a more sensitive imaging method than
qualitative observation for investigating white matter struc-
tures. It has revealed evidence of microstructural disruption
of brain white matter in healthy adults as they age, even in
regions appearing normal on conventional volume imaging
[14]. 1. Introduction The fornix projects from the hippocampal formation to the
hypothalamus, while the cingulum connects the cingulate
and the parahippocampal gyri to the septal cortex. Both the
gray and white matter components of the limbic system have
been studied using MR imaging in several brain disorders,
such as epilepsy [2–4], dementia [5], and schizophrenia [6]. With normal aging, many volumetric studies reported a
reduction in hippocampal, parahippocampal, and cingulated
volumes [7–9] while others did not find evidence for
age-related volume losses in these structures [10, 11]. Fiber tracking studies focused on limbic connections [12]
and potential changes with aging [13] revealed a new
viewpoint for this interesting structure of the human brain. However, to the best of our knowledge none of these studies The limbic system is a group of interconnected structures that
mediate emotions, learning, and memory. It directly connects
the lower and higher brain functions and influences emo-
tions, the visceral responses to those emotions, motivation,
mood, and sensations of pain and pleasure. It is composed of
a group of interconnected gray and white matter structures
that create a loop in each cerebral hemisphere. Papez in
1937 described the set of connections in the limbic system
that link the hippocampus, mamillary bodies, thalamus,
cingulated, and parahippocampal gyrus [1]. The fornix and
the cingulum, the most visible white matter connections of
this circuit, connect cortical and subcortical brain structures. 2 2 The Scientific World Journal Table 1: Age and gender of the subjects. 2.2. MR Imaging Protocol. All subjects were scanned on a
1.5 TMR scanner (Philips Achieva, The Netherlands). Slew
rate 40 mT/m) with an eight-channel head coil. A standard
conventional MR imaging protocol included axial and sagittal
T2-weighted turbo spin-echo (TSE) (TR/TE = 5000/100 ms,
slice thickness (thk): 5 mm), axial fluid-attenuated inversion
recovery (FLAIR) (TR/TE = 6000/120 ms, IR: 2000 ms, thk:
2 mm) sequence, and a T1-weighted 3D magnetization pre-
pared rapid acquisition gradient-echo sequence (MPRAGE)
(TR = 7.2/TE = 3,2 ms, NSA = 1, FOV = 256 mm, slice thk:
1 mm, gap = 0 mm, flip angle = 8∘, matrix = 256 × 256 pixels)
through 160 slices of the entire brain. 1. Introduction The two principal DTI metrics are fractional anisotropy
(FA), which represents the directionality and mean diffusivity
(MD), and the magnitude of water diffusion [15]. The FA as an
indicator of white matter coherence and axonal organization
may be influenced by myelination, orientation, coherence,
packing density, and structural integrity of neural fiber tracts. Highly myelinated fiber bundles with a common orientation
will have high anisotropy and disruption of the myelin
sheath, such as with aging, and can result in increase in
extracellular water content and mean diffusivity [16]. Axonal
damage has been correlated with decreased FA and increased
MD [17]. The architecture of white matter, which restricts
water movement perpendicular to the fiber axis, is especially
suitable for DTI analysis, as it allows three-dimensional (3-
D) characterization of fiber tracts and comparison of white
matter structures between populations. In cooperation with DTI and tractography, the aim of
this study is to characterize the microstructural effects of
aging on the limbic system interconnected components and
relationship between them in course of time. Investigating
the limbic system including several components in a broad
perspective may be more informative in understanding the
effects of aging on emotions, memory, attention, and social
processing. 2.3. Statistical Analysis. Data were analyzed using statistical
software (SPSS, version 16). The level of significance was set
at 𝑃< 0.05 for all tests. We used the Kolmogorov-Smirnov
test to verify the normal distribution of all variant groups. Kruskal-Wallis test was used to examine alterations of the
parameters of the cases changed by age. The Mann-Whitney
𝑈test was performed after the Bonferroni correction to
determine the difference between groups. Cross-table statis-
tics were used to compare categorical variables (K-square,
Fisher). For determining the statistically significant relation-
ship between parameters Spearman correlation analysis was
performed. 2. Materials and Methods 2.1. Subjects. This retrospective study included thirty-one
subjects with no self-reported history of neurological or
psychiatric disease or brain injury, aged 25–70 years (mean ±
standard deviation: 49.39 ± 14.94, 17 males, 14 females). Subjects were approximately equally distributed across the
age range (Table 1). The subjects were recruited from our
data base. To exclude dementia the elder 14 subjects ≥50
years old who have Mini Mental State Examination (MMSE)
score evaluated by physician were included in the study. The
inclusion criterion for minimum MMSE score was chosen 27
points with similar other aging studies [18–20]. 2.4. Results. 3D reconstructions were performed of the
fiber structures of the fornix and the cingulum and the The Scientific World Journal 3 (a)
(b)
(c)
Figure 1: Fiber tracking was performed for 3D segmentation of the two major white matter tracts of the limbic system, the fornix (b), the
cingulum (c), and additionally the parahippocampal gyrus (a). (a) (c) (b) (b) (a) (c) Figure 1: Fiber tracking was performed for 3D segmentation of the two major white matter tracts of the limbic system, the fornix (b), the
cingulum (c), and additionally the parahippocampal gyrus (a). (a)
(b)
(c)
Figure 2: Examples of ROI placements on the hippocampus (a), the amygdala (b), and the parahippocampus (c) obtained from a 33-year-old
man. (a) (b) (c) (a) (b) (c) Figure 2: Examples of ROI placements on the hippocampus (a), the amygdala (b), and the parahippocamp
man. Figure 2: Examples of ROI placements on the hippocampus (a), the amygdala (b), and the parahippocampus (c) obtained from a 33-year-old
man. parahippocampal gyrus for all 31 subjects. Examples of ROI
placements on the hippocampus, the parahippocampus, and
the amygdala obtained from a 33-year-old man are shown
in Figure 2. Diffusivity parameters and fiber characteristics
for the cingulum, the hippocampus, the parahippocampus,
and the amygdala of the left and the right hemispheres were
calculated and evaluated separately. Two-sided paired 𝑡-tests
revealed no differences between the two hemispheres, neither
for diffusivity parameters nor for fiber characteristics. The
mean fiber and nonfiber FA and ADC values for the left and
the right hemispheres were used for statistical evaluation. Correlation related with sex showed a weak negative
relationship between the left hippocampal, the right parahip-
pocampal, and the left cingulum ADC values for men in
comparison with women (𝑃< 0.05). 2. Materials and Methods The ADC values of
hippocampus, parahippocampus, and fornix were found to
be higher in women (Figure 3). In group analysis according to decades, both sides
parahippocampal and cingular FA and ADC values were
statistically different between groups (𝑃< 0.05) that FA
decreased and ADC increased with age (Figure 4). In hippocampal and parahippocampal measurements, FA
values showed negative correlation with ADC values (𝑟=
−0.386, 𝑃
=
0.032) and 13 a positive correlation with
each side. The correlation for the amygdala FA values was
significant at 𝑃< 0.001, 𝑟= 0.662 and not significant for
14 the ADC values. According to the results of Spearman correlation analysis,
a negative correlation was found between age and left hip-
pocampal FA measurements (𝑟= −0.375, 𝑃= 0.038). The
right parahippocampal gyrus FA values showed a moderate
negative correlation with age (𝑟= −0.486, 𝑃= 0.006). There were also weak negative correlations between the left
parahippocampal gyrus, the fornix FA values, and age (𝑃<
0.05). Interestingly, the ADC values of the right amygdala
and the left cingulum showed a decrease with aging while
the ADC values of the left hippocampus increased with age
(𝑟= 0.387, 𝑃= 0.031), as expected. No other statistical
relationship was found between the other parameters and
age. The fiber FA and ADC parameters of the right parahip-
pocampus (𝑟= −0.448, 𝑃= 0.011), fornix (𝑟= −0.605,
𝑃
≤
0.001), and right cingulum (𝑟
=
−0.521, 𝑃
=
0.003) demonstrated a negative correlation. The left cingular
FA negatively correlated with the right cingular ADC. FA
values of parahippocampal fibers for each side showed no
correlation, while cingular fibers correlated significantly (𝑟=
0.373, 𝑃= 0.039). The Scientific World Journal 4 1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
R-Hip-FA
R-Hip-ADC
L-Hip-FA
L-Hip-ADC
R-Phip-FA
R-Phip-ADC
L-Phip-FA
L-Phip-ADC
R-PhipFb-ADC
L-PhipFb-FA
L-PhipFb-ADC
R-Amy-FA
R-Amy-ADC
L-Amy-FA
L-Amy-ADC
For-FA
For-ADC
R-Cin-FA
R-Cin-ADC
L-Cin-FA
L-Cin-ADC
Female
Male
R-PhipFb-FA
Figure 3: The ADC values of hippocampus, parahippocampus, and
fornix were found to be higher in women. a difference from the data reported in previous studies [12,
13, 35]. Concha et al. [12] and Stadlbauer et al. [13] found
higher FA values while Sullivan et al. [35] found lower FA
values in the fornix and cingulum. The ADC values were
in agreement with their results. The observed discrepancies
between studies may be due to clinical differences in the
populations studied as well as methodological differences in
anatomical definitions of these two bundles. 3. Discussion DTI is rapidly becoming a widely available imaging technique
with a myriad of applications. The possibility of discerning
the orientation of white matter bundles and the ability to
reconstruct their 3D structure in vivo has opened the door
to selective studies of fiber tracts both in the healthy and
diseased human brain. In healthy older subjects, anisotropy
reduces and ADC increases in white matter with increasing
age [22]. Pathological white matter axonal degeneration
or demyelination shows reduced anisotropy and increased
mean diffusivity, as, for example, in multiple sclerosis [23],
amyotrophic lateral sclerosis [24], and Alzheimer’s disease
[25]. These changes may reflect demyelination, axonal loss,
or edema [26, 27]. Generally, reduced FA in white matter
suggests less coherence of fibers (e.g., crossing fibers) or
less dense fibers. In contrast, increased ADC may suggest
immaturity or degeneration in this region. it
Looking at the limbic system as a whole, the fornix
includes fibers originating in the hippocampus and termi-
nating in the mammillary bodies and septal nuclei, while
the cingulated and parahippocampal gyri and septal cor-
tex are connected via the cingulum. Left hippocampal FA
values showed a decrease with normal aging as reported
in volumetric studies [38, 39]. The left hippocampus is a
participant in the recall of the spatial memories. When
studying the hippocampal lesions in rats, Eichenbaum [40]
and his team found that the left hippocampus is critical
for effectively combining the “what,” “when,” and “where”
qualities to compose the retrieved memory. This makes the
left hippocampus a key component in the retrieval of spatial
memory. However, Spreng and Mar [41] found that the left
hippocampus is, in fact, a general concentrated region for
binding together bits and pieces of memory composed not
only by the hippocampus, but also by other areas of the brain
to be recalled at a later time. This FA decrement in this region
may be useful to explain the impairment of the memory
functions in elderly people. Several DTI studies, including neonates [28], children
[29], and/or adolescents [30], have evaluated white matter
changes of the brain in the normal aging process. Diffusion-
tensor tractography has been used to delineate the fornix
and/or cingulum in healthy volunteers [31–33], as well as
patients with epilepsy and schizophrenia [12, 34]. Concha
et al. [12] reported the diffusion characteristics of the fornix
and cingulum with CSF suppression in healthy, young adults. Recently, Stadlbauer et al. 2. Materials and Methods Concha et al. interpolated data with eight averages in a scan time of more
than 9 min. The interpolation may have reduced the SNR and
the long scan time may have increased motion artifacts. Both
the SNR and motion artifacts have a strong influence on the
quality of DTI data and the outcome of fiber tracking [36]. i
In the current study, an age-related modest decline of FA
in the fornix but not in the cingulate bundles was observed,
as reported in previous studies [13, 35]. Zhang et al. also
observed no FA changes of the cingulum in elderly controls
while there were reductions especially in the left posterior
cingulate region in mild cognitive impairment patients [37]. Furthermore, significant relative changes per decade of age
were found in cingulum and parahippocampal gyrus FA
measurements. In a retrospective view, the difference was
thought to originate from the results of elderly patients in the
fifth group. Figure 3: The ADC values of hippocampus, parahippocampus, and
fornix were found to be higher in women. 3. Discussion [13] evaluated age-related changes
of the fornix and cingulum with fiber tracking. Sullivan
et al. [35] also mentioned them in a study of lateral and
interhemispheric white matter fiber tracking in normal aging. However, to the best of our knowledge, no study has reported
the diffusion characteristics of the whole components of
the limbic system, including the hippocampus, amygdala,
parahippocampal gyrus, cingulum, and fornix with DTI. In this study the amygdala involved in signaling the cortex
of motivationally significant stimuli such as those related to
reward and fear in addition to social functions showed no
significant differences with aging.h The cingulum that has autonomic functions regulating
heart rate, blood pressure and cognitive, and attentional pro-
cessing showed no difference, but the right parahippocampus
that is connected by the cingulum and plays a role in the
formation of spatial memory showed significantly lower FA
values with aging. Yogarajah et al. found a parahippocampal
FA decrease associated with poorer performance on material
specific memory measures in temporal lobe epilepsy patients
[42]. However, to the best of our knowledge these are the first
results of parahippocampal gyrus tractography in normal
aging. In the present study, we investigated the age-related
changes of quantitative diffusivity parameters and fiber char-
acteristics of the limbic system in healthy volunteers. Some
of our FA values for the fornix and the cingulum showed The Scientific World Journal 5 1.00
20
30
40
50
60
70
80
R-Cin-FA
Age
Group means
5.00
4.00
3.00
2.00
1.00
0.90
0.80
0.70
0.60
0.50
0.40
(a)
20
30
40
50
60
70
80
Age
5.00
4.00
3.00
2.00
1.00
R-Phip-FA
Group means
0.500
0.450
0.400
0.350
0.300
0.250
(b)
20
30
40
50
60
70
80
Age
5.00
4.00
3.00
2.00
1.00
R-Cin-ADC
Group means
0.95
0.90
0.85
0.80
0.75
(c)
20
30
40
50
60
70
80
Age
5.00
4.00
3.00
2.00
1.00
1.300
1.200
1.100
1.000
R-Phip-ADC
Group means
0.900
0.800
0.700
(d)
Figure 4: In group analysis, both sides parahippocampal and cingular FA and ADC values were statistically different between groups (𝑃<
0.05). 3. Discussion 1.00
20
30
40
50
60
70
80
R-Cin-FA
A
0.90
0.80
0.70
0.60
0.50
0.40 20
30
40
50
60
70
80
A
R-Phip-FA
0.500
0.450
0.400
0.350
0.300
0.250 20
30
40
50
60
70
80
A
R-Cin-ADC
0.95
0.90
0.85
0.80
0.75 20
30
40
50
60
70
80
Age
1.300
1.200
1.100
1.000
R-Phip-ADC
0.900
0.800
0.700 R-Cin-ADC (d) (c) Figure 4: In group analysis, both sides parahippocampal and cingular FA and ADC values were statistically different between groups (𝑃<
0.05). 6 The Scientific World Journal 6 G¨unbey, Dr. Fındıkoglu, and Dr. Bulut performed the statis-
tical analysis, the discussion, and the abstract submission. Deterministic tractography methods require threshold
values of FA to define each tract, although this practice may
influence the resulting FA values calculated from the tract. For this study, the threshold was set to voxels with FA values
greater than 0.20, to minimize inclusion of voxels with a
high degree of partial volume contamination, and to avoid
spurious tracts. The effect of choosing a threshold is unlikely
to substantially affect the age-related FA and ADC changes
with respect to the relative differences between structures. FA values derived from tractography are, in general, lower
than FA values derived from ROI analysis due partly to
this floor value, but primarily to variability of FA along the
tracts themselves. ROI analysis defines structures on two-
dimensional maps and generally includes the areas of higher
FA in a particular tract. Tractography, however, includes a
much larger portion of the tract, including the lower FA
values near the ends, and obtains values that are lower overall. This variability in FA measures means that values obtained
using identical methods can be compared, while it is difficult
to compare absolute FA measures from different procedures. Conflict of Interests [13] A. Stadlbauer, E. Salomonowitz, G. Strunk, T. Hammen, and O. Ganslandt, “Quantitative diffusion tensor fiber tracking of age-
related changes in the limbic system,” European Radiology, vol. 18, no. 1, pp. 130–137, 2008. The authors declare that there is no competing financial
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Neuroradiology, vol. 23, no. 9, pp. 1445–1456, 2002. [30] V. J. Schmithorst, M. Wilke, B. J. Dardzinski, and S. K. Holland,
“Correlation of white matter diffusivity and anisotropy with age
during childhood and adolescence: a cross-sectional diffusion-
tensor MR imaging study,” Radiology, vol. 222, no. 1, pp. 212–218,
2002. [31] M. Catani, R. J. Howard, S. Pajevic, and D. K. Jones, “Virtual
in vivo interactive dissection of white matter fasciculi in the
human brain,” NeuroImage, vol. 17, no. 1, pp. 77–94, 2002.
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Regional Public Investment and Regional Economic Growth : Study Using Spatial Econometrics Panel Data Approach
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KASSAOUI Radouane, (MSc en économie des territoires.) Université Mohammed V / Faculté des Sciences Juridiques, Economique et Sociales - Agdal Université Mohammed V / Faculté des Sciences Juridiques, Economique et Sociales - Agdal Déclaration de divulgation : L’auteur n’a pas connaissance de quelconque financement qui
pourrait affecter l’objectivité de cette étude. Conflit d’intérêts : L’auteur ne signale aucun conflit d’intérêts. Conflit d’intérêts : L’auteur ne signale aucun conflit d’intérêts. Pour citer cet article : BOUAMOUD .S & KASSAOUI .R (2023) « Investissement Public Régional et
Croissance Economique Régional : Etude par l’Approche Econométrie Spatiale en Données de Panel »,
African Scientific Journal « Volume 03, Numéro 16 » pp: 354 – 377. Pour citer cet article : BOUAMOUD .S & KASSAOUI .R (2023) « Investissement Public Régional et
Croissance Economique Régional : Etude par l’Approche Econométrie Spatiale en Données de Panel »,
African Scientific Journal « Volume 03, Numéro 16 » pp: 354 – 377. Date de soumission : Janvier 2023
Date de publication : Février 2023 Date de soumission : Janvier 2023
Date de publication : Février 2023 www.africanscientificjournal.com Page a
DOI : 10.5281/zenodo.7705186
Copyright © 2023 – ASJ DOI : 10.5281/zenodo.7705186
Copyright © 2023 – ASJ African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023
Investissement Public Régional et Croissance Economique Régionale :
Etude par l’Approche Econométrie Spatiale en Données de Panel
Regional Public Investment and Regional Economic Growth : Study Using
Spatial Econometrics Panel Data Approach
Auteur 1 : BOUAMOUD Safae,
Auteur 2 : KASSAOUI Radouane,
BOUAMOUD Safae, (MSc en économie des territoires.)
Université Mohammed V / Faculté des Sciences Juridiques, Economique et Sociales - Agdal
KASSAOUI Radouane, (MSc en économie des territoires.)
Université Mohammed V / Faculté des Sciences Juridiques, Economique et Sociales - Agdal
Déclaration de divulgation : L’auteur n’a pas connaissance de quelconque financement qui
pourrait affecter l’objectivité de cette étude. Conflit d’intérêts : L’auteur ne signale aucun conflit d’intérêts. Pour citer cet article : BOUAMOUD .S & KASSAOUI .R (2023) « Investissement Public Régional et
Croissance Economique Régional : Etude par l’Approche Econométrie Spatiale en Données de Panel »,
African Scientific Journal « Volume 03, Numéro 16 » pp: 354 – 377. African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 Investissement Public Régional et Croissance Economique Régionale :
Etude par l’Approche Econométrie Spatiale en Données de Panel
Regional Public Investment and Regional Economic Growth : Study Using
Spatial Econometrics Panel Data Approach Auteur 1 : BOUAMOUD Safae,
Auteur 2 : KASSAOUI Radouane, BOUAMOUD Safae, (MSc en économie des territoires.) KASSAOUI Radouane, (MSc en économie des territoires.) www.africanscientificjournal.com Page a African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 Résumé L’investissement public régional a pris son ampleur après l’adoption de la régionalisation
avancée, est devenu un sujet important de recherche et un instrument des politiques publiques
pour reconfigurer à nouveau la scène politique et économique marocaine. Cet article analyse
l’impact de l’investissement public sur la croissance économique suivant une logique régionale
et spatiale. L’objectif de cette étude est de déceler les effets spatiaux dans l’estimation du
QMLE de Lee and Yu, ceci nous a conduit à traiter la relation entre l’investissement public
régional et la croissance économique régionale sur la période 2015-2019, en se basant
effectivement sur les principes de l’économétrie spatiale. Les résultats obtenus montrent un
effet marginal voire faible de l'investissement public régional, tandis que l’emploi régional
demeure un facteur déterminant de la croissance économique régionale. Quant aux effets de
diffusion spatiale, le produit intérieur brut régional reste un meilleur instrument pour lutter
contre les inégalités spatiales. Mots clés : L’investissement public régional, la croissance économique régionale, la
régionalisation avancée, l’économétrie spatiale en données de panel, les effets de diffusion
spatiale. www.africanscientificjournal.com Introduction Au cours des dernières décennies, le Maroc s'est engagé dans une série de réforme visant
principalement à développer son climat économique et sociale. L'objectif derrière ces réformes
est de faire du territoire marocain un territoire résilient, fort et attractif des investisseurs et
capitaux privés. A cet effet, l'investissement public constitue une priorité des pouvoirs publics
et un instrument essentiel pour conduire une croissance économique inclusive dont le rôle des
régions demeure très important. Actuellement, le débat sur le rôle de l'investissement public et sa gestion dans le but de mener
les grands projets d'infrastructures (Autoroutes, Ports, Aéroports...) s'impose, et surtout après
l'adoption d'un nouveau mode de gouvernance territoriale, qui est la régionalisation avancée. L’objectif ultime de ce chantier est d’assurer un meilleur développement du Maroc et lutter
contre toutes formes de disparité à savoir territoriales ou sociales, tout en optimisant la
répartition de l’effort de l’État sur l’ensemble des territoires (CCR, 2010). Ce principe conduit tout chercheur à se poser la question sur la répartition territoriale et
régionale de l'investissement public ainsi que son impact réel dans la contribution à la création
de richesse sur l'ensemble des régions marocaines. Le conseil économique, social et
environnemental (CESE, 2015) a souligné la répartition inéquitable et non équilibrée des
investissements publics sur les régions marocaines, malgré l'augmentation considérable et
notable des investissements dans le budget général de l’Etat. Le problème persiste toujours
puisque chaque loi de finances ne prend pas en considération ce constat et elle n'essaie pas de
corriger ces antécédents en la matière. Nul ne peut nier la relation entre l’investissement public et la croissance économique,
puisqu’elle a fait l’objet d’une multitude de travaux théoriques et empiriques. Sur le plan
théorique, de nombreux modèles menés par la nouvelle théorie de la croissance endogène
soutiennent l’existence des externalités positives pour l’investissement public et leur rôle
important dans l’épanouissement de l’économie. Du côté des modèles de la croissance exogène,
la contribution de l’investissement public, y compris les politiques publiques restent faibles. Dans ce cas, la croissance économique est déterminée principalement par des facteurs exogènes,
notamment ceux de l’épargne et de la croissance démographique. En ce sens, les études empiriques se multiplient afin de mesurer l’impact des investissements
publics et ses effets. Néanmoins les résultats obtenus ne présentent pas une certaine unanimité. Abstract Regional public investment gained momentum after the adoption of advanced regionalization,
becoming an important research topic and instrument of public policies to reshape the
Moroccan political and economic scene. This article analyzes the impact of public investment
on economic growth following a regional and spatial logic. The objective of this study is to
detect spatial effects in the estimation of Lee and Yu's QMLE by examining the relationship
between regional public investment and regional economic growth over the period 2015-2019
based on the principles of spatial econometrics. The results show a marginal or weak effect of
regional public investment, while regional employment remains a determining factor of
regional economic growth. As for the effects of spatial diffusion, regional GDP remains a better
instrument to combat against spatial inequalities. Keywords : The regional public investment, regional economic growth advanced
regionalization, spatial econometrics in panel data, spatial spillover effects. www.africanscientificjournal.com www.africanscientificjournal.com www.africanscientificjournal.com Page 354 Page 354 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 www.africanscientificjournal.com Introduction www.africanscientificjournal.com www.africanscientificjournal.com Page 356 Page 356 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 Introduction Si le Maroc continue à mener en force ses programmes de développement territorial,
économique et social, la question se pose quant à leur contribution effective dans la croissance Page 355 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 économique dans le but de réduire les inégalités et les disparités régionales, qui reste toujours
une difficulté à surmonter pour les pouvoirs publics. A cet égard, et dans le cadre d'une
coopération régionale, l’interdépendance spatiale des régions peut être un facteur important et
déterminant de l'épanouissement économique régionale, de sorte que les composantes qui
constituent ces régions peuvent interagir entre eux afin d'amortir les effets desdits inégalités. Donc, la diffusion des effets spatiaux entre régions constitue quant à elle, un sujet qui nécessite
d’être analysé et examiné. De ce fait, l’objectif principal de cet article est de déterminer les effets des investissements
publics régionaux sur la croissance économique régionale ainsi que de déceler leur impact
spatial sur la décision économique des 12 régions marocaines. Notre étude prend toute son
importance dans le contexte où il n’existe que peu d’études consacrées à ce sujet et surtout avec
la manière dont notre recherche est conduite. Dans le cadre de cette étude et la modélisation que nous menons à l’aide des techniques de
l’économétrie spatiale, nous allons répondre à la problématique suivante : Quels sont les effets et les répercussions spatiaux de l’investissement public régional sur
la croissance économique des 12 régions marocaines sous l’ère de la régionalisation
avancée ? Pour ce faire, le présent article propose dans sa première partie, une revue de la littérature
économique détaillée sur la relation entre l'investissement public et la croissance économique,
en présentant quelques fondements théoriques et des analyses empiriques réalisées sur le sujet. En deuxième partie, nous menons une analyse descriptive et statistique de l’échantillon retenue
avant de passer aux tests réservés à la détection de l’autocorrélation spatiale. La dernière partie sera consacrée à l’exercice d’une modélisation économétrique spatiale pour
déterminer la relation entre l'investissement public et la croissance économique au niveau
régional à l'aide de la méthode d’estimation de Lee and Yu réservée aux modèles spatiaux en
données de panel. Par la suite nous entamons une discussion des résultats avant de conclure le
travail. www.africanscientificjournal.com 1.1.
Revue de la littérature théorique : www.africanscientificjournal.com www.africanscientificjournal.com Page 357 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 L'étude de Barro (1990) inclut l'investissement public dans son analyse et met l'accent sur son
rôle productif dans la croissance économique. Les dépenses publiques productives qu'il assimile
au capital d'infrastructures publiques, jouent un rôle moteur dans le processus de croissance. La
complémentarité entre capital privé et capital public est attribuée à l'impact positif de ce dernier
sur la productivité des facteurs privés (Barro et Sala I-Martin, 1995). En d'autres termes, le modèle de croissance endogène de Barro (1990) met l'accent sur les
externalités positives générées par les services publics, par exemple les dépenses
d'infrastructures publiques. Les externalités se produisent lorsque les services publics affectent
la productivité du secteur privé et que le secteur privé n'en supporte pas directement les coûts. Ces services publics productifs (infrastructures routières, électricité, eau, etc.) fournis aux
entreprises privées nationales et étrangères réduisent les coûts de production et augmentent la
production. L'existence d'externalités justifie que les États non seulement orientent les acteurs
privés vers des activités porteuses de croissance, mais aussi qu'ils développent des
infrastructures qui augmentent leur productivité. Les auteurs pensent donc que l'investissement public peut entrer dans la fonction productive
des entreprises, rendant les intrants privés plus productifs et stimulants plutôt que d'évincer
l'investissement privé. En supposant que le capital public affecte directement la productivité
des facteurs privés, l'auteur définit une forme fonctionnelle Cobb-Douglas qui inclut le capital
public. Le rendement d'échelle est supposé diminuer du point de vue du secteur privé, mais
reste constant au niveau de l'ensemble de facteurs, de sorte qu'un stock croissant de capital
public stimule un rendement sur l'apport privé. En supposant que les dépenses publiques d'investissement sont financées par les impôts (les
impôts jouent un rôle positif dans la croissance en stimulant le secteur privé), Barro se demande
quel est le niveau optimal de dépenses qui maximise les rendements. Il soutient que le niveau
des dépenses publiques d'investissement qui optimise les taux de croissance est de sorte que le
ratio des dépenses au PIB est le ratio du revenu national revenant au gouvernement lorsque les
services publics sont dans un cadre concurrentiel. A la suite de Barro (1990), Alogoskoufis et Kalyvitis (1996) décrivent un modèle de croissance
endogène avec des solutions qui mettent l'accent sur le rôle du capital public dans la croissance
et l'investissement. 1.1.
Revue de la littérature théorique : La réflexion sur les sources de la croissance économique, notamment sur le rôle du capital dans
la croissance, se développe dans deux directions. Pour Solow (1956), l'investissement public
n'est pas pertinent pour l'analyse de la croissance. A la fin des années 1980, il y a eu une
remontée de la théorie de la croissance endogène, qui considère l'investissement public comme
un facteur de croissance (Barro, 1990). Solow (1956) fonde son analyse sur la flexibilité de la technologie de production. Cette analyse
est basée sur une fonction de production à deux composantes : le capital et le travail. La
croissance suppose le développement du capital par l'investissement et la croissance de la
population active. Mais l'une des conclusions les plus importantes du modèle de Solow est que
la croissance est limitée d'une part par le taux de croissance de la population, une quantité
exogène, et le capital dont la croissance est limitée par la loi des rendements décroissants,
d’autre part. Le modèle de Solow tient également compte des avancées technologiques susceptibles
d'améliorer la productivité des facteurs. Cependant, il s'agit d'un progrès technologique exogène
qui ne peut être expliqué par un modèle. Chez Solow, la croissance est stable si les ratios de
capital sont variables et adaptables. En effet, les variations du ratio K/Y s'adaptent aux
variations des prix relatifs des facteurs de production et remettent automatiquement l'économie
sur le chemin d'une croissance équilibrée. Cela suppose que le fonctionnement du marché ne
soit pas entravé par la rigidité ou l'intervention de l'État. En d'autres termes, le modèle néoclassique de Solow est basé sur l'hypothèse des rendements
décroissants. Il a proposé que le taux de croissance à long terme d'une économie soit déterminé
de manière exogène par le taux de progrès technologique et la démographie. Dans cette
perspective théorique, la politique d'investissement public (infrastructures) ne modifie le taux
de croissance de l'économie que lors du passage à l'équilibre de long terme (état stationnaire). La reprise de l'activité économique due aux politiques expansionnistes n'est que temporaire,
quel que soit l'équilibre de long terme de l'économie. En conséquence, le modèle de Solow ignore non seulement l'interaction entre la croissance
économique et la politique publique, mais aussi l'accumulation de capital et le progrès
technique. Selon Krugman (1987), l'hypothèse des rendements d'échelle décroissants
(l'hypothèse essentielle de la conception néoclassique) est une simplification mathématique de
la modélisation. www.africanscientificjournal.com 1.1.
Revue de la littérature théorique : Ce qui rend ce modèle intéressant, c'est que les mécanismes de transmission
des effets positifs des infrastructures sur la croissance ont rarement fait l'objet de modèles
macroéconomiques. Les auteurs considèrent l'infrastructure comme un bien public dont les Page 358 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 externalités procurent des avantages gratuits aux entreprises et constituent la base de la
croissance autonome à long terme de l'économie. Selon ces auteurs, la politique d'investissement public se prépare selon trois étapes. À un
moment donné, les pouvoirs publics détermineront le ratio du capital public au PIB ; ce modèle
permet de mettre en évidence la dynamique de croissance de long terme tirée par
l'investissement privé. Celle-ci est elle-même une fonction croissante du niveau de capital
public. Les autorités peuvent alors fixer des objectifs pour le taux de croissance du capital public
ou le ratio de l'investissement public au PIB. Dans ce cas, le taux de croissance du capital public
détermine le taux de croissance d'équilibre de l'économie. Après cela, L'investissement privé
augmentera régulièrement car le rapport entre le capital public et le capital privé restera stable
à long terme et la productivité marginale s'améliorera à mesure que l'investissement public
augmentera. A long terme l’investissement privé égalise l’investissement public. www.africanscientificjournal.com 1.2.
Revue de la littérature empirique : Les auteurs supposent
que seul le capital-actions d'une entreprise publique et la présence d'infrastructures routières et
autoroutières peuvent influer directement sur la productivité d'une entreprise privée. Une méthode des moindres carrés généralisés a été utilisée pour estimer un modèle d'équilibre
général qui permet d'identifier le processus d'accumulation du capital (via les déclarations
fiscales). Les auteurs soulignent que la contribution du capital public à la croissance est positive
et significative, mais relativement faible, soit un taux de croissance du PIB de 22%. Dans une autre étude examinant le ralentissement de la croissance de la productivité en France,
Coe et Moghadam (1993) ont étendu la fonction de production à l'ouverture commerciale et aux
stocks de capital de R&D considérés comme des moteurs de la croissance. Estimés via le
processus VAR sur ce modèle à cinq variables (capital public, capital privé, emploi, ouverture
commerciale et stocks de R&D), les auteurs ont trouvé que les élasticités associées aux
variables de capital public et privé étaient de 53%. Ils concluent également que l'ouverture de
l'économie et des camps de R&D ont un effet positif sur la croissance. Les estimations en niveau ont donné des valeurs élevées d’élasticité de la production au capital
public. Selon les économistes, cela indique la présence éventuelle de biais dû à la non-
stationnarité des séries considérées, ce qui conduit à des phénomènes de régression fallacieuse. Pour cette raison, les auteurs ont d'abord proposé des tests de non-stationnarité ou de
cointégration. En plus de ces études empiriques sur des pays individuels, les chercheurs ont utilisé des
techniques de données de panel économétriques pour examiner l'impact de l'investissement
public sur des groupes de pays. Par exemple, Evans et Karras (1994a) considèrent un panel de sept pays de l'OCDE et estiment
la fonction de production avec des différences de premier ordre. L'élasticité estimée de la
production au capital public est relativement élevée à 18% et est significative après exclusion
des effets temporels et individuels. Cependant, les auteurs montrent que ce résultat n'est pas
robuste à l'introduction de certains effets d'une part et à sa spécification (fixe ou aléatoire)
d'autre part. Les auteurs concluent que les pays qui bénéficient d'une forte croissance du PIB
sont ceux dont les capitaux privés et publics sont structurellement abondants. 1.2.
Revue de la littérature empirique : Il existe une abondante littérature empirique sur la relation entre l'investissement public et la
croissance économique. Cependant, les résultats sont légèrement différents selon la définition
statistique du capital, la spécification de la fonction de production et la méthode d'estimation. Dans l'ordre chronologique, nous présentons une série d'études empiriques qui abordent la
question du rôle de l'investissement public dans la croissance économique. Aschauer (1989) utilise des données américaines d'après-guerre pour estimer la fonction Cobb-
Douglas étendue au capital public. En supposant des rendements constants à tous les niveaux
de facteurs, l’auteurs trouve une élasticité de la production au capital de 39%. Il en conclut qu'il existe une forte contribution productive du capital public. Ainsi, selon les
auteurs, la baisse de l'investissement public observée depuis le milieu des années 1970 est la
principale explication de la baisse de la productivité observée sur la même période. En utilisant les mêmes données qu'Aschauer et en suivant la nature des rendements d'échelle,
Munnell (1990a) a trouvé une élasticité entre 31% et 39%. Les auteurs confirment les résultats
d'Aschauer, mais vont plus loin en montrant que la baisse traditionnellement admise de la
productivité du facteur privé est due à la disparition du stock de capital public dans la fonction
de production. Après prise en compte des externalités liées au capital public, les auteurs
montrent que la baisse de la productivité moyenne du travail varie de 1,4% à 0,3% de 1969 à
1987. Finn (1993), notant la forte élasticité d'Aschauer et Munnell, va au-delà de l'approche
d'équilibre partiel des deux auteurs. Il propose une estimation de la contribution productive du www.africanscientificjournal.com Page 359 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 capital public dans le cadre d'un modèle structurel d'équilibre général stochastique dynamique. Il soutient que les estimations élevées d'Aschauer sont dues à la définition du capital public, qui
comprend des éléments improductifs (tels que les musées et les prisons). Les auteurs supposent
que seul le capital-actions d'une entreprise publique et la présence d'infrastructures routières et
autoroutières peuvent influer directement sur la productivité d'une entreprise privée. capital public dans le cadre d'un modèle structurel d'équilibre général stochastique dynamique. Il soutient que les estimations élevées d'Aschauer sont dues à la définition du capital public, qui
comprend des éléments improductifs (tels que les musées et les prisons). www.africanscientificjournal.com 1.2.
Revue de la littérature empirique : Les mêmes résultats ont été apportés par Iaich et Bourouane (2020), sur la période 1991-2017. A l’aide d’un modèle ARDL, les auteurs confirment l’impact non significatif de
l’investissement public à long terme. Pour ce qui est du court terme, l’effet change de signe
d’une année à l’autre. De plus, Azeroual et Oumansour (2019) ont étudié la relation entre l'investissement public et la
croissance économique au Maroc de 1990 à 2015. En utilisant le modèle ARDL, les auteurs
concluent à un effet positif significatif de l'investissement public sur la croissance économique
à court terme. Cependant, à long terme, l'impact de l'investissement public est non signifiant. Les mêmes résultats ont été apportés par Iaich et Bourouane (2020), sur la période 1991-2017. De plus, Azeroual et Oumansour (2019) ont étudié la relation entre l'investissement public et la
croissance économique au Maroc de 1990 à 2015. En utilisant le modèle ARDL, les auteurs
concluent à un effet positif significatif de l'investissement public sur la croissance économique
à court terme. Cependant, à long terme, l'impact de l'investissement public est non signifiant. Les mêmes résultats ont été apportés par Iaich et Bourouane (2020), sur la période 1991-2017. A l’aide d’un modèle ARDL, les auteurs confirment l’impact non significatif de
l’investissement public à long terme. Pour ce qui est du court terme, l’effet change de signe
d’une année à l’autre. Les mêmes résultats ont été apportés par Iaich et Bourouane (2020), sur la période 1991-2017. A l’aide d’un modèle ARDL, les auteurs confirment l’impact non significatif de
l’investissement public à long terme. Pour ce qui est du court terme, l’effet change de signe
d’une année à l’autre. Dans les études empiriques, le résultat récurrent est que l’investissement public a été reconnu,
dans la dernière décennie, comme une variable stratégique des analyses de la production et de
la croissance. Cependant, ces études laissent subsister une grande part d’incertitude. On a pu
vérifier que les estimations portant sur des séries chronologiques nationales, conduisent
fréquemment à des valeurs élevées de l’élasticité de la production par rapport au capital public,
ce qui rejoignent les premiers résultats d’Aschauer. Celles fondées sur des données
différenciées et la recherche de cointégration paraissent mieux étayées. Mais là aussi, les
conclusions sont toujours nuancées selon les pays, les régions et les périodes. 1.2.
Revue de la littérature empirique : Dessus et Herrera (1996), travaillant sur un panel de 28 pays, trouvent une élasticité
significative de 0,26% pour les données en niveau et de 0,18% pour les données en différence www.africanscientificjournal.com www.africanscientificjournal.com www.africanscientificjournal.com Page 360 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 première. Ce résultat doit tenir compte de la possibilité d'un biais d'estimation, car il est difficile
d'établir l'identité des structures de production dans un grand nombre de pays. première. Ce résultat doit tenir compte de la possibilité d'un biais d'estimation, car il est difficile
d'établir l'identité des structures de production dans un grand nombre de pays. Si la plupart des études empiriques sur la relation entre les investissements publics et la
croissance économique est consacrée aux pays développés, il en existe aussi sur le Maroc en
particulier. Si la plupart des études empiriques sur la relation entre les investissements publics et la
croissance économique est consacrée aux pays développés, il en existe aussi sur le Maroc en
particulier. En utilisant un modèle ARDL appliqué à des séries chronologiques annuelles de 1980 à 2014,
Obad et Jamal (2016) ont montré que les dépenses publiques ont un impact négatif sur la
croissance économique au Maroc. Elalaoui et Hefnaoui (2018) ont examiné l'impact des dépenses publiques sur la croissance
économique sur la période 1975-2016. Les résultats de cette étude montrent que les dépenses
publiques ont un impact négatif sur la croissance économique. Cependant, lorsqu'on distingue
investissement public et consommation publique, on constate que cette dernière a un impact
positif sur la croissance économique, alors que l'impact de l'investissement public est négatif. Elalaoui et Hefnaoui (2018) ont examiné l'impact des dépenses publiques sur la croissance
économique sur la période 1975-2016. Les résultats de cette étude montrent que les dépenses
publiques ont un impact négatif sur la croissance économique. Cependant, lorsqu'on distingue
investissement public et consommation publique, on constate que cette dernière a un impact
positif sur la croissance économique, alors que l'impact de l'investissement public est négatif. De plus, Azeroual et Oumansour (2019) ont étudié la relation entre l'investissement public et la
croissance économique au Maroc de 1990 à 2015. En utilisant le modèle ARDL, les auteurs
concluent à un effet positif significatif de l'investissement public sur la croissance économique
à court terme. Cependant, à long terme, l'impact de l'investissement public est non signifiant. www.africanscientificjournal.com 1.2.
Revue de la littérature empirique : Dans le cas du Maroc, toutes les études tendent à montrer qu’il existe un impact significatif sur
la croissance économique exercée par les investissements publics. Ces résultats restent eux
aussi mitigés selon la période étudiée et la technique à laquelle le sujet est modélisé. Cependant,
ces études présentent à nos yeux une limite ; Les données macroéconomiques à l’échelle www.africanscientificjournal.com Page 361 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 nationale ne permettent pas à décrire la situation économique d’une manière désagrégée, c’est-
à-dire, elles ne prennent pas les caractéristiques régionales du thème étudié. nationale ne permettent pas à décrire la situation économique d’une manière désagrégée, c’est-
à-dire, elles ne prennent pas les caractéristiques régionales du thème étudié. La prise en compte de cette limite pourrait contribuer à une meilleure connaissance des effets
des investissements publics régionaux sur la croissance économique régionale au Maroc. A cet
effet, la partie suivante sera consacrée à un exercice de modélisation économétrique pour
déterminer l'effet des investissements publics régionaux sur la croissance économique des
régions tout en considérant les interactions spatiales des variables retenues. 2. Modélisation de l’impact spatial de l’investissement public régional sur la
croissance des régions marocaines 2. Modélisation de l’impact spatial de l’investissement public régional sur la
croissance des régions marocaines En vue d’estimer l’effet de l’investissement public régional sur la croissance économique
régionale et son impact spatial sur les 12 régions marocaines, il est tout d’abord nécessaire de
présenter l’échantillon et les données retenues pour cet exercice, ainsi que la description
statistique de ces derniers. www.africanscientificjournal.com 2.1.
Présentation et analyse de l'échantillon : y
L’échantillon est constitué des données de 12 régions couvrant la période allant de 2015 à 2019,
le choix de cette période est expliqué d’une part par des raison économétriques, étant donné que
le Maroc est constitué de 12 régions donc le nombre d’échantillon n’est pas assez satisfaisant
pour qu’il soit soumis aux régressions économétriques. Et d’autre part les cinq années retenues
sont considérés comme étant cruciales pour juger les premiers résultats après l’adoption du
grand chantier souverain de la régionalisation avancée comme mode de gouvernance. Finalement notre échantillon est constitué de 60 observations, 𝑖 = 1. . . , 12 et t = 1..., 5. La
période entre 2019 et 2021 n’est pas prise en considération compte tenu de la crise sanitaire liée
au COVID-19 et ses répercussions sur l’économie marocaine. Les données servant comme variables sont recueillies principalement à partir des annuaires
statistiques élaborées par le Haut-Commissariat au Plan et le Ministère de l’Economie et de
Finance. Les annuaires contiennent des informations sur la production nationale,
l’investissement public, l’éducation, la démographie et le marché de travail. Le tableau ci-dessous donne un aperçu des données. L’activité économique est captée par le
produit intérieur brut (PIB). L’investissement en capital indique le montant de la croissance de
l’investissement public en capital (par exemple, bâtiments, équipements et autres stocks). Les
dépenses d’investissement public en éducation sont les dépenses totales d’éducation, avec
soustraction des dépenses de fonctionnement dans le secteur d’éducation. La population active www.africanscientificjournal.com www.africanscientificjournal.com Page 362 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 est mesurée par la part de la population active dans la population totale et enfin l’indice de Gini
pour mesurer l’effet des inégalités territoriales. Tableau
N°1 :
Présentation
des
variables www.africanscientificjournal.com Source : Réalisé par les auteurs www.africanscientificjournal.com Page 364 Page 364 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023
Figure N°1 : L’évolution de l’indice de Gini entre 2015 et 2019 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023
Figure N°1 : L’évolution de l’indice de Gini entre 2015 et 2019 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
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Vol : 3 Numéro 16 Février Figure N°1 : L’évolution de l’indice de Gini entre 2015 et 2019 Source : Réalisé par les auteurs, logiciel STATA Source : Réalisé par les auteurs, logiciel STATA Une analyse de corrélation doit être effectué entre les variables explicatives, cela permet de
repérer des corrélations éventuelles entre les variables exogènes afin d’identifier et éviter la
multi colinéarité entre les variables. Source : Réalisé par les auteurs Source : Réalisé par les auteurs Avant de passer à l’étape du choix du modèle d’économétrie spatiale adéquat à la problématique
étudiée, il s’avère important d’analyser statistiquement les données et explorer les données en
main, pour ce faire le logiciel STATA et le logiciel SPSS peuvent fournir des informations
facilitant l’analyse des variables. Le tableau ci-dessous affiche les statistiques descriptives des variables : www.africanscientificjournal.com www.africanscientificjournal.com Page 363 Page 363 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 Tableau N°2 : Les statistiques descriptives des variables
Source : Réalisé par les auteurs, logiciel STATA Tableau N°2 : Les statistiques descriptives des variables Tableau N°2 : Les statistiques descriptives des variables Source : Réalisé par les auteurs, logiciel STATA Source : Réalisé par les auteurs, logiciel STATA On remarque que la valeur moyenne du PIB se situe à 88586 MDH entre les régions marocaines. On remarque que la valeur moyenne du PIB se situe à 88586 MDH entre les régions marocaines. On peut aussi remarquer l’étendue du PIB qui indique que la création de la richesse connaît des
grandes disparités spatiales. L’investissement public régional enregistre une valeur minimale
de 576 MDH et une valeur maximale de 39731 MDH dans l’ensemble des régions de
l’échantillon, les dépenses d’investissement en éducation enregistrent en moyenne 224 MDHS
dans les 12 régions étudiées et on constate qu’il existe une grande dispersion entre la valeur
minimale des dépenses d’éducation (1,96 MDHS) et la valeur maximale qui est proche de
570,34 MDHS. Ce constat confirme l’idée que les disparités régionales persistent au sein du
territoire marocain. D’ailleurs l’indice de Gini résume et synthétise ce constat, ce dernier affiche
une valeur moyenne de 0,44 ce qui signifie que la croissance économique n’a pas profité à tous. Le graphique montre l’évolution de cet indice dans l’ensemble des régions marocaines entre la
période 2015-2019. www.africanscientificjournal.com Tableau N°3 : la matrice de corrélation entre les variables explicatives Tableau N°3 : la matrice de corrélation entre les variables explicatives www.africanscientificjournal.com Page 3
Tableau N°3 : la matrice de corrélation entre les variables explicatives
Source : Réalisé par les auteurs, logiciel SPSS Tableau N 3 : la matrice de corrélation entre les variables explicatives Source : Réalisé par les auteurs, logiciel SPSS www.africanscientificjournal.com Page 365 Page 365 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 La matrice de corrélation ci-dessus montre qu’il existe une faible corrélation significative entre
la majorité des variables, ce qui signifie que l’introduction de ces quatre variables comme
variables exogènes n’entrainera pas de problème de multi colinéarité. Le test de corrélation de Pearson peut lui aussi s’effectuer entre la variable dépendante et les
variables indépendantes pour déterminer la force et la direction de la relation entre ces deux
types de variables. La matrice ci-après montre qu’il existe une forte corrélation positive et significative et donc une
très forte relation entre la variable dépendante (PIB) et la variable indépendante (Inv), ce qui
entrainera une augmentation du PIB si l’Inv augmente, et vice versa. La corrélation reste
significative et positive mais modéré entre le PIB et Inv-Edu ainsi que pour le PIB et Popact. Pour la variable Gini, on constate que la force de la relation est faible. www.africanscientificjournal.com www.africanscientificjournal.com 2.2.
Analyse spatiale et présentation du modèle spatial Dans cette section, nous prenons à rappeler les modèles de panel spatial : le modèle spatial
autorégressif (SAR), le modèle d’erreur spatial (SEM) et le modèle spatial de Durbin (SDM). Le modèle spatial autorégressif examine principalement si la variable dépendante a des
phénomènes de diffusion dans une région. Comme point de départ, il faut considérer
l’estimation d’un modèle autorégressif spatial simple de la forme suivante :
𝑌= 𝜌𝑊𝑌+ 𝑋𝛽+ 𝜇+ 𝜂+ 𝜀 www.africanscientificjournal.com www.africanscientificjournal.com Page 366 Page 366 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 Où Y est la variable dépendante, X est la variable explicative exogène de la matrice 𝑛∗𝑘, 𝜌 est
le coefficient d’autocorrélation spatiale, qui montre les effets des retombées des régions
voisines sur la région en tant que telle, W est une matrice spatiale pondérée d’ordre n*n,
généralement, on utilise une matrice de contiguïté, 𝜇 est le vecteur de l’effet individuel, 𝜂 est le
vecteur de l’effet du temps, et 𝜀 est un terme d’erreur aléatoire. Le paramètre 𝛽 reflète l’effet
de la variable indépendante X sur la variable dépendante. Le modèle d’erreur spatiale peut être modélisé comme : 𝑌= 𝑋𝛽+ 𝑢 𝑌= 𝑋𝛽+ 𝑢 Source : Codifier la structure de voisinage, INSEE Source : Codifier la structure de voisinage, INSEE Avec 𝑢= 𝜆𝑊𝑢+ 𝜇+ 𝜂+ 𝜀 Où 𝜀 est le vecteur d’erreur aléatoire de la distribution normale. 𝜆 est le coefficient autorégressif
spatial des résidus de régression, qui mesure l’effet du choc de la variable dépendante répandu
des régions voisines à la région en tant que 𝑢 reflète l’erreur spatialement corrélé. Où 𝜀 est le vecteur d’erreur aléatoire de la distribution normale. 𝜆 est le coefficient autorégressif
spatial des résidus de régression, qui mesure l’effet du choc de la variable dépendante répandu
des régions voisines à la région en tant que 𝑢 reflète l’erreur spatialement corrélé. Le modèle spatial de Durbin ne diffère pas des autres présentés ci-dessus, il prend la forme
suivante : 𝑌= 𝜌𝑊𝑌+ 𝑋𝛽+ 𝜃𝑊𝑋+ 𝜇+ 𝜂+ 𝜀 𝑌= 𝜌𝑊𝑌+ 𝑋𝛽+ 𝜃𝑊𝑋+ 𝜇+ 𝜂+ 𝜀 Avec 𝜃 qui représente les interactions spatiales exogènes. Les autres variables et paramètres du
modèle SEM et SDM sont les mêmes que dans le modèle SAR. Avec 𝜃 qui représente les interactions spatiales exogènes. Les autres variables et paramètres du
modèle SEM et SDM sont les mêmes que dans le modèle SAR. Comme les éléments d’effet spatial sont inclus dans les modèles de panel spatial, le modèle
économique classique est complété par les effets spatiaux de la variable dépendante pour
obtenir des estimations plus précises. En outre, les coefficients du modèle de panel spatial sont généralement mesurés par l’estimation
du maximum de vraisemblance (EMV) (Elhorst 2003 ; LeSage, Pace 2009). Cependant notre
travail consiste à utiliser la méthode d’estimation de Lee et Yu (2010) pour estimer l’effet fixe
du modèle afin d’éviter une estimation incohérente du paramètre de variance étant donné que
l’intervalle de temps est court. Avant de procéder aux estimations, la première étape consiste à choisir le type de voisinage, ce
dernier impactera les résultats et par conséquent les décisions prises lors du test
d’autocorrélation spatiale. Il existe notamment deux types de voisinage, à savoir la contiguïté et la distance. Le choix de
voisinage dépend aux données utilisées. Nos données portent sur des zones géographiques, donc
la contiguïté reste le meilleur choix en matière de voisinage car cette notion repose sur celle de
frontière partagée. www.africanscientificjournal.com www.africanscientificjournal.com Page 367 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 2.2.1. La matrice de contiguïté : Cette sous-section introduit les différentes notions de contiguïté. La contiguïté au sens de Rook
stipule que les voisins possèdent au moins un segment de frontière commune, cela correspond
aux déplacements de la "Tour" du jeu d’échecs. Figure N°2 : la contiguïté Rook Figure N 2 : la contiguïté Rook Source : Codifier la structure de voisinage, INSEE Figure N°3 : la contiguïté Queen Figure N°3 : la contiguïté Queen S
C difi
l
t
t
d
i i
INSEE S
C difi
l
t
t
d
i i
INSEE Source : Codifier la structure de voisinage, INSEE Pour que deux zones soient voisines au sens de la contiguïté Queen, il suffit qu’elles partagent
un point de frontière commune. Cela correspond aux déplacements de la "Reine" du jeu
d’échecs. 2.2.2. Tests d’autocorrélation spatiale et diagramme de Moran La démarche de l’estimation nécessite tout d’abord, la détection d’une éventuelle
autocorrélation spatiale à l’aide de l’indice de Moran, ce dernier aide à déterminer la structure
spatiale des variables. Pour ce faire le logiciel Geoda peut servir à calculer les indices de Moran
pour chaque variable ainsi que de tracer les diagrammes pour conclure le type d’autocorrélation
spatiale. Le tableau présente les résultats du test d’autocorrélation spatiale pour la matrice de
contiguïté de type « Queen ». Ce type de matrice est fréquemment utilisé lors du traitement des
données spatiales. www.africanscientificjournal.com Page 368
Tableau N°5 : Les résultats du test de l’autocorrélation spatiale
Var
Indice de Moran Espèrance Variance
Zscore Le modèle SEM : ln 𝑃𝐼𝐵= 𝛼1 ln 𝐼𝑛𝑣+ 𝛼2 ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝛼3 ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝛼3𝐺𝑖𝑛𝑖+ 𝑢
𝑢= 𝜆𝑊𝑢+ 𝜇+ 𝜂+ 𝜀 ln 𝑃𝐼𝐵= 𝛼1 ln 𝐼𝑛𝑣+ 𝛼2 ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝛼3 ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝛼3𝐺𝑖𝑛𝑖+ 𝑢
𝑢= 𝜆𝑊𝑢+ 𝜇+ 𝜂+ 𝜀 Tableau N°5 : Les résultats du test de l’autocorrélation spatiale www.africanscientificjournal.com Page 368 Page 368 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 PIB
0,262
-0,09
0,03
2,00
Inv
0,236
-0,09
0,03
1,85
InvEdu
0,669
-0,09
0,03
4,30
Popact
0,223
-0,09
0,03
1,78
Gini
0,323
-0,09
0,03
2,34
Source : Calculé par les auteurs logiciel Geoda Source : Calculé par les auteurs, logiciel Geoda Les résultats confirment l’existence d’une autocorrélation spatiale au niveau de la majorité des
variables, notamment pour le produit intérieur brut de chaque région, les dépenses
d’investissement en matière d’éducation et l’indice de GINI, et ce après la vérification des seuils
de significativité du test statistique de Moran. Donc on conclut qu’il existe une interdépendance
spatiale entre les régions. D’ailleurs les diagrammes de Moran proposent une répartition des
régions au sein des quatre quadrants qui mérite d’être examiné. Figure
N°4 :
La
synthèse
des
résultats
des
diagrammes
de
Moran résultats
des
diagrammes
de
Moran des www.africanscientificjournal.com Page 369 Page 369 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 Vol : 3, Numéro 16 , Février 2023 Source : Elaboré par les auteurs, logiciel Geoda www.africanscientificjournal.com Page 370 Page 370 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 3. Estimation du modèle spatial et discussion des résultats Les résultats du test d’autocorrélation spatiale confirment une interdépendance spatiale des
régions. Dans ce cas, le passage à l’étape de la spécification du modèle spatiale est nécessaire
afin de révéler l’effet des variables indépendantes et notamment l’investissement public
régional sur la croissance économique, ainsi de déceler leur impact spatial. A cet effet, notre étude revisite le modèle de production Cobb-Douglas avec matrice spatiale
pour décrire l’impact de chaque variable exogène de manière complète. La production
économique régionale (Y) est modélisée en fonction de l’investissement public régional (K),
de la main d’œuvre (L) et l’investissement public en matière d’éducation à l’aide d’une fonction
Cobb-Douglas. La fonction de production Cobb-Douglas s’écrit comme suit : 𝑌= 𝐾𝛼1𝐿𝛼2𝐴𝛼3 Avec A est conçu comme un facteur exogène. Donc cette variable peut être influencée par la
politique économique, le climat économique ou même l’environnement… Avec A est conçu comme un facteur exogène. Donc cette variable peut être influencée par la
politique économique, le climat économique ou même l’environnement… La fonction devient comme suit après l’introduction du logarithme : ln 𝑌= 𝛼1 ln 𝐾+ 𝛼2 ln 𝐿+ 𝛼3 ln 𝐴+ 𝜀 ln 𝑌= 𝛼1 ln 𝐾+ 𝛼2 ln 𝐿+ 𝛼3 ln 𝐴+ 𝜀 Et comme on est dans une logique et une étude qui porte sur des données spatiales, et après
l’introduction des variables, les trois modèles spatiaux deviendraient idéalement de la forme
suivante : Le modèle SAR : ln 𝑃𝐼𝐵= 𝜌𝑊ln 𝑃𝐼𝐵+ 𝛼1 ln 𝐼𝑛𝑣+ 𝛼2 ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝛼3 ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝛼3𝐺𝑖𝑛𝑖+ 𝜀
Le modèle SEM : ln 𝑃𝐼𝐵= 𝜌𝑊ln 𝑃𝐼𝐵+ 𝛼1 ln 𝐼𝑛𝑣+ 𝛼2 ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝛼3 ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝛼3𝐺𝑖𝑛𝑖+ 𝜀
Le modèle SEM : www.africanscientificjournal.com www.africanscientificjournal.com Le modèle SDM : Tandis que, l’estimation du modèle par la
méthode des Moindres Carrés ordinaires confirme la relation positive entre l’investissement
public régional et le niveau économique régional et ceci avec une significativité au seuil de 1%. Les dépenses d’investissement en éducation et la part de population active affichent des
coefficients positifs et significatifs au seuil de 5% dans la majorité des modèles sauf dans le
modèle d’erreur spatial. Quant à la variable Gini, nous notons que les effets générés par cette
variable ne sont pas significatifs. Les dépenses d’investissement en éducation et la part de population active affichent des
coefficients positifs et significatifs au seuil de 5% dans la majorité des modèles sauf dans le
modèle d’erreur spatial. Quant à la variable Gini, nous notons que les effets générés par cette
variable ne sont pas significatifs. En appliquant l’approche ELHORST, les résultats sur le critère d’Akaike nous ramènent à
retenir le modèle spatial de Durbin, car il présente un meilleur pouvoir explicatif du sujet. A cet
effet, nous procédons à une interprétation économique de l’ensemble des coefficients de ce
modèle. La plupart des coefficients du modèle retenu sont significativement non nulles au seuil de 5%,
à l’exception du coefficient de l’investissement public en infrastructures, cela peut s’expliquer
par le manque d’impact spatial exercé par cette variable, car même si elle arrive à expliquer la
croissance économique régionale au Maroc, mais elle n’arrive pas à contribuer directement à la
générer. La croissance économique régionale est créée par d’autres facteurs régionaux, cette
conclusion nous conduit à penser aux investissements privés et le rôle des centres régionaux
d’investissement à stimuler et dynamiser ce volet. L’effet spatial des dépenses d’investissement
en éducation demeure limité sur le produit intérieur brut régional (0,02), alors que la grande
part contribution dans la création de richesse dans l’ensemble des régions marocaines dépend
de la part de population active (0,47). Quant à l’interaction spatiale exogène, les variables dont les coefficients sont significatifs ont
tendance à diminuer la décision économique dans les régions voisines. Donc, les retombées
spatiales sont négatives et qu’en conséquence chaque région doit compter sur ces propres
ressources sans avoir recours aux celles des régions proches. Le modèle SDM : ln 𝑃𝐼𝐵= 𝜌𝑊ln 𝑃𝐼𝐵+ 𝛼1 ln 𝐼𝑛𝑣+ 𝛼2 ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝛼3 ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝛼3𝐺𝑖𝑛𝑖+ 𝜃1𝑊ln 𝐼𝑛𝑣
+ 𝜃2𝑊ln 𝑃𝑜𝑝𝑎𝑐𝑡+ 𝜃3𝑊ln 𝐼𝑛𝑣𝐸𝑑𝑢+ 𝜃3𝑊𝐺𝑖𝑛𝑖+ 𝜀 Avec PIB est le produit intérieur brute régional, Inv représente l’investissement public en
infrastructure, InvEdu sont les dépenses d’investissement public en matière d’éducation et
Popact est la population active par région. Et afin d’identifier l’impact de la disparité régionale,
le modèle est soutenu par la variable GINI pour capter les effets des inégalités territoriales et
leur impact spatial sur les régions. Comme déjà cité, nous allons procéder à estimer les trois types de modèles spatiaux par la
méthode d’estimation de Lee and Yu (2010). Cette méthode est souvent utilisée lorsque Page 371 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 l’intervalle de temps ou T est petit, dans le but d’obtenir des estimations cohérentes. Et selon
l’approche ELHORST, le modèle spatial possédant le meilleur pouvoir explicatif est celui qui
affiche la plus faible valeur d’Akaike parmi les modèles. Tableau N°6 : Les modèles estimés (a-spatial, spatiaux)
Source : Calculé par les auteurs, logiciel STATA Tableau N°6 : Les modèles estimés (a-spatial, spatiaux) Source : Calculé par les auteurs, logiciel STATA Page 372 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 Les résultats d’estimation des modèles fournissent des coefficients de détermination R²
supérieur à 0,50 ou 50%, cela confirme que les variables retenues arrivent à expliquer plus de
50% la variable dépendante. Les estimations SAR, SDM et SEM affichent le même niveau
d’impact de l’investissement public régional sur la croissance économique régionale, il est
faible, positif mais non significatif au seuil de 10%. Tandis que, l’estimation du modèle par la
méthode des Moindres Carrés ordinaires confirme la relation positive entre l’investissement
public régional et le niveau économique régional et ceci avec une significativité au seuil de 1%. Les résultats d’estimation des modèles fournissent des coefficients de détermination R²
supérieur à 0,50 ou 50%, cela confirme que les variables retenues arrivent à expliquer plus de
50% la variable dépendante. Les estimations SAR, SDM et SEM affichent le même niveau
d’impact de l’investissement public régional sur la croissance économique régionale, il est
faible, positif mais non significatif au seuil de 10%. www.africanscientificjournal.com Le modèle SDM : Pour ce qui est de l’interaction spatiale endogène, le coefficient « Rho » renvoie à la dépendance
spatiale entre la variable endogène de chaque région, celui-ci est positif et significatif, ce qui
signifie que la décision économique dans une région dépend fortement de celle des régions de
voisinage. Ce résultat rejoint le principe et l’idée de l’interrégional marocain et la coopération www.africanscientificjournal.com www.africanscientificjournal.com Page 373 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 Vol : 3, Numéro 16 , Février 2023
interrégionale marocaine. En effet, les territoires peuvent surmonter leur obstacle au
développement économique lorsque les décideurs régionaux unissent leurs forces. Ainsi, une
région peut se développer grâce à la coordination voire la coopération avec d’autres régions. L’analyse des effets des investissements publics sur les économies des régions marocaines nous
a permis de déduire, d’abord, que les dépenses en matière d'éducation et l’investissement en
capital public ont un impact positif mais très faible sur la croissance économique régionale
compte tenu de leur retombée spatiale. Le problème derrière ce constat est le mal arbitrage des
dépenses d’investissement public, la concentration des grands projets dans le triangle (Tanger-
Rabat- Casa) ainsi que leur orientation vers les secteurs non-créatifs de richesses. D’ailleurs,
les résultats des interactions spatiales montrent que le recours vers les principes de la
régionalisation avancé et la coopération interrégionale ont bénéficié aux régions de se
collaborer entre elles dans l’objectif de réaliser leur développement économique. interrégionale marocaine. En effet, les territoires peuvent surmonter leur obstacle au
développement économique lorsque les décideurs régionaux unissent leurs forces. Ainsi, une
région peut se développer grâce à la coordination voire la coopération avec d’autres régions. L’analyse des effets des investissements publics sur les économies des régions marocaines nous
a permis de déduire, d’abord, que les dépenses en matière d'éducation et l’investissement en
capital public ont un impact positif mais très faible sur la croissance économique régionale
compte tenu de leur retombée spatiale. Le problème derrière ce constat est le mal arbitrage des
dépenses d’investissement public, la concentration des grands projets dans le triangle (Tanger-
Rabat- Casa) ainsi que leur orientation vers les secteurs non-créatifs de richesses. D’ailleurs,
les résultats des interactions spatiales montrent que le recours vers les principes de la
régionalisation avancé et la coopération interrégionale ont bénéficié aux régions de se
collaborer entre elles dans l’objectif de réaliser leur développement économique. www.africanscientificjournal.com Le modèle SDM : L’analyse des effets des investissements publics sur les économies des régions marocaines nous
a permis de déduire, d’abord, que les dépenses en matière d'éducation et l’investissement en
capital public ont un impact positif mais très faible sur la croissance économique régionale
compte tenu de leur retombée spatiale. Le problème derrière ce constat est le mal arbitrage des
dépenses d’investissement public, la concentration des grands projets dans le triangle (Tanger-
Rabat- Casa) ainsi que leur orientation vers les secteurs non-créatifs de richesses. D’ailleurs,
les résultats des interactions spatiales montrent que le recours vers les principes de la
régionalisation avancé et la coopération interrégionale ont bénéficié aux régions de se
collaborer entre elles dans l’objectif de réaliser leur développement économique. www.africanscientificjournal.com Page 374 Page 374 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 www.africanscientificjournal.com BIBLIOGRAPHIE Alogoskoufis, G. & Kalyvitis, S. (1996). Public Investment and Endogenous Growth in a Small
Open Economy. Social Science Research Network. Alogoskoufis, G. & Kalyvitis, S. (1996). Public Investment and Endogenous Growth in a Small
Open Economy. Social Science Research Network. Aschauer, D. A. (1989). Does public capital crowd out private capital ? Journal of Monetary
Economics, 24(2), 171-188. Aschauer, D. A. (1989). Does public capital crowd out private capital ? Journal of Monetary
Economics, 24(2), 171-188. AZEROUAL M. et OUMANSOUR N. (2019). Investissements publics et croissance
économique au Maroc : une évaluation par l’approche ARDL Bound Testing. Les cahiers du
plan. Numéro spécial. Volume 2, pp : 66-79. AZEROUAL M. et OUMANSOUR N. (2019). Investissements publics et croissance
économique au Maroc : une évaluation par l’approche ARDL Bound Testing. Les cahiers du
plan. Numéro spécial. Volume 2, pp : 66-79. Barro, R. J. (1990). Government Spending in a Simple Model of Endogeneous Growth. Journal
of Political Economy, 98(5, Part 2), S103-S125. Barro, R. J. (1990). Government Spending in a Simple Model of Endogeneous Growth. Journal
of Political Economy, 98(5, Part 2), S103-S125. Barro, R. J & Sala-I-Martin, X. (1995). Technological diffusion, convergence and growth. Research Papers in Economics. Coe, D. T. & Moghadam, R. A. (1993b). Capital and Trade as Engines of Growth in France :
An Application of Johansen’s Cointegration Methodology. Staff papers, 40(3), 542. Coe, D. T. & Moghadam, R. A. (1993b). Capital and Trade as Engines of Growth in France :
An Application of Johansen’s Cointegration Methodology. Staff papers, 40(3), 542. Commission consultative de la régionalisation (2010). Rapport sur la régionalisation avancée
soumis à la haute attention de sa Majesté le roi Mohammed VI 2010. Livre I, Royaume du
Maroc. Commission consultative de la régionalisation (2010). Rapport sur la régionalisation avancée
soumis à la haute attention de sa Majesté le roi Mohammed VI 2010. Livre I, Royaume du
Maroc. Commission consultative de la régionalisation (2010). Rapport sur la régionalisation avancée
soumis à la haute attention de sa Majesté le roi Mohammed VI, 2010. Livre II Rapports
thématique, Royaume du Maroc. Commission consultative de la régionalisation (2010). Rapport sur la régionalisation avancée
soumis à la haute attention de sa Majesté le roi Mohammed VI, 2010. Livre II Rapports
thématique, Royaume du Maroc. Commission consultative de la régionalisation (2010). Rapport sur la régionalisation avancée
soumis à la haute attention de sa Majesté le roi Mohammed VI, 2010. Conclusion Les résultats des estimations nous ont conduit à confirmer l’existence d’une relation positive
entre les investissements public régionaux et la croissance économique régionale. Ceci dit,
l’investissement public régional reste incapable de contribuer à la création de la richesse
économique régionale, vu sa faiblesse en termes de diffusion des effets spatiales. L'impact positif de l'investissement public sur l’activité économique est une preuve empirique
spécifique des nouvelles théories de la croissance (théories de la croissance endogène, avec
Barro 1990 ; Barro et Sala-I-Martin, 1995). Les dépenses publiques régionales en
investissement sont censées avoir un effet positif et un impact spatial sur l’économie régionale. En marge de ces résultats, nous pensons que l'État a désormais la lourde responsabilité de
promouvoir l'investissement public ainsi que de l’orienter vers les secteurs productifs, cela dans
la mesure où il permet de tirer profit de ces ressources publiques. De ce fait, la distinction entre les secteurs de l’investissement public peut faire l’objet d’un
prolongement de ce travail. La désagrégation de l’investissement public régional en secteur
peut conduire à dégager les secteurs stimulateurs de la croissance économique régionale. En guise de conclusion, l’investissement public régional marocain ne peut à lui seul, répondre
aux défis de la croissance économique et améliorer le climat économique et social, il faut
combiner un ensemble de facteurs pour y arriver. Parmi ces facteurs, on cite le rôle de
l’investissements privé, ce dernier revêt lui aussi une importance particulière dans le
développement socioéconomique du pays qui place les citoyens au cœur du développement
régional à travers l’amélioration de la qualité des prestations offertes, la création des
opportunités d’emploi et la réduction des disparités spatiales. Cette importance accordée à l’investissement privé régional comme un moteur de
développement socio-économique a poussé le Maroc à créer en 2002 au niveau de chaque
région, des nouveaux organismes nommés « Centres Régionaux d’investissement » et a lancé
en 2022 une réforme de la charte d’investissement de 1995 pour but de la promotion de
l’investissement et de la simplification des procédures de création d’entreprises. www.africanscientificjournal.com Page 375 Page 375 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 African Scientific Journal
ISSN : 2658-9311
Vol : 3, Numéro 16 , Février 2023 www.africanscientificjournal.com www.africanscientificjournal.com BIBLIOGRAPHIE Livre III La
régionalisation avancée au service du développement économique et social, Royaume du
Maroc. Conseil Economique, Social et Environnemental (2016). Rapport : Exigences de la
régionalisation avancée et défis de l’intégration. Auto-Saisine n°22/2016. régionalisation avancée et défis de l’intégration. Auto-Saisine n°22/2016. Dessus, S. & Herrera, R. (1996). Le rôle du capital public dans la croissance des pays en
développement au cours des années 80. OECD Development Centre working papers. Dessus, S. & Herrera, R. (1996). Le rôle du capital public dans la croissance des pays en
développement au cours des années 80. OECD Development Centre working papers. ELALAOUI J. et HEFNAOUI A. (2018). L’impact des dépenses publiques sur la croissance
économique : approche par le modèle ARDL cas du Maroc. Revue du contrôle de la
comptabilité et de l’audit. Numéro 6, pp : 638-653. ELALAOUI J. et HEFNAOUI A. (2018). L’impact des dépenses publiques sur la croissance
économique : approche par le modèle ARDL cas du Maroc. Revue du contrôle de la
comptabilité et de l’audit. Numéro 6, pp : 638-653. Elhorst JP (2003). Specifcation and estimation of spatial panel data models. International
Regional Science Review 26 : 244-268. www.africanscientificjournal.com www.africanscientificjournal.com www.africanscientificjournal.com Page 376 African Scientific Journal
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Vol : 3, Numéro 16 , Février 2023 Finn, M. B. (1993). Is all government capital productive. Economic Quarterly, 53-80. Finn, M. B. (1993). Is all government capital productive. Economic Quarterly, 53-80. IAICH M.H & BOUROUANE B. (2020) « Investissement public et croissance économique au
Maroc : approche par modèle ARDL », Revue du contrôle, de la comptabilité et de l’audit
« Volume 4 : numéro 3 » pp : 467-485 IAICH M.H & BOUROUANE B. (2020) « Investissement public et croissance économique au
Maroc : approche par modèle ARDL », Revue du contrôle, de la comptabilité et de l’audit
« Volume 4 : numéro 3 » pp : 467-485 Karras. G & Evans. P. G. (1994). Are Government Activities Productive ? Evidence from a
Panel of U.S. States. The Review of Economics and Statistics, 76(1), 1. Karras. G & Evans. P. G. (1994). Are Government Activities Productive ? Evidence from a
Panel of U.S. States. The Review of Economics and Statistics, 76(1), 1. Krugman, P. (1987). The narrow moving band, the Dutch disease, and the competitive
consequences of Mrs. Thatcher. Journal of Development Economics, 27(1-2), 41-55. Lee, L. & Yu, J. (2010). Estimation of spatial autoregressive panel data models with fixed
effects. Journal of Econometrics, 154(2), 165-185. LeSage, J ,& Pace, R. K. (2009). Introduction to Spatial Econometrics. Chapman and Hall/CRC
eBooks. Munnell, A. H. (1990). How Does Public Infrastructure Affect Regional Economic
Performance. New England Economic Review, 34, 11-33. OBAD J. et JAMAL Y. (2016). L’impact des dépenses publiques sur la croissance économique
au Maroc : Application de l’approche ARDL. International Journal of Innovation and Applied
Studies. Vol 16. N° 2, pp : 444-45. Solow, R. M. (1956). A Contribution to the Theory of Economic Growth. Quarterly Journal of
Economics, 70(1), 65. www.africanscientificjournal.com Page 377
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Mining the mutanome: developing highly personalized Immunotherapies based on mutational analysis of tumors
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* Correspondence: Woverwijk@mdanderson.org
1Department of Melanoma Medical Oncology, The University of Texas M.D,
Anderson Cancer Center, Houston, TX, USA
Full list of author information is available at the end of the article REVIEW Open Access Open Access © 2013 Overwijk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Abstract T cells can mediate remarkable tumor regressions including complete cure in patients with metastatic cancer. Genetic alterations in an individual’s cancer cells (the mutanome) encode unique peptides (m-peptides) that can be
targets for T cells. The recent advances in next-generation sequencing and computation prediction allows, for the
first time, the rapid and affordable identification of m-peptides in individual patients. Despite excitement about the
extended spectrum of potential targets in personalized immunotherapy, there is no experience or consensus on
the path to their successful clinical application. Major questions remain, such as whether clinical responses to
cytokine therapy, T cell transfer, and checkpoint blockade are primarily mediated by m-peptide-specific reactivity,
whether m-peptides can be effectively used as vaccines, and which m-peptides are most potently recognized. These and other technological, immunological and translational questions will be explored during a 1-day
Workshop on Personalized Cancer Immunotherapy by the Society for Immunotherapy of Cancer, directly before the
Annual Meeting, on November 7, 2013 at the National Harbor, MD near Washington, DC. Keywords: Mutanome, Mutation, Neoantigen, Peptide epitope, T lymphocyte, Checkpoint blockade, Exome,
Vaccine, Next-generation sequencing, Omics Introduction specific antigens that are different from “shared” anti-
gens, which are expressed in tumors from multiple
patients and are typically normal, non-mutated self-
proteins. In particular, mutanome-encoded peptides
(hereafter called m-peptides) may evoke a more vigor-
ous T cell response due to a lack of thymic tolerance
against them, and this immunity may be restricted
to tumors, since the mutated gene product is only
expressed in tumors [9]. Mining the mutanome: developing highly
personalized Immunotherapies based on
mutational analysis of tumors Mining the mutanome: developing highly
personalized Immunotherapies based on
mutational analysis of tumors Willem W Overwijk1*, Ena Wang2, Francesco M Marincola2,3, Hans-Georg Rammensee4, Nicholas P Restifo5,6
and for the Organizing Committee of the 2013 SITC Workshop on Personalized Immunotherapy Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 The cancer mutanome: is it important? Peptides encoded by mutated genes in cancer cells have
long been recognized as potential T cell targets, yet they
were not pursued for personalized cancer therapy due
to time and cost constraints on their identification. The
recent arrival of next-generation sequencing and bio-
informatics approaches allows, for the first time, the
rapid and affordable elucidation of an individual cancer
patient’s genome, exome, epigenome and transcriptome
at the single nucleotide level [1]. This in turn enables
the identification of patient-specific omic alterations
that can function as unique therapeutic targets such
as neoantigens [2-8] (Figure 1). The collective of an
individual patient’s tumor-specific alterations and muta-
tions, the so-called mutanome, thus encodes patient- These assumptions have not been rigorously tested,
and our understanding of, for example, m-peptide-spe-
cific peripheral tolerance and T cell cross-reactivity
with wild-type peptides is limited at best. Yet several
very exciting recent reports on the power of immunity
against m-peptides to shrink tumors in mice and pa-
tients suggest the importance of understanding the
biology of m-peptides and their application in immuno-
therapy. For example, a tantalizing possibility is that
the remarkable clinical response rates to adoptively
transferred T cells, and to CTLA-4 and/or PD(L)-1 Page 2 of 4 Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Figure 1 Highly personalized medicine. Inexpensive and highly available DNA sequencing can revolutionize cancer immunotherapy by
enabling highly personalized approaches involving the identification of new tumor-associated antigens. The expressed genes from a patient’s
tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that could potentially bind to any given patient’s HLA
molecules could be predicted using peptide prediction algorithms (e.g. http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm. Or http://www-bimas.cit.nih.gov/molbio/hla_bind). If peptides derived from mutant proteins are found to capable of forming new HLA-restricted
target structures, the candidate peptides can be used in one of at least several ways: 1) “fish out” or sort cells for relevant antigens (such as those
specific for driver oncogenes) using tetramer like reagents; 2) use the candidate peptides to stimulate T cell clonotypes already present in a
patient’s tumor or in their peripheral blood; 3) use antigens to elicit new T cell receptors in mice that are transgenic for human MHC molecules;
and 4) to immunize patients against antigens. The cancer mutanome: is it important? If the T cells generated are specific for a patient’s tumor, they can be expanded and adoptively
transferred if they are of human origin, or used as a source of TCR for gene engineering approaches. Figure 1 Highly personalized medicine. Inexpensive and highly available DNA sequencing can revolutionize cancer immunotherapy by
enabling highly personalized approaches involving the identification of new tumor-associated antigens. The expressed genes from a patient’s
tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that could potentially bind to any given patient’s HLA
molecules could be predicted using peptide prediction algorithms (e.g. http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm. Or http://www-bimas.cit.nih.gov/molbio/hla_bind). If peptides derived from mutant proteins are found to capable of forming new HLA-restricted
target structures, the candidate peptides can be used in one of at least several ways: 1) “fish out” or sort cells for relevant antigens (such as those
specific for driver oncogenes) using tetramer like reagents; 2) use the candidate peptides to stimulate T cell clonotypes already present in a
patient’s tumor or in their peripheral blood; 3) use antigens to elicit new T cell receptors in mice that are transgenic for human MHC molecules;
and 4) to immunize patients against antigens. If the T cells generated are specific for a patient’s tumor, they can be expanded and adoptively
transferred if they are of human origin, or used as a source of TCR for gene engineering approaches. Figure 1 Highly personalized medicine. Inexpensive and highly available DNA sequencing can revolutionize cancer immunotherapy by
enabling highly personalized approaches involving the identification of new tumor-associated antigens. The expressed genes from a patient’s
tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that could potentially bind to any given patient’s HLA
molecules could be predicted using peptide prediction algorithms (e.g. http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm. Or http://www-bimas.cit.nih.gov/molbio/hla_bind). If peptides derived from mutant proteins are found to capable of forming new HLA-restricted
target structures, the candidate peptides can be used in one of at least several ways: 1) “fish out” or sort cells for relevant antigens (such as those
specific for driver oncogenes) using tetramer like reagents; 2) use the candidate peptides to stimulate T cell clonotypes already present in a
patient’s tumor or in their peripheral blood; 3) use antigens to elicit new T cell receptors in mice that are transgenic for human MHC molecules;
and 4) to immunize patients against antigens. Review
R Recent studies on mutanome-specific anti-tumor immunity
The systematic study of the mutanome has only just
begun. Importantly, it appears that different cancer types
and histologies harbor different numbers of mutations. Those with high numbers of mutation include cutaneous
melanoma, smoking-induced lung cancer, and colon
cancer, in particular hypermutators [9,14]. This is likely
related to the high exposure of the respective cells of
origin to mutagenic and inflammatory stimuli: UV light,
cigarette smoke and food-contained mutagenic and in-
flammatory compounds. It is interesting that melanoma
and lung cancer both respond particularly well to PD-1
immunotherapy, possibly implying that m-peptides are
particularly involved in the therapeutic T cell response
to this therapy [15,16]. Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Page 3 of 4 checkpoint blockade, are mediated in part, or even primar-
ily, by m-peptide-specific T cells [10,11]. M-peptide-specific
T cells present before treatment may be a required sub-
strate for successful therapy with cytokines and checkpoint
blockade. Thus, an exciting possibility is that peptide-based
vaccination [12,13] with m-peptides could induce tumor-
specific T cells that are lacking in patients who have failed
to respond to checkpoint blockade, and convert these pa-
tients into responders. M-peptides may also prove to be
major natural targets for tumor-specific TIL [6], and could
be used for ex vivo expansion of patient-derived T cells
(TIL or PBMC) before adoptive T cell therapy. In addition,
m-peptides
could
be
efficiently
targeted
with TCR-
transduced T cells. Finally, m-peptides may be useful in im-
mune monitoring, to evaluate whether m-peptide-specific
immunity correlates with response or disease recurrence
after immunotherapy. Thus, understanding mutanome-
encoded m-peptides as a target for anti-tumor T cells is a
new frontier for cancer immunotherapy. of established B16 melanomas [17]. Robbins et al. similarly
studied melanomas from 3 patients who responded to ther-
apy with ex-vivo expanded autologous tumor-infiltrating
lymphocytes (TIL), and identified a total of 7 unique m-
peptides that appeared to be processed and presented by
autologous tumor cells and were recognized by the autolo-
gous TIL [11]. These three studies used exome sequencing and com-
puter algorithm-guided peptide epitope prediction to iden-
tify MHC Class I-binding m-peptides that were processed
and presented by tumor cells and recognized by tumor-
specific T cells, thus validating the approach. However,
other approaches exist, such as the direct elution of pep-
tides from the surface of tumor cells, or even from circulat-
ing tumor-derived HLA complexes, and their subsequent
identification by microcapillary chromatography/tandem
mass spectrometry [18]. In combination with tumor exome
data, or after in silico subtraction of peptides isolated from
normal cells, tumor-expressed m-peptides can be identi-
fied, including peptides that undergo post-translational
processing, and that would not be identified using genetic
approaches alone. The cancer mutanome: is it important? If the T cells generated are specific for a patient’s tumor, they can be expanded and adoptively
transferred if they are of human origin, or used as a source of TCR for gene engineering approaches. Figure 1 Highly personalized medicine. Inexpensive and highly available DNA sequencing can revolutionize cancer immunotherapy by
enabling highly personalized approaches involving the identification of new tumor-associated antigens. The expressed genes from a patient’s
tumor can be sequenced to identify candidate mutant T cell epitopes. Relevant epitopes that could potentially bind to any given patient’s HLA
molecules could be predicted using peptide prediction algorithms (e.g. http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm. Or http://www-bimas.cit.nih.gov/molbio/hla_bind). If peptides derived from mutant proteins are found to capable of forming new HLA-restricted
target structures, the candidate peptides can be used in one of at least several ways: 1) “fish out” or sort cells for relevant antigens (such as those
specific for driver oncogenes) using tetramer like reagents; 2) use the candidate peptides to stimulate T cell clonotypes already present in a
patient’s tumor or in their peripheral blood; 3) use antigens to elicit new T cell receptors in mice that are transgenic for human MHC molecules;
and 4) to immunize patients against antigens. If the T cells generated are specific for a patient’s tumor, they can be expanded and adoptively
transferred if they are of human origin, or used as a source of TCR for gene engineering approaches. Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Conclusions 9. Heemskerk B, Kvistborg P, Schumacher TN: The cancer antigenome. EMBO J 2013, 32:194–203. 9. Heemskerk B, Kvistborg P, Schumacher TN: The cancer antigenome. EMBO J 2013, 32:194–203. Mutanome-based personalized immunotherapy:
open questions With the technology to efficiently mine the mutanome
in a high-throughput fashion only just becoming avail-
able, many questions remain regarding its application in
personalized immunotherapy. Some of these are: 1. What are the possible and most important uses of
m-peptides? 2. Are m-peptides superior to shared/self-peptides as
therapeutic targets for anti-tumor T cells? 3. What fraction of m-peptides is actually processed
and presented in the context of MHC molecules on
tumor cells? In mice, methylcholanthrene-induced sarcomas likewise
harbor a large number of mutations, and a subset of these
tumors are particularly immunogenic: they will grow in
immunodeficient but not in immunocompetent mice. Matsushita et al. analyzed the mutanome of one such im-
munogenic tumor by exome sequencing and MHC binding
algorithms and identified a point mutation in the spectrin-
β2 gene that resulted in an m-peptide with greatly in-
creased MHC Class I binding. This peptide proved to be a
dominant tumor antigen that caused the complete, spon-
taneous, CD8+ T cell-mediated regression of this tumor in
immunocompetent mice [14]. Using a similar approach,
Castle et al. analyzed the mutanome of the widely used
B16 melanoma cell line and tested 50 MHC-binding m-
peptides, 16 of which were immunogenic and 11 of which
preferentially recognized the mutant peptide over the wild-
type counterpart. Importantly, they showed that vaccin-
ation with 2 of those suppressed the growth suppression 4. What fraction of these peptides evokes a T cell
response in the host (spontaneously or after
vaccination)? 5. How frequently do T cells against m-peptides cross-
react with the corresponding wild-type peptides
expressed on normal cells? 6. Are m-peptides the dominant peptides recognized in
spontaneous tumor-specific immune responses? 7. Can m-peptide-based vaccines induce therapeutic
immune responses, alone or as part of combination
therapies? 8. Are m-peptides the dominant peptides recognized in
therapy-induced immune responses (e.g. checkpoint
blockade, TIL, IL-2, IFN-α, radiation, immunogenic
chemotherapies). 9. What “pipeline” allows the shortest time from fresh
tumor sample to validated m-peptide? Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Overwijk et al. Journal for ImmunoTherapy of Cancer 2013, 1:11
http://www.immunotherapyofcancer.org/content/1/1/11 Page 4 of 4 Page 4 of 4 10. What are the (dis)advantages of sequencing exomes
vs. RNA? 11. What are the best approaches for filtering the
numerous sequencing errors before declaring a
somatic mutation? 12. What peptide prediction algorithms are best at
predicting MHC-binding peptides from exome data? 13. References What are safety and regulatory issues in designing
personalized cancer vaccines against never-before
targeted, patient-specific antigens? 7. Segal NH, et al: Epitope landscape in breast and colorectal cancer. Cancer Res 2008, 68:889–892. 7. Segal NH, et al: Epitope landscape in breast and colorectal cancer. Cancer Res 2008, 68:889–892. 8. Pieper R, et al: Biochemical Identification of a Mutated Human Melanoma
Antigen Recognized by CD4(+) T Cells. J Exp Med 1999, 189:757–766. 8. Pieper R, et al: Biochemical Identification of a Mutated Human Melanoma
Antigen Recognized by CD4(+) T Cells. J Exp Med 1999, 189:757–766. Received: 22 July 2013 Accepted: 22 July 2013
Published: 29 July 2013 The 2013 society for immunotherapy of cancer workshop
on personalized immunotherapy Bassani-Sternberg M, et al: Soluble plasma HLA peptidome as a potential
source for cancer biomarkers. Proc Natl Acad Sci USA 2010, 107:18769–18776. source for cancer biomarkers. Proc Natl Acad Sci USA 2010, 107:1876 Competing interests
The authors declare t Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Abbreviations
TIL T
i fil doi:10.1186/2051-1426-1-11
Cite this article as: Overwijk et al.: Mining the mutanome: developing
highly personalized Immunotherapies based on mutational analysis of
tumors. Journal for ImmunoTherapy of Cancer 2013 1:11. TIL: Tumor-infiltrating lymphocytes; PBMC: Peripheral blood mononuclear
cells; TCR: T cell receptor; MHC: Major histocompatibility complex;
HLA: Human leukocyte antigen. The 2013 society for immunotherapy of cancer workshop
on personalized immunotherapy 10. Wolchok JD, et al: Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med 2013. 10. Wolchok JD, et al: Nivolumab plus Ipilimumab in Advanced Melanoma. N Engl J Med 2013. y
The mining of the mutanome has just begun, and many
questions regarding methodology, application and future
opportunities remain open and ready for debate. With
many groups currently engaging this area, it is a prime time
to come together and share data, viewpoints and best prac-
tices, forge collaborations, and chart a course for the imme-
diate future. The Society for Immunotherapy of Cancer
invites you to attend their Workshop on Personalized Im-
munotherapy, directly before the Annual Meeting, on Nov
7, 2013 at the National Harbor, MD near Washington, DC. Specifics can be found at the Society website at www. sitcancer.org/2013/workshop. Abstracts and posters are
welcome through August 26, 2013, and platform presenta-
tions will be selected. 11. Robbins PF, et al: Mining exomic sequencing data to identify mutated
antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 2013, 19:747–752. 11. Robbins PF, et al: Mining exomic sequencing data to identify mutated
antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 2013, 19:747–752. 12. Walter S, et al: Multipeptide immune response to cancer vaccine IMA901
after single-dose cyclophosphamide associates with longer patient
survival. Nat Med 2012, 18:1254–1261. 13. Hailemichael Y, et al: Persistent antigen at vaccination sites induces
tumor-specific CD8(+) T cell sequestration, dysfunction and deletion. Nat Med 2013, 19:465–472. 14. Matsushita H, et al: Cancer exome analysis reveals a T-cell-dependent
mechanism of cancer immunoediting. Nature 2012, 482:400–404. 14. Matsushita H, et al: Cancer exome analysis reveals a T-cell-dependent
mechanism of cancer immunoediting. Nature 2012, 482:400–404. 15. Topalian SL, et al: Safety, activity, and immune correlates of anti-PD-1
antibody in cancer. N Engl J Med 2012, 366:2443–2454. 15. Topalian SL, et al: Safety, activity, and immune correlates of anti-PD-1
antibody in cancer. N Engl J Med 2012, 366:2443–2454. 16. Hamid O, et al: Safety and Tumor Responses with Lambrolizumab
(Anti-PD-1) in Melanoma. N Engl J Med 2013. 16. Hamid O, et al: Safety and Tumor Responses with Lambrolizumab
(Anti-PD-1) in Melanoma. N Engl J Med 2013. 17. Castle JC, et al: Exploiting the mutanome for tumor vaccination. Cancer Res 2012, 72:1081–1091. 17. Castle JC, et al: Exploiting the mutanome for tumor vaccination. Cancer Res 2012, 72:1081–1091. 18. Authors’ contributions
d WWO, EW, FMM, HGR and NPR conceived the Workshop idea and topics and
participated in writing the manuscript. All authors read and approved the
final manuscript. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: References 1. Khalili JS, Hanson RW, Szallasi Z: In silico prediction of tumor antigens
derived from functional missense mutations of the cancer gene census. Oncoimmunology 2012, 1:1281–1289. 1. Khalili JS, Hanson RW, Szallasi Z: In silico prediction of tumor antigens
derived from functional missense mutations of the cancer gene census. Oncoimmunology 2012, 1:1281–1289. 11. What are the best approaches for filtering the
numerous sequencing errors before declaring a
somatic mutation? 2. Restifo NP, Dudley ME, Rosenberg SA: Adoptive immunotherapy for cancer:
harnessing the T cell response. Nat Rev Immunol 2012, 12:269–281. 2. Restifo NP, Dudley ME, Rosenberg SA: Adoptive immunotherapy for cancer:
harnessing the T cell response. Nat Rev Immunol 2012, 12:269–281. 3. Wolfel T, et al: A p16INK4a-insensitive CDK4 mutant targeted by cytolytic
T lymphocytes in a human melanoma. Science 1995, 269:1281–1284. 3. Wolfel T, et al: A p16INK4a-insensitive CDK4 mutant targeted by cytolytic
T lymphocytes in a human melanoma. Science 1995, 269:1281–1284. 12. What peptide prediction algorithms are best at
predicting MHC-binding peptides from exome data? y
p
y
,
4. Coulie PG, et al: A mutated intron sequence codes for an antigenic
peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA 1995, 92:7976–7980. y
p
y
,
4. Coulie PG, et al: A mutated intron sequence codes for an antigenic
peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA 1995, 92:7976–7980. 13. What is the impact of the immunoproteasome,
peptide splicing, and post-translational modification
on the actual expression of predicted m-peptides on
tumor cells? 5. Echchakir H, et al: A point mutation in the alpha-actinin-4 gene generates
an antigenic peptide recognized by autologous cytolytic T lymphocytes
on a human lung carcinoma. Cancer Res 2001, 61:4078–4083. 5. Echchakir H, et al: A point mutation in the alpha-actinin-4 gene generates
an antigenic peptide recognized by autologous cytolytic T lymphocytes
on a human lung carcinoma. Cancer Res 2001, 61:4078–4083. g
6. Lennerz V, et al: The response of autologous T cells to a human
melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci USA
2005, 102:16013–16018. 6. Lennerz V, et al: The response of autologous T cells to a human
melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci USA
2005, 102:16013–16018. 14. What are safety and regulatory issues in designing
personalized cancer vaccines against never-before
targeted, patient-specific antigens? 14. Mutanome-based personalized immunotherapy:
open questions What is the impact of the immunoproteasome,
peptide splicing, and post-translational modification
on the actual expression of predicted m-peptides on
tumor cells? 14. What are safety and regulatory issues in designing
personalized cancer vaccines against never-before
targeted, patient-specific antigens? 10. What are the (dis)advantages of sequencing exomes
vs. RNA? Author details
1 1Department of Melanoma Medical Oncology, The University of Texas M.D,
Anderson Cancer Center, Houston, TX, USA. 2Infectious Disease and
Immunogenetics Section, Department of Transfusion Medicine, Clinical
Center, National Institutes of Health, Bethesda, MD, USA. 3Sidra Medical and
Research Centre, Doha, Qatar. 4Department of Immunology, Institute for Cell
Biology, University of Tübingen, Tübingen, Germany. 5Center for Cancer
Research, National Cancer Institute, National Institutes of Health, Bethesda,
MD, USA. 6NIH Center for Regenerative Medicine, National Institutes of
Health, Bethesda, MD, USA. • Convenient online submission • Thorough peer review Received: 22 July 2013 Accepted: 22 July 2013
Published: 29 July 2013
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JORGE CASCALES RIBERA1
Raewyn Connell: una vida atravesada por el género
Raewyn Connell: a life crossed by gender
Hay veces que pienso que académicamente vivimos la investigación como devotos aficionados de fútbol. Investigar, razonar académicamente, implica dialogar y construir, incluso sin conocer personalmente, afinidades y reconocimiento
con personas que también han reflexionado sobre lo que estamos investigando.
Si nos paramos unos segundos a pensarlo encontraremos nombres de autores y
autoras que aparecen repetidamente en nuestras bibliografías, en nuestros textos
y en nuestras conversaciones formando parte de aquello sobre lo que dialogamos.
Cuando evocamos sus teorías exhibimos una cierta emoción que los identifica y
de la misma forma que uno verbaliza ser más de Messi que de Cristiano Ronaldo,
académicamente también decimos de forma coloquial que somos más de Foucault
que de Bourdieu, más de Butler que de Fraser, más de Celia Amorós que de Luce
Irigaray…; y aunque lo neguemos por cuestiones epistémicas, más que nos pese,
todas y todos tenemos nuestras preferencias para explicar la realidad.
Raewyn Connell probablemente sea una de las personalidades que más
evocamos aquellas personas que nos dedicamos a los estudios de masculinidades.
Con una prolífica producción académica de más de 30 años de recorrido y un
significativo número de traducciones en varios idiomas aparece en incontables
textos y discusiones. Socióloga de profesión, Raewyn Connell ha dedicado su vida a
la investigación y a la docencia académica en universidades como la de Melbourne,
Macquarie, California, Sud África, Harvard o Sídney donde actualmente es
profesora emérita en dicha universidad. Premiada por la American Sociological
Association por sus contribuciones a los estudios de género y por la Asociacion
Sociológica Australiana por sus aportaciones sobre su tierra natal, ha profundizado
en temáticas como la educación, la globalización, el neoliberalismo, la colonialidad,
el trabajo intelectual o en su último libro la producción del conocimiento desde la
universidad. Sin embargo, por aquello que más se la conoce es por aportaciones
a los estudios de género y a los estudios de las masculinidades, espacio donde
se la reconoce como una de las principales impulsoras y pioneras en este tipo de
estudios. No obstante, cuando verbalizamos yo soy más de Connell, como en
cualquier espacio de reconocimiento, siempre hay algo que va más allá que un mero
currículum, siempre aparece un matiz emocional que hace que académicamente
nos situemos frente a su espejo.
1
Universitat de València, jorge.cascales@uv.es
Asparkía, 35; 2019, 171-176 - ISSN: 1132-8231 - e-ISSN: 2340-4795 - DOI: http://dx.doi.org/10.6035/Asparkia.2019.35.9
172
Jorge Cascales Ribera
La historia de vida de las personas forma parte de esta construcción emocional
del reconocimiento. Raewyn Connell nace un 3 de Enero de 1944 en Sídney (Australia) en el seno de una familia con raíces irlandesas, escocesas y galesas, poseyendo un
cuerpo varón y siendo llamado Robert W. Connell. Como parte de toda una generación de postguerras y transiciones políticas destacará durante sus años de estudiante
por su activismo político y participación en el movimiento New Left o movimiento de
la nueva izquierda de los años 60’ y 70’, empapándose de una nueva idea de militancia y activismo social. Unas nuevas fórmulas que irán más allá de las formas clásicas
de reivindicación sindical o política. Las luchas por los derechos civiles contra el
elitismo, las luchas contra la opresión por cuestiones de raza, sexo, clase social o género, o las reivindicaciones contra la guerra y contra la violencia social e institucional
se vislumbrarán constantemente en sus análisis y en su relato de vida. A finales de
los años 60 el feminismo aparecerá públicamente en Australia y Connell, alentado e
influenciado por su futura esposa Pam Benton, se acercará al movimiento empapándose de las cuestiones de género y de la lucha por la emancipación de las mujeres.
Así pues, Raewyn Connell participará, en la medida de lo posible, en la militancia
feminista siendo esto una constante en toda su obra.2
Análogamente a todo esto, Raewyn Conell posee una posición epistemológica
muy particular, y es que después de que el cáncer se llevase a su mujer tras una relación de 21 años,3 anuncia públicamente su condición transexual, cría a su hija como
padre soltero y una vez su hija llega a la adultez decide dar un paso importante en
su identidad de género y hacer la transición de hombre (antes Robert W. Connell) a
mujer (actualmente Raewyn Connell). La propia Connell relata que, si no hubiese
sido por el apoyo de su mujer, probablemente, no habría sobrevivido a la realidad
transexual que le había tocado vivir, sintiéndose mujer en el cuerpo de un hombre.
Este hecho identitario atravesará, no solamente la vida personal de la autora, sino
también su mirada y sus intereses, haciendo mella en su producción académica. Autoras como Sara Martín se aventurarán a situar la transexualidad de Raewyn Connell
y su transición como una paradoja epistomológica que modifica “de manera harto
interesante su visión de la masculinidad y el género” (Martín, 2007: 94). En este sentido, la propia Connell en la entrevista realizada en 2011 para el periódico de La Vanguardia explicitará dicho posicionamiento epistemológico relatando que su posición
como varón antes de la transición le había permitido experimentar el “acceso a ciertos privilegios que tienen los hombres en términos de carrera profesional y autoridad
social, pero siempre en peligro por ser un tipo raro de hombre”.4
2
Pam Benton, como la describe Raewyn Connell, fue una mujer feminista, activista del movimiento
de mujeres, psicóloga, investigadora social, escritora y funcionaria pública. Ella será una de las
fundadoras de la Red de Mujeres Mayores en Australia y formará parte en la creación del primer
centro de salud para mujeres en el sur de Australia. Al mismo tiempo participará tras una mirada
feminista de transformación social en el desarrollo de políticas de igualdad en Nueva Gales del Sur.
En la Enciclopedia de las Mujeres y el Liderazgo en la Australia del Siglo XX se describe a Pam Benton
como una mujer talentosa, querida, solidaria y madre de su hija.
3 Connell, tras su muerte, editará y publicará sus escritos en la obra Kept on Dancing: writting by Pam (1998).
4 Entrevista publicada en el diario La Vanguardia – Raewyn Connell (antes Robert William Connell)
experta en masculinidad. Fecha de la publicación: 16-11-2011.
Asparkía, 35; 2019, 171-176 - ISSN: 1132-8231 - e-ISSN: 2340-4795 - DOI: http://dx.doi.org/10.6035/Asparkia.2019.35.9
Raewyn Connell: una vida atravesada por el género
173
Aunque siempre ha destacado por su activismo militante y académico en temas
tan variados como la igualdad de género, la erradicación de la violencia, la justicia
educativa, la lucha contra el VIH o la lucha por los derechos de las personas trans,
el tema central que la hará ser conocida internacionalmente a nivel académico
serán sus estudios sobre la construcción social de la masculinidad. Para Raewyn
Connell, y así lo expone en su obra Gender and Power (1987), siempre ha sido central
dar una respuesta, llegar a comprender, el funcionamiento del orden de género en
su conjunto. Como evidencia la propia autora en una entrevista realizada en 2011
por La Independent
…si queremos entender el orden de género patriarcal, así como entender
las vidas de los grupos oprimidos por él, tenemos que entender los grupos
privilegiados por él. Necesitamos entender cómo funciona el género para ellos
y la manera en la que “hacen género” desde la educación y la amistad hasta la
violencia de género y la guerra.5
Sin embargo, la autora, ante la incidencia continuada de textos e investigaciones
destinadas a indagar en los colectivos más oprimidos, desfavorecidos o explotados,
esta termina por depositar su mirada en las estructuras dominantes, produciendo
una amplia literatura en este sentido. Un ejemplo de esto es su obra Ruling Class,
Ruling Culture (1977) donde profundiza en el análisis estructural de la clase social australiana como categoría social. A partir de este momento, involucrada en
proyectos de investigación dentro del marco escolar, Raewyn Connell empieza a
preguntarse sobre las relaciones que se generan entre las diferentes formas de masculinidad.
Su principal obra, Masculinities, saldrá en 1995. Dicha obra en 2011 llega a ser
referenciada solo para lengua inglesa en más de 4000 publicaciones, y por la cual
recibirá un premio de la American Sociological Society llevándola a colaborar en diversas agencias de Naciones Unidas para abordar asuntos sobre la masculinidad,
la violencia y la construcción de la paz. Los textos de Connell alrededor del debate
sobre la identidad masculina, y en concreto su obra Masculinities (1995), convertirán a la autora en una de las pioneras y referentes en la creación de los estudios de
masculinidades. En sus investigaciones Connell fragmentará el sujeto homogéneo
y universal masculino (el sujeto hombre) para hablar de masculinidades en plural,
ofreciendo una propuesta de clasificación de las masculinidades según su posición
en referencia a la construcción social de masculinidad (hegemónica, cómplice, disidente, marginal), llevándole a visibilizar la diversidad de hombres que existen
en el orden de género patriarcal. De esta forma, a partir de sus investigaciones,
cuestionará la esencia masculina y todo tipo de obras o discursos que seccionen
5
Raewyn Connell: “Involucrarse profundamente en la crianza de una persona nueva lleva a que las
personas quieran con menos probabilidad matar y mutilar a otras”. Escrito por Elena Ledda en La
Independent. Agència de noticies en visió de gènere el 08 de Octubre de 2011 [www.laindependent.cat].
Asparkía, 35; 2019, 171-176 - ISSN: 1132-8231 - e-ISSN: 2340-4795 - DOI: http://dx.doi.org/10.6035/Asparkia.2019.35.9
174
Jorge Cascales Ribera
el orden de género desde una esencia inmanente de masculinidad y feminidad.6
Así pues, a partir del texto Towards a New Sociology of Masculinity (Carrigan, Connell y Lee, 1985) se acercará el concepto gramsciano de hegemonía en correlación
con la masculinidad. Esta idea la conducirá a reflexionar sobre una subjetividad
masculina patriarcal a la que aspirar, reflejarse o rechazar llevándola a plantear
el controvertido concepto de “masculinidad hegemónica” desde el que situar a la
masculinidad más patriarcal y desde donde repensar la re-negociación identitaria
(situada cultural e históricamente), y con ello, la articulación de unos “dividendos
patriarcales” y la “justicia de género” ante la propia masculinidad.
La disección de las masculinidades y la configuración auto-referencial de una
masculinidad hegemónica patriarcal dibujará desde un ideario feminista una
masculinidad a la que no aspirar, posibilitando no solamente la visibilidad de las
masculinidades disidentes o marginadas que eran oprimidas por la propia etiqueta
de masculinidad (varones homosexuales, mujeres trans, hombres sin techo, etc.),
sino también, la aparición de la capacidad de articulación de masculinidades contrahegemónicas con consciencia disidente. Hombres que en la práctica convergen con
los feminismos y se impregnan de un ideario pensado para una igualdad real entre
hombres y mujeres. Varones que actúan desde sus prácticas cotidianas y desde
la reivindicación social de la justicia de género y la igualdad para posibilitar un
cambio real en el orden de género.
El giro discursivo que producen las teorías de Raewyn Connell servirá para
producir un cambio de consciencia social y visibilizar un campo de estudio que
hasta el momento había sido precariamente explorado.7 Sin embargo, tal cual se va
avanzando con las investigaciones de masculinidades y se va produciendo discurso de los hombres en la igualdad y en el feminismo el concepto de “masculinidad
hegemónica” de Connell será puesto en cuestión. El hecho de entender la masculinidad hegemónica como una forma de estructura u orden social se interpretará
como el establecimiento de un modelo fijo, ahistórico, que no puede responder a
la diversidad que se producen entre las categorías marcadas y ante la definición de
los cambios de lo femenino, lo masculino, el género, el sexo o el cuerpo. Además,
no llegará a conectar con los procesos que se derivan de las prácticas individuales,
y con ello, con la capacidad de agencia que se articula alrededor de la reproducción
de ciertas prácticas sociales. Con ello, a dicha teoría se le sumará el controvertido
riesgo de construir un marco auto-referencial de masculinidad hegemónica que
produzca y configure un imaginario irreal y complejo de hombres tras un ideario
feminista que están libres de prácticas patriarcales; y al mismo tiempo, el riesgo
de producir un espacio de prácticas y discursos de vanguardia que haga que no
se cuestione su propia masculinidad y que planteen como absoluta su forma de
6
La propia autora, en alguna de sus entrevistas, cuestionará el valor de verdad de las obras que
explicitan que el orden de género se fundamenta en una complementariedad esencializada y opuesta
entre hombres y mujeres como seres desiguales en esencia y sin posibilidad de cambio social.
7 Los estudios de masculinidades son anteriores a la obra de Connell, sin embargo, la autora será
una de las grandes impulsoras de dichos estudios. Destacar autores y autoras como Josep Vicent
Marqués, Elisabeth Badinter, Donald H. Bell, David Gillmore, Pierre Bourdieu, Michael Kimmel,
Harry Brod, Bob Pease, Jeff Hearn, Victor Seidler o Michael Kaufman entre otros.
Asparkía, 35; 2019, 171-176 - ISSN: 1132-8231 - e-ISSN: 2340-4795 - DOI: http://dx.doi.org/10.6035/Asparkia.2019.35.9
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https://openalex.org/W3099104443
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https://iris.unipa.it/bitstream/10447/448757/2/TEDESCO_2020.pdf
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Ancora sulla fortuna de La Fuerza lastimosa nell’opera del Seicento: Alfonso I di Matteo Noris (Venezia Napoli Palermo)
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Studi e saggi
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cc-by
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Anna Tedesco, University of Palermo, Italy, anna.tedesco@unipa.it, 0000-0002-1199-0287
FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup_best_practice)
Anna Tedesco, Ancora sulla fortuna de La fuerza lastimosa nell’opera del Seicento: Alfonso I di
Matteo Noris (Venezia Napoli Palermo), pp. 179-204, © 2020 Author(s), CC BY 4.0 International, DOI
10.36253/978-88-5518-150-1.09, in Fausta Antonucci, Salomé Vuelta García (edited by), Ricerche
sul teatro classico spagnolo in Italia e oltralpe (secoli XVI-XVIII), © 2020 Author(s), content CC BY
4.0 International, metadata CC0 1.0 Universal, published by Firenze University Press (www.fupress.
com), ISSN 2704-5919 (online), ISBN 978-88-5518-150-1 (PDF), DOI 10.36253/978-88-5518-150-1 ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA
NELL’OPERA DEL SEICENTO: ALFONSO I DI MATTEO NORIS
(VENEZIA NAPOLI PALERMO) Anna Tedesco 1 Se ne veda l’edizione critica in L. de Vega Carpio, La fuerza lastimosa, in
Alberola (1998: I, 69-243). 2 Sulla fortuna della Fuerza lastimosa in Italia rimando innanzitutto agli studi
di Carmen Marchante Moralejo e Salomé Vuelta García che forniscono un inqua-
dramento generale della questione: la prima nella sua tesi dottorale sulle traduzio-
ni e gli adattamenti italiani di Lope de Vega (poi in volume Marchante Moralejo,
2007), la seconda nei suoi lavori sulla presenza del teatro spagnolo sulla scena fio-
rentina, in particolare Vuelta García (2006). Cfr. inoltre Antonucci (2014) ed il pre-
zioso catalogo di Profeti (2009). Sulla presenza negli scenari dei comici cfr. Blundo
(1999) e Antonucci (2017). Sulla figura di Fivizzani si veda anche Símini (2011). Su
Celano, cfr. Navarra (1919); Vaiopoulos (2003: 88-89) e Trecca (2016). Sull’utilizzo
di sequenze della Fuerza lastimosa nei drammi per musica, si vedano (per Euripo)
Badolato (2011), e per il Giasone Antonucci (2012: 266-269), e Antonucci, Bianconi
(2013). Su Salvi, cfr. Giuntini (1994: 15-21), che identifica nell’adattamento edito
a Firenze nel 1691 la fonte del libretto di Salvi. Sull’opera a Livorno, Porta (1995). Introduzione Le ricerche di Fausta Antonucci, Carmen Marchante e Salomé Vuel-
ta hanno già messo in luce la grande diffusione di cui godette in Italia La
fuerza lastimosa di Lope de Vega, composta intorno al 1599 e stampata
nel 1609 nella Segunda parte de las comedias de Lope de Vega (A. Martín,
Madrid)1: non solo tracce intertestuali di questo lavoro si ritrovano nel più
celebre dramma per musica del Seicento, il Giasone di Giacinto Andrea
Cicognini (1649), e nell’Euripo di Giovanni Faustini dello stesso anno, ma
pure ne vennero ricavati quattro adattamenti in prosa, uno scenario dei
comici dell’Arte conservato nella Biblioteca Casanatense ed un altro dram-
ma per musica: La forza compassionevole di Antonio Salvi, rappresenta-
to a Livorno nel 1694, e dedicato al granprincipe Ferdinando de Medici. g
p
p
Con questo contributo mi propongo di aggiungere un nuovo tassello
alla storia della fortuna italiana della Fuerza lastimosa, discutendo un ul-
teriore dramma per musica da essa derivato, che venne rappresentato in
tre diversi luoghi e occasioni: si tratta di Alfonso primo di Matteo Noris,
intonato da Carlo Francesco Pollarolo per il Teatro San Salvatore di Vene-
zia nel Carnevale 1694 e ripreso con modifiche a Napoli e a Palermo, con
il titolo Alfonso sesto re di Castiglia e nuove musiche. Il dramma di Noris,
a mio avviso chiaramente derivato dalla comedia di Lope, si differenzia
tuttavia da essa e dagli adattamenti italiani già conosciuti. Nei paragrafi
che seguono discuterò i rapporti tra il libretto, la comedia spagnola e gli ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 180 adattamenti italiani, esaminerò le circostanze delle tre rappresentazioni
del dramma per musica ed infine illustrerò le fonti musicali pervenute si-
no a noi. La Tavola 1 elenca le derivazioni finora note: adattamenti italiani, esaminerò le circostanze delle tre rappresentazioni
del dramma per musica ed infine illustrerò le fonti musicali pervenute si-
no a noi. La Tavola 1 elenca le derivazioni finora note: Titolo (autore, luogo e data di stampa o data presunta per i manoscritti)
La forza lastimosa (scenario, raccolta Ciro Monarca dell’Opere regie 1640-1650)
Giasone (G.A. Cicognini, Venezia 1649)
Euripo (G Faustini Venezia 1649) Euripo (G. Faustini, Venezia 1649) La violenza lacrimevole, overo il traditor fortunato (P.P. Todini, Roma 1664)
La forza compassionevole (M. Stanchi o P. Susini?, ms. anonimo, Firenze 1673-1681;
stampa Firenze 1691) La violenza lacrimevole, overo il traditor fortunato (P.P. 4 Ho consultato l’esemplare della Biblioteca Nazionale Braidense di Milano
(Corniani Algarotti Racc. Dramm. 512). Il riferimento storico sarebbe ad Alfonso
I d’Aragona e Navarra, detto il Battagliero (sp. el Batallador), poi re di Castiglia in
seguito al matrimonio con Urraca. Introduzione gg
p
• per quanto riguarda i testi per musica, il dramma di Salvi discende
dall’adattamento fiorentino del 1691, invece i due libretti veneziani
del Giasone e dell’Euripo rielaborano molto liberamente solo alcune
sequenze della comedia, intrecciandole ad altri spunti drammatici, se-
condo una tecnica tipica dei poeti per musica di metà Seicento3. gg
p
per quanto riguarda i testi per musica, il dramma di Salvi discende
dall’adattamento fiorentino del 1691, invece i due libretti veneziani
del Giasone e dell’Euripo rielaborano molto liberamente solo alcune
sequenze della comedia, intrecciandole ad altri spunti drammatici, se-
condo una tecnica tipica dei poeti per musica di metà Seicento3. L’opera di Noris-Pollarolo, rappresentata nello stesso anno in cui il
dramma per musica di Antonio Salvi andò in scena a Livorno (1694), si
colloca sullo stesso versante degli altri due libretti veneziani: oltre a tace-
re, anzi a nascondere, la fonte, la manipola e la trasforma, utilizzandone
solo il nodo principale. 3 Per il caso di Cicognini, si veda, oltre ai saggi sopra citati, Tedesco (2012). Introduzione Todini, Roma 1664)
La forza compassionevole (M. Stanchi o P. Susini?, ms. anonimo, Firenze 1673-1681;
stampa Firenze 1691) Non ha cuore chi non sente pietà (G. Fivizzani ante 1689; da cui derivano F. Scarnelli, Bologna 1689; G. Fivizzani, Lucca 1699; A. Perrucci, Chi non ha cuore
non ha pietà, overo la Rosaura, Napoli 1719) Il vero consigliere del suo proprio male (C. Celano, Napoli 1692) Il vero consigliere del suo proprio male (C. Celano, Napoli 1692) La forza compassionevole (A. Salvi, Livorno 1694) Riassumo brevemente quanto reso noto dalle precedenti ricerche2: Riassumo brevemente quanto reso noto dalle precedenti ricerche2:
lo scenario della raccolta Ciro monarca (1640/1650 circa) e l’adatta • lo scenario della raccolta «Ciro monarca» (1640/1650 circa) e l’adatta-
mento di Todini (1664) sono i più antichi e i più vicini al testo originale. In particolare sono gli unici a mantenere la prima jornada della comedia
eliminata negli altri testi italiani al fine di rispettare l’unità di tempo. • lo scenario della raccolta «Ciro monarca» (1640/1650 circa) e l’adatta-
mento di Todini (1664) sono i più antichi e i più vicini al testo originale. In particolare sono gli unici a mantenere la prima jornada della comedia
eliminata negli altri testi italiani al fine di rispettare l’unità di tempo. gi
p
p
• un adattamento in prosa (conservato in tre manoscritti) venne recita-
to a Firenze dagli Accademici Imperfetti tra il 1673 ed il 1681 e ripreso
nell’Accademia dei Sorgenti nel 1691. L’edizione apparsa a Firenze ad
istanza dei Sorgenti viene attribuita da una fonte coeva a uno «Stan-
chi istrione» (ma è stata avanzata anche la paternità di Pietro Susini,
traduttore di diversi testi drammatici spagnoli). p g
)
• l’adattamento degli Imperfetti è alla base di quello di Fivizzani, sacer-
dote di Lucca, scritto anteriormente al 1689, ma dato alle stampe solo p g
)
• l’adattamento degli Imperfetti è alla base di quello di Fivizzani, sacer-
dote di Lucca, scritto anteriormente al 1689, ma dato alle stampe solo 181 Anna Tedesco nel 1699. Da esso derivano gli adattamenti di Scarnelli e Perrucci. Essi
condividono con quello di Celano del 1692 l’eliminazione della prima
jornada, trasformata in antefatto e l’amplificazione dell’elemento co-
mico (invece la comedia di Lope manca della figura del gracioso). In
Celano gli eventi dell’antefatto vengono svelati solo all’inizio del terzo
atto, creando una maggiore attesa nello spettatore. Alfonso primo di Matteo Noris e La fuerza lastimosa Nella premessa il librettista dichiara di aver tratto il suo soggetto dal
tomo II, libro IV, cap. 18° dell’Historia della perdita e riacquisto della Spa-
gna del gesuita nativo di Castellamare di Stabia, Bartolomeo De Rogatis
(Noris, 1694: 5)4. Pubblicato nel 1648 a Napoli, l’Historia ebbe numerose
edizioni successive ed è già stata indicata da Paolo Fabbri come una delle
fonti dei libretti di soggetto spagnolo (Fabbri, 1990: 285). gg
p g
(
)
Cosa si narra nel cap. 18° della Historia? Dell’amore di Semena (italia-
nizzazione di Jimena o Ximena), sorella del re Alfonso II, per Sancho Díaz
conte di Saldaña. La principessa s’innamora perdutamente di lui, lo con-
vince con suppliche e minacce a diventare suo amante e ne ha in segreto un
figlio, Bernardo. Certa del perdono del fratello, gli rivela la loro relazione,
ma il re, lungi dal perdonarla, la fa chiudere in un convento e fa accecare
ed imprigionare l’amante. Il perdono arriverà solo per i meriti militari del
figlio illegittimo, e solo quando il conte è in punto di morte (o addirittura
morto, secondo alcune versioni) (De Rogatis, 1648: 696-714). Si tratta di un
soggetto largamente presente nell’epica medievale spagnola. Il figlio dei due
amanti, Bernardo del Carpio, è il protagonista di diversi testi epici (che lo
vedono partecipare alla battaglia di Roncisvalle) e di testi drammatici, tra ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 182 cui due di Lope de Vega, uno attribuito con certezza (El casamiento en la
muerte) e uno dubbio (Las mocedades de Bernardo del Carpio)5. p
Il dramma di Noris però, si allontana dalla sua pretesa fonte: legami
con Rogatis sono l’ambientazione vagamente spagnola (l’opera si svolge in
Cantabria), il nome del re (che in Rogatis è Alfonso II il casto6) e la passione
di una principessa per un uomo non adeguato al suo rango, che la spinge
ad un amore clandestino. Tutto il resto invece non è solo «finzione» come
sostenuto da Noris, bensì rielaborazione della Fuerza lastimosa. E chissà
che l’autore non si riferisca cripticamente a questo, quando nella dedica,
in un linguaggio piuttosto circonvoluto, si accusa di un «furto»: «Io die-
tro la mano di Prometeo, di furto tingendo la penna in questo abisso di
luce, indoro la mia industriosa ambizione, e insigno del nome di V. Ecc. la prima pagina del drama presente». 5 La prima apparve nella Parte Primera de comedias de Lope de Vega (1604); la
seconda nella Parte 29 de Diferentes Autores (1634).
6 Alfonso II il Casto (sp. el Casto) re delle Asturie e di León (Oviedo 759 - ivi
842). Figlio di Fruela I assassinato nel 768, gli succedette effettivamente nel 789,
all’abdicazione di Bermudo I. Vinse i Mori nella battaglia di Lutos, ma fu sconfitto
l’anno seguente (795). 5 La prima apparve nella Parte Primera de comedias de Lope de Vega (1604); la
seconda nella Parte 29 de Diferentes Autores (1634). Alfonso primo di Matteo Noris e La fuerza lastimosa p
p g
p
Noris infatti attinge al motivo principale de La fuerza lastimosa, ossia
l’amore di una principessa, figlia del re d’Irlanda (l’infanta Dionisia) per
un uomo di condizione inferiore (il conte Enrique) e l’inganno perpetrato
ai loro danni. L’infanta dà al giovane un appuntamento notturno; tuttavia
per uno stratagemma del duca Otavio, pure innamorato di lei, Enrique vie-
ne incarcerato e Dionisia passa la notte col duca pensando che sia il conte. Resosi conto di essere stato ingannato, quest’ultimo fugge (prima jornada). Quando, dopo alcuni anni, torna in Irlanda ammogliato con Isabela, figlia
del conte di Barcellona, Dionisia folle di gelosia confessa al padre quanto
avvenuto con una lettera; questi convoca il conte e gli chiede di scegliere
una punizione per l’uomo che si sia macchiato di una colpa siffatta. Enri-
que, ignaro di tutto, suggerisce che il colpevole uccida la moglie legittima in
modo da poter sposare la donna offesa; proprio quello che il re immediata-
mente gli ordina di fare. Non avendo il coraggio di uccidere la moglie, che
pure si offre di morire per la sua salvezza, Enrique affida questo compito al
marchese Fabio che propone di abbandonarla su una barca in avaria. Posta
in barca, Isabela viene fortunosamente salvata proprio dal vero seduttore
dell’Infanta, il duca Otavio (seconda jornada). Il conte sprofonda nel dolo-
re e nella pazzia, che gli impediscono di sposare l’infanta. Intanto, appresa
la tragica sorte della figlia, il conte di Barcellona, col nipote primogenito
don Juan, muove col suo esercito contro il regno d’Irlanda. Il re risolve di
consegnare loro Enrique. Isabela, che nel frattempo ha appreso la verità da
Otavio, raggiunge le truppe spagnole travestita da uomo e si accusa di esse-
re il seduttore di Dionisia, costringendo Otavio a rivelare la verità. Isabela
ed Enrique sono così ricongiunti mentre Dionisia sposa Otavio. 183 Anna Tedesco I personaggi del dramma di Noris sono: Alfonso re delle Asturie e sua
figlia Gelinda, il principe Enrico vassallo di Alfonso, marito di Attilia fi-
glia del re delle Gallie, Teoderico; Ariene, che dissimulata nelle vesti del
paggio Alindo serve Gelinda, e suo fratello Gubaldo, consigliere di Al-
fonso; il figlioletto di Enrico e Attilia, Erenio; infine il loro servo Zelto. 7 I travestimenti maschili di personaggi femminili sono frequenti sia nel teatro
del siglo de oro che nell’opera italiana coeva. Anche gli altri adattamenti italiani de
La fuerza lastimosa utilizzano questo espediente: nello scenario della Casanatense
Isabella, sedotta da Enrico nove anni prima, lo segue travestita da pellegrino, cfr.
Vuelta García (2006: 178). Nella versione di Scarnelli, nel II atto Isabella si presenta
a corte come Florante e la principessa se ne innamora credendola un uomo. Cfr.
Marchante Moralejo (2007: 229). Alfonso primo di Matteo Noris e La fuerza lastimosa Enrico è oggetto della passione della principessa Gelinda, così come En-
rique lo è dell’infanta nella Fuerza; Attilia equivale a sua moglie Isabela
mentre Gubaldo, innamorato di Gelinda, si comporta come il duca Ota-
vio, riuscendo ad introdursi nelle stanze della principessa e a trascorrere
la notte con lei. Il dramma per musica riduce il numero dei personaggi
da venti a nove, eliminando diversi personaggi secondari come Clenar-
do, consigliere del re, il marchese Fabio (di cui Gubaldo assume alcune
funzioni), gli altri figli di Enrique e i criados del duque Otavio; allo stesso
tempo introduce un personaggio che non trova corrispondenza nella co-
media: si tratta di Ariene, che nelle vesti maschili di Alindo serve Gelinda
come paggio. Sorella di Gubaldo (che però non conosce la sua vera identi-
tà) è stata precedentemente amata dal re Teoderico e vuole riconquistar-
lo; la sua presenza alla corte di Alfonso si spiega proprio con il desiderio
di impedire le imminenti nozze tra Teoderico e Gelinda. A questo scopo
lei stessa, nell’antefatto, ha introdotto Gubaldo nelle stanze della princi-
pessa. Il servo Zelto riassume in sé le figure dei vari criados: un servo di
questo nome ricorre in diversi drammi per musica veneziani coevi, quali
La pace tra Tolomeo e Seleuco (1691), L’Ibraim sultano (1691), Nerone fatto
Cesare (1692), Il Domizio (1696). La Tavola 2 elenca in corrispondenza i personaggi della Fuerza lasti-
mosa e quelli di Alfonso primo. L’introduzione del personaggio di Ariene, e di conseguenza di una ter-
za coppia di amanti formata da lei e dal re Teoderico, è la differenza più
evidente tra il plot di Noris e quello di Lope: la fanciulla ricopre un ruolo
familiare agli spettatori del melodramma secentesco, quello della donna
abbandonata che sotto vesti maschili va in cerca dell’amante traditore. Ol-
tre a scelte drammaturgiche, la presenza di Ariene potrebbe essere dovuta
alla necessità di impiegare una cantante della compagnia, tuttavia questo
ruolo en travesti ricorda anche il travestimento di Isabela, che nella ter-
za jornada si presenta in veste di soldato e si accusa di essere il seduttore
dell’infanta mostrando l’anello che la principessa ha donato a Otavio, co-
stringendo questi a confessare la verità7. 4
ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 184 Tavola 2. 8 Lo apprendiamo dal suo dialogo con Ariene: Gubaldo «Ma che fra l’ombre de
la notte oscura / da Gelinda creduto il prence Enrico / Gelinda io già godei / noto è
ad alcun?». Ariene «Chi può saperlo? io solo / (perché non sia di Teoderico sposa) /
te, in loco del ritroso / prencipe, a sua beltà barbaro e crudo / guidai dentro le soglie». Ariene Ariene
Come te n’avvedesti? Gelindai Gelinda
Alindo o fido Alindo,
se già strinsi notturna, ed al terz’anno
oggi più mesi aggiungo,
Enrico il mio tiranno; in aureo cerchio
se mi diè fè di sposo; e come, stelle,
come del franco sire
incontrerò gli amplessi? pensa Ariene
(Destin pria dammi morte.) Gelinda iraconda e con furore
O Enrico, o fiero
mostro dell’alma mia: sì di repente
lasciasti me? Prendesti
di consorte e di padre
d’un’altra in braccio i titoli amorosi? Alindo: ah ben m’avvidi,
che, quando ei mi stringeva, in quel momento
perfido, ingannatore
concepia nel diletto il tradimento. Alfonso primo di Matteo Noris e La fuerza lastimosa Comparazione tra i personaggi de La fuerza lastimosa e di Alfonso primo
La fuerza lastimosa
Alfonso primo
Belardo, criado del Conde Enrique
Zelto servo [di Enrico e Attilia]
Celinda, dama de la Infanta
-
Clenardo, secretario del Rey
-
Don Juan niño, hijo de Doña Isabela y
del Conde Enrique
Erenio fanciullo, loro figlio [di Enrico
e Attilia]
Doña Isabela, mujer del Conde Enrique
Attilia figlia di Teoderico
El Capitán Carlos, capitán, español
-
El Conde de Barcelona
Teoderico re delle Gallie [padre
d’Attilia]
El conde Enrique
Enrico principe vassallo d’Alfonso
marito d’Attilia
El duque Octavio
Gubaldo principe fratello di Ariene, suo
[di Alfonso] consigliero
El marqués Fabio
-
El Rey de Irlanda
Alfonso I re dell’Asturia
Fenicio, soldado, español
-
Hortensio, criado del Conde Enrique
-
Infanta Dionisia
Gelinda sua figlia
Lucindo, soldado, español
-
Pescadores (2)
-
Polibio [Filipo] criado del Duque Otavio
-
Tereo, criado del Duque Octavio
-
Villanos (2)
-
-
Ariene con nome di Alindo in abito da
paggio, servo di Gelinda 185 Anna Tedesco Come avviene in quasi tutti gli adattamenti italiani, Noris abolisce la
prima jornada ma, al contrario di quanto fa, ad esempio, Celano, egli in-
forma già nel I atto lo spettatore degli eventi accaduti: nella scena ix Gu-
baldo rivela di essere lui il seduttore8, mentre nella scena iii Gelinda narra
l’incontro notturno col suo amante ad Ariene/Alindo che in alcuni a parte
svela il suo amore per Teoderico: ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 186 Il I atto segue abbastanza fedelmente la seconda jornada della come-
dia, dalla scena in cui Dionisia scaccia via i musicisti che vorrebbero ralle-
grarla fino al momento in cui il re ordina ad Enrico di uccidere la moglie9. Come in Lope, è proprio Enrico (nella scena ultima) a consigliare al re di
far assassinare la moglie del seduttore da quest’ultimo, in modo che possa
sposare la principessa. Altre scene del I atto del libretto sono chiaramente
derivate dall’ipotesto: il colloquio tra la moglie di Enrico e la principes-
sa che ne ravviva la gelosia (scena vii), la confessione di questa al padre
attraverso una lettera (scena xv), che però non è scritta in presenza del re
ma a lui consegnata da Ariene. Il II e il III atto di Alfonso primo invece si
allontanano sia da Lope sia dagli altri possibili ipotesti: Enrico comuni-
ca sì alla moglie che dovrà morire ma in effetti non la uccide né affida ad
altri il compito di farlo. Attilia rimane a corte, interrogandosi sulla fedel-
tà del marito, mentre Gelinda fomenta la sua gelosia e addirittura attenta
alla sua vita (III, ix). La differenza più rilevante, oltre all’assenza del per-
sonaggio di don Juan quale giovane capitano, sta proprio nel ruolo di At-
tilia che è molto meno eroica di Isabela e soprattutto non ha alcun ruolo
nello svelamento della verità. Il lieto fine è raggiunto grazie ad Ariene che
rivela la propria identità e reclama Teoderico: Enrico viene scagionato e
riunito ad Attilia, Gelinda sposa Gubaldo e Ariene si ricongiunge a Teo-
derico. L’inganno perpetrato dai due fratelli, Ariene e Gubaldo, è giusti-
ficato perché motivato dalla salvaguardia dell’onore della donna: «piaga
d’onor / perché si sani / tutto lice tentar».fi Uno dei momenti più efficaci della Fuerza lastimosa è il momento in
cui Isabela, che sta per essere uccisa dal marito, chiede che i figli vengano
affidati a suo padre e supplica di rivederli un’ultima volta (Fuerza lasti-
mosa, II, vv. 1862-1873); tale momento aveva ispirato una delle scene più
celebri del Giasone di Cicognini e Cavalli (Antonucci, 2012: 268). La se-
quenza corrispondente (II, vi) è in Noris molto più asciutta: è Enrico a
dire alla moglie: «Pria che tu muoia / non vuoi, che almen ti abbracci? /
Veder non vuoi la prole?» ma non c’è nessuna invocazione della sposa ai
figli che non sono presenti. 9 L’ira di Dionisia è in Lope causata dal soggetto della canzone che narra di
Olimpia abbandonata da Bireno; nelle versioni italiane (tranne che in quella di
Todini) la canzone s’incentra sul mito di Arianna e Teseo (Vuelta García, 2006: 178,
188). Nei drammi tutti cantati di Noris e di Salvi la canzone è assente ma il riferi-
mento mitologico rimane: in Salvi è effigiato nelle pitture di una sala che viene de-
scritta (Marchante Moralejo, 2007: 267), in Noris nelle parole di Ariene a Teoderico
(I, xv): «Giurerei ch’è timore / l’angoscia dell’infanta; e ch’ella teme / o per novelli
amori o per antichi / ritrovar nel marito / la sorte d’Arianna». Gelinda
f
l Gelinda
Senti: fra l’ombre cieche, a l’or, che seco
io mi giacea, lo interrogai più volte
di suo amor, di sua fede. Ei muto a le dimande
non proferì parola;
m’abbracciò frettoloso;
se n’andò appena giunto; e dal mio seno
quando partir il cor fellon prefisse,
con voce che, crudele,
appena intesi, appena addio mi disse. piange pp
g
quando partir il cor fellon prefisse, q
p
con voce che, crudele, 8 Lo apprendiamo dal suo dialogo con Ariene: Gubaldo «Ma che fra l’ombre de
la notte oscura / da Gelinda creduto il prence Enrico / Gelinda io già godei / noto è
ad alcun?». Ariene «Chi può saperlo? io solo / (perché non sia di Teoderico sposa) /
te, in loco del ritroso / prencipe, a sua beltà barbaro e crudo / guidai dentro le soglie». ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 10 L’adattamento di Noris si avvicina in questa scelta a quello di Celano. Cfr.
Trecca (2016: 287). ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO Il pathos è recuperato nella scena ix del III at-
to, dove Gelinda si accinge a pugnalare Attilia, dopo averle fatto scrivere
con l’inganno una lettera di addio. Attilia si accomiata dal figlio non con
un’aria, ma con un recitativo, accompagnato da una pantomima indicata
da dettagliate didascalie (nella scena corrispondente del libretto di Napoli
il passo è tagliato tranne i primi due versi). 187 Anna Tedesco [Gelinda] se le avventa per ferirla, ella le tiene il braccio.ii [Gelinda] se le avventa per ferirla, ella le tiene il braccio. Attilia Aspetta, fin che al figlio
doni l’ultimo amplesso. La stessa Gelinda va a prendere Erenio, e con fretta lo guida
a lei. Gelinda Eccolo, stringi e bacia a un tempo stesso. Posto un solo ginocchio a terra dice ad Erenio Attilia. Attilia Erenio. Erenio le getta con impeto le braccia al collo e la bacia. Attilia O abbracciamenti o baci. Gelinda staccando Erenio da Attilia le dice:
Gelinda Basta. Si avventa per ferirla, la ferma Attilia. Attilia Dhe, un sol momento ancora
Dona a l’amor di figlio. Erenio si rubba dalla mano di Gelinda e corre di nuovo ad
abbracciare e baciare Attilia. O labbra, o bocca. Difficile dire se Noris conoscesse direttamente il testo di Lope o abbia
attinto ad uno dei rifacimenti italiani: come in questi ultimi gli avveni-
menti della prima jornada non sono rappresentati. Questa scelta, indot-
ta dal rispetto delle regole aristoteliche, è dovuta, a mio parere, anche
al desiderio di rendere Enrico del tutto innocente: egli non ha mai ri-
cambiato l’amore della principessa, né trama l’omicidio della moglie. Lo
spettatore può dunque condividere senza remore il suo dolore10. D’altra
parte Noris non utilizza elementi del testo fonte ripresi negli altri adat-
tamenti, quali il fallito annegamento di Isabela e l’arrivo dell’esercito
guidato dal figlio bambino, e ne aggiunge invece di nuovi come la gelo-
sia di Attilia e l’impazienza di Teoderico di concludere le nozze con Ge-
linda. Una debole spia della conoscenza de La fuerza lastimosa da parte
del drammaturgo sta nel nome scelto per la principessa, Gelinda, simile
a quello della dama di Dionisia, Celinda nel testo di Lope. Nessun altro
adattamento usa questo nome, mentre tutti concordano nel mantenere
quello di Enrique/Enrico. Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia 11 Per la data di rappresentazione, si veda Selfridge-Field (2007: 208). La studio-
sa propone di identificare il dedicatario di Alfonso primo con Juan Antonio Moles,
«Spanish special envoy». Non possono esserci dubbi che si tratti invece del diplo-
matico ceco Nostitz-Rieneck. Giovanni Moles, dedicatario di Sigismondo al dia-
dema nel 1696 (Selfridge-Field, 2007: 216), era un nobile napoletano sposato con
Maddalena Trivulzio dell’importante casata milanese. Era figlio di Francesco Moles
duca di Parete, uomo politico e diplomatico legato a Carlo II, che nel 1696 dava ini-
zio alla sua ambasceria a Venezia. Cfr. Miletti (2011), voce «Moles, Francesco». Su
Nostitz-Rieneck, cfr. Krueger (2009: 104 sgg.; 138). 12 Avviso da Venezia, 6 febbraio in: Avvisi italiani, ordinarii e straordinarii dell’anno
1694, Corriere ordinario, n. 14, 17 febbraio 1694. Raccolta della Österreichische National
Bibliothek di Vienna. Da notare che l’avviso cita il titolo dell’opera aggiungendo al nome
del re «di Castiglia», che non compare sul frontespizio del libretto, come nell’edizione
napoletana; inoltre utilizza l’altro nome con cui era conosciuto il teatro, S. Luca. 13 Se si guarda alla cronologia teatrale di Venezia, Napoli e Palermo non sono
pochi i titoli che dalla città lagunare si spostano al Sud: nello stesso 1694 un vecchio
dramma per musica dello stesso Noris, Il Bassiano, derivato peraltro da El mayor
imposible di Lope, venne intonato a Napoli da Alessandro Scarlatti. Anche gli altri
due drammi per musica andati in scena al S. Salvatore in quella stagione, Laodicea e
Berenice e La moglie nemica, vennero ripresi rispettivamente a Napoli il primo (1701,
musica di A. Scarlatti) e a Palermo il secondo (1698). Sui rapporti tra il Bassiano e il
testo fonte cfr. Profeti (2009: 446-448) e Badolato (2016). Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia Alfonso primo andò in scena il 28 gennaio 1694. La dedica, firmata da
Noris, è rivolta a «Antonio Gio. del Sacro Romano Impero conte di Nostis
e Arienck, consigliere di S.M.C. cameriero e assessore nella cancelleria auli-
ca di Boemia», da identificarsi col conte Anton Johan von Nostitz-Rieneck ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 188 (1659-1736), diplomatico e collezionista d’arte11. Un avviso ci ragguaglia
del successo dello spettacolo, dovuto soprattutto all’allestimento scenico. Il
testo prevede infatti ben sedici mutazioni di scena (tra cui sei sale dedicate
agli dèi Venere, Marte, Lucina, Giove, Sole, Mercurio, la cui descrizione è
derivata, a dire di Noris, da quella delle famose sale del palazzo di Lucullo –
forse ripresa da Plutarco), oltre ad una prima scena di contenuto allegorico
che anticipa il contenuto del dramma. Riporta l’avviso, datato 6 febbraio: Continua poi con tutta quietezza il Carnevale, non ostante l’infinito
numero di maschere; ed è andata sulla scena l’opera nuova nel Teatro
Vendramino di S. Luca, che ha il titolo di Alfonso primo re di Castiglia,
e riesce di tutta sodisfazzione, sendovi bellissime scene, machine e
apparenze. E ha gran concorso tanto di questa nobiltà, quanto ancora
de i prencipi e cavalieri forestieri, che qui si trovano e vanno capitando
giornalmente da varie parti12. Non sappiamo se tra i «prencipi e cavalieri forestieri» che videro l’o-
pera di Noris e Pollarolo a Venezia e la apprezzarono ci fosse qualche na-
poletano; esisteva peraltro una fitta rete di scambi di cantanti e partiture
da una parte all’altra della penisola, che aveva in Venezia uno dei centri
di irradiazione13. Sta di fatto che nel novembre dello stesso anno Alfonso
primo venne ripreso sotto il titolo di Alfonso il sesto re di Castiglia a Napo-
li per il compleanno del re di Spagna Carlo II e due anni dopo a Palermo 12 Avviso da Venezia, 6 febbraio in: Avvisi italiani, ordinarii e straordinarii dell’anno
1694, Corriere ordinario, n. 14, 17 febbraio 1694. Raccolta della Österreichische National
Bibliothek di Vienna. Da notare che l’avviso cita il titolo dell’opera aggiungendo al nome
del re «di Castiglia», che non compare sul frontespizio del libretto, come nell’edizione
napoletana; inoltre utilizza l’altro nome con cui era conosciuto il teatro, S. Luca. 14 La derivazione di Alfonso il sesto re di Castiglia dal precedente libretto vene-
ziano è stata indicata da Bianconi (1979: 89). A sua volta Maria Grazia Profeti inse-
risce Alfonso il sesto nel catalogo dei libretti derivati dal teatro aureo da lei realizzato
nel 2009, senza indicare uno specifico ipotesto ed invitando a compierne un esame
più accurato. Cfr. Profeti (2009: 373; 458).i 15 Alfonso il sesto re di Castiglia (1694). La dedica è firmata dall’impresario Nicolò
Serino, il 6 novembre 1694. Ho utilizzato l’esemplare della Biblioteca Universitaria di
Bologna, segnato A.V.Tab.I.E.III.29a.2. Il personaggio storico sarebbe Alfonso VI il
Valoroso (sp. el Bravo), n. 1040 ca. -m. 1º luglio 1109, re di Castiglia e León. fi
g
17 Lettere di Del Giudice a Medinaceli, Napoli 4 e 7 dicembre 1694, conservate
a Toledo presso la Fundación Casa Ducal de Medinaceli, Archivo Histórico, Fondo
“Correspondencia diplomatica IX duque de Medinaceli”, segnatura Leg. 24 r 5. 16 Lo stesso Perrucci si attribuisce il prologo dell’opera intitolata Alfonso nel suo trat-
tato Dell’arte rappresentativa premeditata e all’improvviso (1699: 176, Parte I, Regola, xv).
Cfr. anche Griffin (1993: 209), che ipotizza che il prologo sia stato musicato da Scarlatti. 14 La derivazione di Alfonso il sesto re di Castiglia dal precedente libretto vene-
ziano è stata indicata da Bianconi (1979: 89). A sua volta Maria Grazia Profeti inse-
risce Alfonso il sesto nel catalogo dei libretti derivati dal teatro aureo da lei realizzato
nel 2009, senza indicare uno specifico ipotesto ed invitando a compierne un esame
più accurato. Cfr. Profeti (2009: 373; 458).
15 Alfonso il sesto re di Castiglia (1694). La dedica è firmata dall’impresario Nicolò
Serino, il 6 novembre 1694. Ho utilizzato l’esemplare della Biblioteca Universitaria di
Bologna, segnato A.V.Tab.I.E.III.29a.2. Il personaggio storico sarebbe Alfonso VI il
Valoroso (sp. el Bravo), n. 1040 ca. -m. 1º luglio 1109, re di Castiglia e León.
16 Lo stesso Perrucci si attribuisce il prologo dell’opera intitolata Alfonso nel suo trat-
tato Dell’arte rappresentativa premeditata e all’improvviso (1699: 176, Parte I, Regola, xv).
Cfr. anche Griffin (1993: 209), che ipotizza che il prologo sia stato musicato da Scarlatti.
17 Lettere di Del Giudice a Medinaceli, Napoli 4 e 7 dicembre 1694, conservate
a Toledo presso la Fundación Casa Ducal de Medinaceli, Archivo Histórico, Fondo
“Correspondencia diplomatica IX duque de Medinaceli”, segnatura Leg. 24 r 5. Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia 13 Se si guarda alla cronologia teatrale di Venezia, Napoli e Palermo non sono
pochi i titoli che dalla città lagunare si spostano al Sud: nello stesso 1694 un vecchio
dramma per musica dello stesso Noris, Il Bassiano, derivato peraltro da El mayor
imposible di Lope, venne intonato a Napoli da Alessandro Scarlatti. Anche gli altri
due drammi per musica andati in scena al S. Salvatore in quella stagione, Laodicea e
Berenice e La moglie nemica, vennero ripresi rispettivamente a Napoli il primo (1701,
musica di A. Scarlatti) e a Palermo il secondo (1698). Sui rapporti tra il Bassiano e il
testo fonte cfr. Profeti (2009: 446-448) e Badolato (2016). 189 Anna Tedesco per il compleanno della regina14. A Napoli la rappresentazione avvenne
il 6 novembre nel palazzo reale e come di consueto in queste occasioni fu
preceduta da un prologo encomiastico e accompagnata da rinfreschi, così
come descritto nella Gazzetta di Napoli: Per coronare sì nobil festeggiamento, fu la stessa sera ivi rappresentato
in musica un Melodrama bellissimo & universalmente applaudito;
intitolato Alfoso [sic] re di Castiglia, e vi fu un prologo, o sia
introduzione di nobilissima inventione, e furono dispensati in immensa
copia rinfreschi pretiosissimi di tutte le sorti, riuscendo ricco, allegro e
plausibilissimo questo Real festino (Griffin, 1993: 208-209). Il libretto tace il nome del poeta, del compositore e degli interpre-
ti15. Nella dedica firmata dall’impresario Nicolò Serino si dichiara che
il soggetto è stato scelto dal dedicatario, il viceré conte di Santo Stefano
(Santisteban). Egli avrebbe dato ordine di «ravvivare sulle scene la me-
moria del giustissimo Alfonso sesto re di Castiglia», argomento quanto
mai adeguato per celebrare il compleanno di un monarca ispano, cui si
augurava lunga vita e numerosa prole. Anche il prologo, che si può attri-
buire alla penna di Andrea Perrucci, auspica l’arrivo del sospirato erede:
la stessa Monarchia di Spagna chiede conto alla dea Giunone dell’infer-
tilità della coppia reale, ed insieme interrogano Lucina, protettrice dei
parti. A sua volta essa consiglia di rivolgersi al Sole, che sorge e che li
rassicura, promettendo la nascita di «tanti eroi dal sangue austriaco»16. Un riferimento alla rappresentazione napoletana si trova nel carteggio
tra il cardinale Francesco del Giudice e il duca di Medinaceli, ambasciatore
del re di Spagna a Roma. Il cardinale si trova a Napoli ospite del viceré, che
lo conduce a teatro secondo le abitudini dell’epoca. 21 Essi erano: Pietro Antonio Fidi (castrato contralto, Totila), Giuseppe Trivelli
(castrato contralto, Vitige), Giuseppe Cesareo (basso, Belisario), Agata Carani
(soprano, Lepido), Anna Alfonso (soprano, Climene); Giuseppe Acciaro (bas- Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia L’opera non riscuote il
suo apprezzamento: in una prima lettera la definisce addirittura «descon-
soladisima» (tristissima) mentre la seconda rappresentazione gli appare più
riuscita. Neanche Giudice fa menzione di autori o cantanti17. Il libretto tace il nome del poeta, del compositore e degli interpre-
ti15. Nella dedica firmata dall’impresario Nicolò Serino si dichiara che
il soggetto è stato scelto dal dedicatario, il viceré conte di Santo Stefano
(Santisteban). Egli avrebbe dato ordine di «ravvivare sulle scene la me-
moria del giustissimo Alfonso sesto re di Castiglia», argomento quanto
mai adeguato per celebrare il compleanno di un monarca ispano, cui si
augurava lunga vita e numerosa prole. Anche il prologo, che si può attri-
buire alla penna di Andrea Perrucci, auspica l’arrivo del sospirato erede:
la stessa Monarchia di Spagna chiede conto alla dea Giunone dell’infer-
tilità della coppia reale, ed insieme interrogano Lucina, protettrice dei
parti. A sua volta essa consiglia di rivolgersi al Sole, che sorge e che li
rassicura, promettendo la nascita di «tanti eroi dal sangue austriaco»16. p
g
Un riferimento alla rappresentazione napoletana si trova nel carteggio
tra il cardinale Francesco del Giudice e il duca di Medinaceli, ambasciatore
del re di Spagna a Roma. Il cardinale si trova a Napoli ospite del viceré, che
lo conduce a teatro secondo le abitudini dell’epoca. L’opera non riscuote il
suo apprezzamento: in una prima lettera la definisce addirittura «descon-
soladisima» (tristissima) mentre la seconda rappresentazione gli appare più
riuscita. Neanche Giudice fa menzione di autori o cantanti17. ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 190 Due anni più tardi, il medesimo dramma musicale veniva ristampato a
Palermo per un allestimento che celebrava il compleanno di Maria Anna di
Palatinato-Neuburg regina di Spagna e che probabilmente venne realizzato
nel Palazzo reale nel corso di festeggiamenti per la guarigione del re da una
lunga malattia18. Infatti l’erudito palermitano Mongitore, che descrive tali
feste nel suo diario, fa cenno anche ad una commedia (per musica presu-
mibilmente) che venne rappresentata a palazzo il 30 ottobre: si tratta pro-
babilmente del nostro Alfonso il sesto (il compleanno della regina cadeva il
28 ottobre e la dedica del libretto al viceré di Sicilia, Pedro Colón duca di
Veraguas, reca la stessa data). Indi in palazzo si disposero comedie in musica, festini e balli, che
riuscirono di comune sodisfazione. 18 Alfonso il sesto re di Castiglia (1696). Esemplare conservato a Palermo, Biblioteca
Centrale della Regione Siciliana Alberto Bombace e segnato MISC. A.74.11. 20 Il prologo napoletano del 1694 venne peraltro utilizzato a Palermo l’anno
dopo per l’opera Nerone fatto Cesare, rappresentata per un altro compleanno reale.
Cfr. Lo Verde (2017: 71). 19 Mongitore (1871: 148-149). Su questi festeggiamenti cfr. Domínguez (2017). 18 Alfonso il sesto re di Castiglia (1696). Esemplare conservato a Palermo, Biblioteca
Centrale della Regione Siciliana Alberto Bombace e segnato MISC. A.74.11.
19 Mongitore (1871: 148-149). Su questi festeggiamenti cfr. Domínguez (2017).
20 Il prologo napoletano del 1694 venne peraltro utilizzato a Palermo l’anno
dopo per l’opera Nerone fatto Cesare, rappresentata per un altro compleanno reale.
Cfr. Lo Verde (2017: 71).
21 Essi erano: Pietro Antonio Fidi (castrato contralto, Totila), Giuseppe Trivelli
(castrato contralto, Vitige), Giuseppe Cesareo (basso, Belisario), Agata Carani
(
L
id ) A
Alf
(
Cli
) Gi
A
i
(b Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia E la nobiltà tutta si fece vedere in
segno di giubilo vestita con bizzarre gale, da molti anni a questa parte
non viste in Palermo; e il viceré fece a tutti dispensare copiosi rinfreschi. Una comedia fu fatta a 30 del presente mese d’ottobre19. L’opera si apriva anch’essa con un prologo, interpretato da Sicilia, Febo, Ibe-
ria, Giove, Giunone e Fato. Come di prammatica in componimenti del gene-
re, Giove e Giunone vi annunciano la guarigione del re e formulano l’augurio
di un erede. Il tutti finale «Quante stelle il Ciel rinserra / Quanti fiori ornan
la terra» riecheggia un’aria contenuta nel prologo napoletano «Quante stelle
in ciel risplendono / Quanti fiori ornano il suol», prova che l’anonimo poeta
palermitano avesse sott’occhio il libretto stampato a Napoli due anni prima,
del quale riprende con lievi varianti anche il testo di dedica e l’argomento20. q
p
g
Per quanto riguarda i cantanti impiegati e l’autore delle musiche, il
libretto non li nomina; Pietro Antonio Fidi che firma la dedica è un can-
tante attivo a Palermo alla fine del Seicento nell’ambito dell’Unione dei
musici di Santa Cecilia, un’associazione di mutuo soccorso che esercitò
un importantissimo ruolo nella disseminazione dell’opera veneziana a
Palermo, anche in quanto proprietaria del primo teatro d’opera di Sicilia,
il Teatro Santa Cecilia. Nello stesso anno quel teatro, di cui Fidi era im-
presario, inscenò un vecchio dramma di Matteo Noris, Totila, col titolo
modificato in Totila in Roma e le musiche di Francesco Gasparini: è pro-
babile che gli interpreti del Totila in Roma – a noi noti attraverso il libretto
– abbiano partecipato alla rappresentazione di Alfonso il sesto a palazzo21. 191 Anna Tedesco Il confronto tra i libretti stampati per le tre rappresentazioni fa emer-
gere modifiche consuete nel passaggio di un melodramma da una piaz-
za all’altra, dovute alla necessità di adeguarsi a nuovi cantanti e al gusto
del pubblico locale. In particolare possiamo notare che, come avveniva in
quegli anni, a Napoli il personaggio del servo Zelto assume maggior ri-
levanza e gli vengono assegnate alcune arie. A Palermo l’aspetto comico
è ancora più spiccato, dato che a Zelto viene affiancata la serva Lisetta. 23 Tale circostanza è già stata notata da Termini (1970: 219): «Alfonso Primo,
the extant Pollarolo opera on a Noris libretto, on the other hand, is totally devoid of
humor and extremely long-winded in explanations of the complex entanglements
of the story which result in an unusually large number of consecutive recitative
scenes». Si veda inoltre Termini (1970: 467) sulla posizione delle arie. so, Servio), Maria Rosa Gasparini (Marzia), Oliviero Matraia (castrato contralto,
Publicola), Rosa Russo (Cleria), Paolo Chirico (Desbo). Il libretto si conserva nella
Biblioteca universitaria di Bologna, ai segni A.V.Tab.I.F.III.59.4. Si può ipotizzare
che Fidi abbia interpretato Enrico, Agata Carani Ariene, ruolo en travesti, Chirico
il servo Zelto, Trivelli o Matraia l’altro ruolo per un cantante evirato, Gubaldo. Sul
Teatro Santa Cecilia, cfr. Tedesco (1992). 22 Cfr. Giacobello (1994). Se così fosse, la cantante che interpretò Attilia potreb-
be essere stata sua moglie Maria Rosa Borrini. Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia A
parte la presenza di quest’ultima, il libretto palermitano incorpora sostan-
zialmente le modifiche effettuate in quello di Napoli e dunque presumi-
bilmente venne usata la stessa musica; tuttavia quasi tutte le arie destinate
al personaggio di Attilia (che è la sorella di Teoderico e non la figlia come
nel libretto veneziano) vengono sostituite, probabilmente perché inadatte
alla cantante che avrebbe dovuto eseguirle. Si potrebbe ipotizzare per esse
la paternità di un musicista locale, se non addirittura quella del già citato
Gasparini che si trovava a Palermo nel marzo 169622. Il confronto tra i libretti stampati per le tre rappresentazioni fa emer-
gere modifiche consuete nel passaggio di un melodramma da una piaz- p
Nello spostamento da Venezia a Napoli, un’aria viene sostituita (Ge-
linda, II,i; Venezia “Imminente è il mio periglio”; Napoli “Per uscir da un
laberinto”) e ben ventidue nuove arie vengono aggiunte. Si tratta di un nu-
mero cospicuo, cosa che fa ipotizzare il coinvolgimento di un altro poeta
(forse lo stesso Perrucci?) e di un altro musicista attivi a Napoli ma stimo-
la anche delle riflessioni sulla struttura drammaturgica del testo di Noris. Sembrerebbe che i revisori lo abbiano ritenuto scarso di arie e abbiano di
conseguenza provveduto a farcirlo, addirittura inserendo due nuove arie
nella stessa scena, o in scene dove un’aria era già prevista. Il testo originale
di Noris presenta in effetti delle scene prive di arie e con lunghe tirate in
recitativo23; le arie sono poste in genere ma non esclusivamente in chiusura,
con il rientro del personaggio tra le quinte, secondo la tecnica che lo stesso
Noris definì «inanellatura delle scene» (prefazione a L’odio e l’amor, Vene-
zia 1702), ossia la liaison des scènes derivata dal teatro francese. Il libretto
napoletano, al contrario, interrompe frequentemente l’azione per permet-
tere al personaggio di esprimere il proprio stato d’animo attraverso un’aria. Emblematico il finale dell’Atto I che contiene uno degli snodi fondamentali ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 192 dell’intreccio: il re ha appena mostrato ad Enrico la lettera che lo accusa di
aver sedotto la principessa e gli dà ordine di eseguire quanto ha consigliato
lui stesso, ossia l’uccisione della propria moglie. Enrico rimane impietrito
e dopo aver letto ad alta voce la prima riga della lettera «si ammutisce, e
con atto di stupore entra», ossia esce di scena. Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia Alfonso
Senti: Siloe de Medi
tiene una figlia; a questa
prence di lui vassallo
pegno diè di consorte. L’abbracciò, la lasciò: cercasi il modo
onde viva redento il regio onore. Enrico
È facile signore. Alfonso
Come? Enrico
Il prence vassallo
sposi la regal donna. Alfonso
D’altra, s’egli è marito? Enrico
Mora la moglie. Alfonso
Questa
è suo conforto e vita. Napoli, I, ultima
Enrico
Il foglio che di Murcia
mi destina al comando ha ne la destra. Felice Enrico. Napoli, I, ultima
Enrico
Il foglio che di Murcia
mi destina al comando ha ne la destra. Felice Enrico. Da Venezia a Napoli a Palermo: Alfonso il sesto re di Castiglia Il libretto di Napoli, peraltro
molto meno accurato nelle didascalie sceniche, gli fornisce invece un’aria
dal contenuto piuttosto generico “Stelle fiere, che mai sarà?”. Venezia, I, ultima
Enrico
Il foglio che di Murcia
mi destina al comando ha ne la destra. Felice Enrico. Alfonso si volta e gli dice ridendo
Alfonso
Enrico
Enrico inchinato gli bacia la mano
di tua virtù prudente
ho d’uopo in questo punto. Enrico
La mente umilio a la sovrana legge. Alfonso
Siloe di Media il re di Alfonso amico
ricerca in questo foglio alto consiglio;
arduo, perch’io lo vedo,
o Solon dell’impero a te lo chiedo. Enrico
Sol chi nel mondo è Giove errar non può. Alfonso
Senti: Siloe de Medi
tiene una figlia; a questa
prence di lui vassallo
pegno diè di consorte. L’abbracciò, la lasciò: cercasi il modo
onde viva redento il regio onore. Enrico
È facile signore. Alfonso
Come? Enrico
Il prence vassallo
sposi la regal donna. Alfonso
D’altra, s’egli è marito? Enrico
Mora la moglie. Alfonso
Questa
è suo conforto e vita. Napoli, I, ultima
Enrico
Il foglio che di Murcia
mi destina al comando ha ne la destra. Felice Enrico. Alfonso
Enrico
di tua virtù prudente
ho d’uopo in questo punto. Enrico
La mente umilio a la sovrana legge. Alfonso
Siloe di Media il re di Alfonso amico
ricerca in questo foglio alto consiglio;
arduo, perch’io lo vedo,
o Solon dell’impero a te lo chiedo. Enrico
Sol chi nel mondo è Giove errar non può. Alfonso
Senti: Siloe de Medi
tiene una figlia; a questa
prence di lui vassallo
pegno diè di consorte. L’abbracciò, la lasciò: cercasi il modo
onde viva redento il regio onore. Enrico
È facile signore. Alfonso
Come? Enrico
Il prence vassallo
sposi la regal donna. Alfonso
D’altra, s’egli è marito? Enrico
Mora la moglie. Alfonso
Questa
è suo conforto e vita. Venezia, I, ultima
Enrico
Il foglio che di Murcia
mi destina al comando ha ne la destra. Felice Enrico. Alfonso si volta e gli dice ridendo
Alfonso
Enrico
Enrico inchinato gli bacia la mano
di tua virtù prudente
ho d’uopo in questo punto. Enrico
La mente umilio a la sovrana legge. Alfonso
Siloe di Media il re di Alfonso amico
ricerca in questo foglio alto consiglio;
arduo, perch’io lo vedo,
o Solon dell’impero a te lo chiedo. Enrico
Sol chi nel mondo è Giove errar non può. Alfonso Enrico
di tua virtù prudente
ho d’uopo in questo punto. Enrico Tu prendi, gli dà la lettera e a momenti
esequisci. Enrico doppo letta la prima riga della lettera
dice
Enrico
Mio re…
Alfonso
Chiudi quel labro. Qui a momenti la moglie
a te verrà: esequisci
fellone il tuo consiglio
o su la moglie uccisa
sbranato avrai dinanzi a gli occhi il figlio. parte sdegnato
Legge forte Enrico la lettera
Enrico
“Fé di sposo mi dié tra l’ombre Enrico”
Io? Fé di sposo? Enrico? i
ti
tt di t p
t Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. Tu prendi, gli dà la lettera e a momenti
esequisci
Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. Tu prendi, e a momenti esequisci. Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. Tu prendi, e a momenti esequisci. Enrico
Mio re…
Alfonso
Chiudi quel labro. Qui a momenti la moglie
a te verrà: esequisci
fellone il tuo consiglio
o su la moglie uccisa
sbranato avrai dinanzi a gli occhi il figlio. Enrico
“Fé di sposo mi dié tra l’ombre Enrico”
Io? Fé di sposo? Enrico? Stelle fiere, che mai sarà? Congiurar sopra le sfere
contro me forse volete? E mutandovi in comete
armare i vostri rai di crudeltà? Stelle ecc. Enrico Enrico
Sol chi nel mondo è Giove errar non può. 193 Anna Tedesco 193
Anna Tedesco
Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. Tu prendi, gli dà la lettera e a momenti
esequisci. Enrico doppo letta la prima riga della lettera
dice
Enrico
Mio re…
Alfonso
Chiudi quel labro. Qui a momenti la moglie
a te verrà: esequisci
fellone il tuo consiglio
o su la moglie uccisa
sbranato avrai dinanzi a gli occhi il figlio. parte sdegnato
Legge forte Enrico la lettera
Enrico
“Fé di sposo mi dié tra l’ombre Enrico”
Io? Fé di sposo? Enrico? si ammutisce e con atto di stupore entra. Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. Tu prendi, e a momenti esequisci. Enrico
Mio re…
Alfonso
Chiudi quel labro. Qui a momenti la moglie
a te verrà: esequisci
fellone il tuo consiglio
o su la moglie uccisa
sbranato avrai dinanzi a gli occhi il figlio. Enrico
“Fé di sposo mi dié tra l’ombre Enrico”
Io? Fé di sposo? Enrico? Stelle fiere, che mai sarà? Congiurar sopra le sfere
contro me forse volete? E mutandovi in comete Enrico
E di sua vita egli in pena del fallo
il carnefice sia. Alfonso
Qual colpa danna
la semplice in amor donna innocente? Enrico
Onor offeso è giudice inclemente. Alfonso
Dunque al nume d’onore
tal vittima destini? Enrico
È tale il mio consiglio. Alfonso
È atroce ed empio. Enrico
Ai ministri del re serva d’esempio. Alfonso
Ma se tu fossi il grande
disonorato Re, così faresti? Enrico
Certo così farei. Alfonso
Soldati: voi quest’usci custodite. ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 194 Per far posto alle arie, il recitativo viene spesso abbreviato; tuttavia
l’eliminazione di alcuni versi non si deve solo a ragioni di brevità quanto
alla necessità di censurare passi che alla corte napoletana suonavano for-
se troppo scabrosi e sessualmente espliciti. Si veda l’esempio seguente, in
cui il re si rivolge alla figlia dopo averne scoperto il segreto: Venezia, II, 2
Napoli, II, 2
Alfonso
Quando
Col franco re, con Teoderico, deggio
stringerti in sacro nodo,
Tu, che un altro ti strinse in chiuso soglio
Temeraria mi scopri. De l’onor tuo, de l’onor mio nemica,
Di nascosto, furtiva
Uno a te diseguale
Abbracciasti notturna? Un suddito del trono? Un vassallo del regno? Un, che, infame per l’opre, è prence indegno? Or di te che far deggio? Tu dillo: qui fra poco
Per annodarti sposa
Verrà il re Teoderico. Che gli dirò? che sei
Non più vergine? e donna
Senza marito? narrerò gli amplessi,
Che a l’amator occulta
Desti fra l’ombre? Io gl’Imenei promisi. Giurai le nozze, e le firmai ne’ fogli. Dir ciò che sei non deggio,
Mancar di fé non posso: audace, indegna
Figlia di Alfonso, Astrea, per sì gran colpa
Scarsa è di pene atroci, ed in sua mano
Folgor non ha che basti ‘l Giove ispano. Alfonso
Col re britanno Teoderic
stringerti in sacro nodo,
Tu, che un altro ti strinse in
Temeraria mi scopri. Or di te che far deggio? Tu dillo: qui fra poco
Per annodarti sposa
Verrà il re Teoderico. Dir ciò che sei non degg
Mancar di fé non posso:
Figlia di Alfonso. Astrea,
Scarsa è di pene atroci; e
Folgor non ha che basti Venezia, II, 2
Alfonso Napoli, II, 2 Venezia, II, 2 Venezia, II, 2
Alfonso
Quando
Col franco re, con Teoderico, deggio
stringerti in sacro nodo,
Tu, che un altro ti strinse in chiuso soglio
Temeraria mi scopri. De l’onor tuo, de l’onor mio nemica,
Di nascosto, furtiva
Uno a te diseguale
Abbracciasti notturna? Un suddito del trono? Un vassallo del regno? Un, che, infame per l’opre, è prence indegno? Or di te che far deggio? Tu dillo: qui fra poco
Per annodarti sposa
Verrà il re Teoderico. Che gli dirò? che sei
Non più vergine? e donna
Senza marito? narrerò gli amplessi,
Che a l’amator occulta
Desti fra l’ombre? Io gl’Imenei promisi. Enrico Enrico
“Fé di sposo mi dié tra l’ombre Enrico”
Io? Fé di sposo? Enrico? Stelle fiere, che mai sarà? Congiurar sopra le sfere
contro me forse volete? E mutandovi in comete
armare i vostri rai di crudeltà? Stelle ecc. ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO Giurai le nozze, e le firmai ne’ fogli. Dir ciò che sei non deggio,
Mancar di fé non posso: audace, indegna
Figlia di Alfonso, Astrea, per sì gran colpa
Scarsa è di pene atroci, ed in sua mano
Folgor non ha che basti ‘l Giove ispano. Alfonso Alfonso Quando
Col re britanno Teoderico, io deggio
stringerti in sacro nodo,
Tu, che un altro ti strinse in chiuso soglie [sic]
Temeraria mi scopri. Or di te che far deggio? Tu dillo: qui fra poco
Per annodarti sposa
Verrà il re Teoderico. Or di te che far deggio? Tu dillo: qui fra poco
Per annodarti sposa
Verrà il re Teoderico. Tu dillo: qui fra poco Per annodarti sposa Verrà il re Teoderico. Che gli dirò? che sei Non più vergine? e donna Senza marito? narrerò gli amplessi, Che a l’amator occulta Dir ciò che sei non deggio,
Mancar di fé non posso: audace indegna
Figlia di Alfonso. Astrea, per sì gran colpa
Scarsa è di pene atroci; ed in sua mano
Folgor non ha che basti il Giove Ispano. La stessa pruderie o una sorta di rispetto per la fonte letteraria da cui il
libretto si dice derivato, la Historia della perdita e riacquisto della Spagna,
emerge anche dalla premessa dell’autore della versione napoletana, molto
più lunga e articolata dell’altra. Semena dell’Argomento di Venezia «indu-
ce» l’amante «ad abbracciarla» e «s’ingravida». L’infanta napoletana è invece
«troppo proclive agli strali di Cupido». Nel libretto di Napoli l’argomento
riassume dettagliatamente quanto avviene nel I atto della Fuerza lastimosa,
che – ricordo – viene eliminato nel libretto di Noris. Inoltre si cerca una
maggiore verisimiglianza storica: Gubaldo rappresenterebbe il conte Rai-
mondo di Borgogna, reale marito dell’Urraca storica. 195 Anna Tedesco 24 Alfonso primo (1694). Cfr. Termini (1970: 288; 353-354). La partitura di
Pollarolo appartenne alla collezione del cardinale Ottoboni, cfr. Lindgren, Murata
(2018: 23-24). È curioso osservare che, stando ad una annotazione dei giornali di
Conforto, Ottoboni potrebbe aver assistito ad una recita di Alfonso il sesto, re di
Castiglia, dato che si trovava a Napoli alla fine del 1694. gi
25 I manoscritti in questione appartengono a raccolte di musica italiana conser-
vate rispettivamente nella Bibliothèque nationale de France, département Musique
(Rés. Vmf. ms. 35 e Rés. Vmf. ms. 88); nella Sächsische Landesbibliothek - Staats-,
und Universitätsbibliothek di Dresda (Mus. 1-J-2,3) e nella Biblioteca Nacional de
España (M/2246). Alcune arie sono testimoniate da più di un manoscritto. Un altro
gruppo di otto arie, il cui testo coincide col libretto di Noris, si conserva presso la
Biblioteca Apostolica Vaticana (Barb.lat. 4131) ma le arie ivi contenute corrispon-
dono all’intonazione di Pollarolo. Si veda Lindgren, Murata (2018: 17-20). Ringrazio
Margaret Murata per l’aiuto prestato alle mie ricerche. Fornisco in appendice l’elen-
co delle arie collegate alle due intonazioni dell’opera che ho finora reperito, riman-
dandone l’esame ad altra occasione. ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 196 Napoli Dice il Rogatis To. 2 L. 4 n. 18 che Alfonso
primo re dell’Asturia ebbe una sorella
nominata Semena. Questa s’invaghì
d’un Grande suo vassallo. Segli scoperse
amante; egli repugnò, infine Semena lo
indusse ad abbracciarla, e s’ingravidò
di nascosto del fratello. L’aver avuto una figlia di nome Urraca
Alfonso il sesto re di Castiglia, e questa
troppo proclive agli strali di Cupido (al
riferire del Padre Rogatis al 3. tomo delle
Historie di Spagna) ha dato il motivo
all’autore del presente melodramma,
radolcendo il nome di Urraca in quello
di Gelinda, d’intrecciarvi i seguenti
verisimili. Io ho cambiato il nome di Semena in
Gelinda per accomodarlo alla musica,
& quello di sorella in figlia di Alfonso,
perché meglio mi cadde in acconcio. Dalle stanze famose di Lucullo Romano
ho preso l’esempio delle sei sale.i Che Gubaldo (in questo mutando ancora
il nome di Raimondo conte di Borgogna)
acceso dell’infanta Gelinda, l’avesse per
mezo d’Alindo (creduto di lei paggio)
goduta tra gli orrori della notte col nome
di Enrico principe di Candespina, di cui
vivea ardentemente innamorata Gelinda,
e che lasciato le avesse in pegno d’averla
sposata un aureo cinto, con una gemma
in essa impressavi l’imagine d’Enrico, da
questi a caso ad Alindo dato, all’ora che
si partì per Bertagna fuggendo gli amori
importuni dell’Infanta. Quello vi è più è finzione per far intreccio
maggior al Drama. Sta sano. Che Alindo fusse Ariene sorella di
Gubaldo, ma da lui non conosciuta (per
essersi allontanato fanciullo da Borgogna)
e che venuta in Castiglia fuggendo il
disprezzo di Teoderico Re di Bertagna,
havesse machinato di far godere al fratello
l’Infanta, per impedire di Teoderico
le nozze, da cui stata era all’ora che fu
lasciata dal genitore estinto sotto la di
lui cura, con fede di sposo, e goduta, e
poi discacciata. p
Ch’Enrico avesse in Londra sposata
Attilia di Teoderico germana, da cui ne
avesse ottenuto tre figli, e per per suo
mezzo, Alfonso avesse trattato le nozze
di Teoderico con Gelinda. E che Teoderico debellato l’Eusino,
approdasse con l’armata navale nei lidi
d’Iberia, sì per dar soccorso ad Alfonso,
contro i suoi rebelli, come per isposare
Gelinda, precedendoli con la sposa
Enrico, per effettuarne le nozze. pf
Da questi verisimili premesse s’intreccia
la peripezia del drama, che prende dal
regnante Alfonso il nome. 26 Nel suo importante saggio, cui rinvio per la storia della collezione Prunières
e per notizie sui copisti e sui manoscritti, l’autore non elenca né discute in dettaglio
le arie provenienti da Alfonso il sesto. Le fonti musicali In quanto alle musiche che intonarono il testo nelle tre rappresentazioni,
la partitura veneziana di Pollarolo si conserva in una biblioteca california-
na24. Considerati i molti testi di arie nuovi presenti nel libretto stampato a
Napoli, è inevitabile pensare che in quella città il testo venne intonato ex
novo, in tutto o in parte. La corrispondenza testuale tra il libretto di Napoli
e quello di Palermo, oltre ai legami politici tra le due città, rende probabi-
le che in Sicilia si utilizzasse la versione musicale napoletana, tranne che
per poche arie aggiunte o cambiate, quelle di Lisetta e di Attilia già citate. p
p
gg
q
g
L’autore delle nuove arie intonate a Napoli potrebbe essere Alessandro
Scarlatti, ch’era allora maestro della cappella reale e dunque deputato a
comporre per le feste di corte. Non ci sono però documenti amministra-
tivi che dimostrino il suo coinvolgimento e Ulisse Prota Giurleo, nel suo
classico libro sui teatri di Napoli, ha ipotizzato che l’autore delle musiche
possa essere il più giovane Francesco Mancini (Prota Giurleo, 2002: 377,
vol. III). Tuttavia alcune arie dell’opera testimoniate in diverse raccolte
rendono molto plausibile l’attribuzione a Scarlatti, come ora vedremo. Si
tratta di tredici arie conservate in quattro manoscritti, due a Parigi, uno
a Dresda e il quarto a Madrid25. Solo recentemente alcune arie contenu-
te nel manoscritto Rés. Vmf. ms. 35 della collezione parigina sono state
identificate come provenienti da Alfonso il sesto ed attribuite a Scarlatti
(cfr. Ruffatti, 2015: 196)26. Le altre arie, contenute in un altro volume del-
la stessa collezione appartenuta al musicologo francese Henry Prunières,
e nelle raccolte di Dresda e Madrid, non erano finora state identificate,
oppure erano state attribuite a Pollarolo. Ritengo probabile l’attribuzio-
ne a Scarlatti perché le arie in questione si trovano all’interno di raccolte gi
25 I manoscritti in questione appartengono a raccolte di musica italiana conser-
vate rispettivamente nella Bibliothèque nationale de France, département Musique
(Rés. Vmf. ms. 35 e Rés. Vmf. ms. 88); nella Sächsische Landesbibliothek - Staats-,
und Universitätsbibliothek di Dresda (Mus. 1-J-2,3) e nella Biblioteca Nacional de
España (M/2246). Alcune arie sono testimoniate da più di un manoscritto. Un altro
gruppo di otto arie, il cui testo coincide col libretto di Noris, si conserva presso la
Biblioteca Apostolica Vaticana (Barb.lat. 4131) ma le arie ivi contenute corrispon-
dono all’intonazione di Pollarolo. Si veda Lindgren, Murata (2018: 17-20). Le fonti musicali Ringrazio
Margaret Murata per l’aiuto prestato alle mie ricerche. Fornisco in appendice l’elen-
co delle arie collegate alle due intonazioni dell’opera che ho finora reperito, riman-
dandone l’esame ad altra occasione. 26 Nel suo importante saggio, cui rinvio per la storia della collezione Prunières
e per notizie sui copisti e sui manoscritti, l’autore non elenca né discute in dettaglio
le arie provenienti da Alfonso il sesto. 197 Anna Tedesco di provenienza napoletana, che contengono arie di altre opere di Scarlatti
rappresentate negli stessi anni. Per quanto riguarda i manoscritti parigi-
ni, la questione dirimente è il fatto che il testo di quattro arie si trovi solo
nel libretto napoletano ed una quinta aria presenti un testo leggermente
diverso da quello intonato a Venezia, cosa che esclude per esse la paterni-
tà di Pollarolo. Le arie cui mi riferisco sono le seguenti: 1. “Deh perché sì dispietate”
(Gubaldo - NA III,6). F-Pn (Rés. Vmf. ms. 88, c. 5v). 2. “Dove mi lasci o sposo?”
(Attilia - NA II, 7). F-Pn (Rés. Vmf. ms. 88, c. 5r). 3. “Io non so, se nel mio petto”
(Ariene - NA I, 8). F-Pn (Rés. Vmf. ms. 88, c. 8v). 4. “Miei pensieri date a l’armi”
(Attilia - III,3)
F-Pn (Rés. Vmf. ms. 88, cc. 6r-6v27). 5. “No, che non v’è del mio
più crudo affanno”
(Gubaldo - NA 1,8)
F-Pn (Rés. Vmf. ms. 35, cc. 5r-7r). Le prime quattro sono copiate di seguito nello stesso manoscritto (Rés. Vmf. ms. 88) ma solo la n. 3 viene associata al titolo di un’opera, Alfonzo
re d’Asturia. La quinta aria si trova nel manoscritto segnato Rés. Vmf. ms. 35, e spicca sia per la presenza di un magnifico capolettera ornato, sia per
l’indicazione del nome dell’interprete «s.r Nicolino», da identificarsi con
il celebre castrato Nicola Grimaldi. Altre due arie dello stesso manoscritto
presentano queste caratteristiche: l’aria di Ariene “Adorata cara speranza”
e quella di Enrico “Adorate luci care”, entrambe interpretate da due star
dell’epoca «s.ra Vittoria», Vittoria Tarquini detta la Bombace, e «s.r Mat-
teo», il celebre soprano Matteo Sassano detto Matteuccio28. Il manoscritto
sopra citato contiene inoltre altre quattro arie per le quali viene indicata
la provenienza: «L’Alfonzo 1694». Gli altri due volumi che conservano arie provenienti da Alfonso il sesto
re di Castiglia sono uno a Dresda e l’altro a Madrid: il primo appartenne al
musicista romano Antonio Agostino de Rossi (fl. 27 L’incipit dell’aria intonata a Venezia è “Pensieri date all’armi”. Si veda l’elenco
delle arie in appendice. Vengono indicate in grassetto le quattro arie presenti solo
nel libretto napoletano.
28 Grazie alle ricerche di Sarah Iacono su Pirro e Demetrio, sappiamo che
Matteuccio cantò a Napoli in quella stagione e che fu impiegato anche al Palazzo
reale, così come l’architetto Filippo Schor, probabile autore delle scenografie. Cfr.
Iacono (2008). 28 Grazie alle ricerche di Sarah Iacono su Pirro e Demetrio, sappiamo che
Matteuccio cantò a Napoli in quella stagione e che fu impiegato anche al Palazzo
reale, così come l’architetto Filippo Schor, probabile autore delle scenografie. Cfr.
Iacono (2008). 27 L’incipit dell’aria intonata a Venezia è “Pensieri date all’armi”. Si veda l’elenco
delle arie in appendice. Vengono indicate in grassetto le quattro arie presenti solo
nel libretto napoletano. Le fonti musicali 1709-1755), violoncelli-
sta della Hofkapelle, e passò poi nella collezione reale; in esso si conserva-
no anche alcune arie da un’altra opera di Scarlatti, Le nozze con l’inimico,
overo L’Analinda andata in scena al Teatro San Bartolomeo di Napoli il
23 gennaio 1695. Il secondo volume fa parte di un gruppo di manoscritti ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 198 di origine italiana oggi conservati alla Biblioteca Nacional de España, che
appartennero probabilmente a diplomatici spagnoli di stanza a Napoli alla
fine del Seicento: i due possessori più probabili sono il conte de Santiste-
ban e il duca di Medinaceli, entrambi viceré di Napoli (cfr. Anglés, Subirá,
1946-1949: 412, vol. I)29. Le opere testimoniate nel volume che contiene le
arie da Alfonso il sesto (segnato M/2246) sono in gran parte di Scarlatti, e
furono tutte rappresentate a Napoli negli anni Novanta del Seicento: La
Teodora Augusta, 1692; Gerone tiranno di Siracusa, 1692; Il Pirro e Deme-
trio, 1694; Massimo Puppieno, 1695; Nerone fatto Cesare, 1695; Le nozze
con l’inimico, o vero L’Analinda, 1695; La caduta de’ Decemviri, 1697; Il
prigioniero fortunato, 1698. 29 Sulla provenienza di questo gruppo di manoscritti si veda Domínguez (2009). Conclusioni In conclusione, oltre ad aver individuato in Alessandro Scarlatti il pro-
babile autore delle arie aggiunte in Alfonso il sesto re di Castiglia, spero di
aver dimostrato la derivazione del dramma per musica Alfonso primo di
Matteo Noris, nelle sue diverse versioni, dalla comedia La fuerza lastimo-
sa di Lope de Vega, per quanto probabilmente per il tramite di un adatta-
mento italiano preesistente. p
Resta da comprendere quali siano i motivi della rinnovata fortuna della
Fuerza lastimosa nell’anno 1694, con ben due drammi per musica (quello
di Salvi e quello di Noris) ispirati ad essa e a quanto pare del tutto indi-
pendentemente l’uno dall’altro: ulteriore prova, comunque, dell’enorme
vitalità del teatro del Fénix de los ingenios. 29 Sulla provenienza di questo gruppo di manoscritti si veda Domínguez (2009). 199 Anna Tedesco Appendice Appendice
Matteo Noris
Alfonso I (Venezia 28.1.1694, musica di Carlo Francesco Pollarolo)
Alfonso il sesto re di Castiglia (Napoli 6.11.1694, musica di
Alessandro Scarlatti)
Elenco delle arie identificate
Incipit
Personaggio,
atto, scena
Organico,
tempo
tonalità
Autore
Fonti musicali Note
1. “Adorata
cara
speranza”
(Ariene - II,
14; NA II,
15). S, b.c. Allegro C
Do M
Scarlatti
D-Dl Mus.1-
J-2,3,
102c-104r. E-Mn M/2246,
55v-56r. F-Pn, Rés. Vmf. ms.35, cc. 1r-3r. Interprete
indicata in
F-Pn: S.ra
Vittoria
[Tarquini]
2. S, b.c. 3/4 Sol M
Pollarolo
I-Rvat, Barb. Lat 4131 cc. 9-10v
3. “Adorate
luci care”
(Enrico -
I,13; NA I,
12). S, b.c. Allegro 3/8
La M
Scarlatti
E-Mn -
M/2246, c. 54v-55r. F-Pn, Rés. Vmf. ms. 35,
cc. 9r-11v. Interprete
indicato
in F-Pn:
S.r Matteo
[Sassano]
4. “Amor
tu mi
tradisti”
(Gelinda - I,
2; NA I, 1). S, b.c. C Sol M
Scarlatti
E-Mn M/2246,
cc. 52v-53r. F-Pn, Rés. Vmf. ms. 35,
cc. 3v-4v. 5. “Come
s’uccida e
s’impiaghe”
(Teoderico -
I, 12)
S. b.c. C Si
bemolle M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 83-84v
6. “Chi
fingere più
sa”
(Gelinda -
III, 3). S, b.c. Allegro C
Do M
Scarlatti
D-Dl Mus.1-
J-2,3, cc. 106
r-107v. 7. “Deh
perché sì
dispietate”
(Gubaldo -
NA III,6). S, b.c. Adagio C
Re M
Scarlatti
F-Pn Rés. Vmf. ms. 88 c. 5v. 8. “Disarmi
il tuo
consiglio”
(Gelinda
- II,12)
S b.c. 12/8 Do M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 59r-60v. Manca nel
libretto
consultato. Cfr. Lindgren-
Murata p. 21. ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO ANCORA SULLA FORTUNA DE LA FUERZA LASTIMOSA NELL’OPERA DEL SEICENTO 200 00
NCOR
SU
OR UN
U R
S
OS
N
O
R
S
C N O
Incipit
Personaggio,
atto, scena
Organico,
tempo
tonalità
Autore
Fonti musicali Note
9. “Dove mi
lasci o
sposo?”
(Attilia - NA
II, 7). S, b.c. C Si m
Scarlatti
F-Pn Rés. Vmf. ms. 88 c. 5r. 10. “Gelosa più
non sono”
(Attilia - I,
14)
S, b.c. 3/8 Do M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 3-4v. Anche I-PAc
11. “Gelosia la
vuoi con
me”
(Attilia - 1,8;
NA I, 7). S, b.c. Largo assai
C
Re m
Scarlatti
D-Dl, Mus.1-
J-2,3, cc. 101r-102r
E-Mn M/2246,
cc. 53v-54r. 12. “Io non so,
se nel mio
petto”
(Ariene - NA
I, 8). S, b.c. Minuet 3/8
Si m
Scarlatti
F-Pn, Rés. Vmf. Appendice ms 88,
c. 8v. 13. “Labro
dolce”
(Gubaldo -
1,9; NA 1,8). S, b.c. A tempo
giusto 12/8
Sol m
Scarlatti
E-Mn M/2246,
cc. 56v-59r. F-Pn, Rés. Vmf. ms. 35,
cc. 12r-15r. In F-Pn:
formato
simile a
quello delle
tre arie che
presentano
un
capolettera
ornato, che
qui manca
come se il
manoscritto
dovesse
essere
ultimato. 14. C, b.c. 12/8 Re M
Pollarolo
I-Rvat, Barb. Lat 4131 cc. 13-14v. 15. “Miei
pensieri
date a
l’armi”
(Attilia
- III,3). S, b.c. C Re M
Scarlatti
F-Pn, Rés. Vmf. ms. 88,
cc. 6r-6v. [Venezia:
Pensieri date
all’armi]
16. “No, che
non v’è
del mio
più crudo
affanno”
(Gubaldo -
NA 1,8). S, b.c. 3/8 La m
Scarlatti
F-Pn, Rés. Vmf. ms. 35,
cc. 5r-7r. Capolettera
ornato. Interprete
indicato in
F-Pn: S.r
Nicolino
[Grimaldi] Incipit
Personaggio,
atto, scena
Organico,
tempo
tonalità
Autore
Fonti musicali Note
9. “Dove mi
lasci o
sposo?”
(Attilia - NA
II, 7). S, b.c. C Si m
Scarlatti
F-Pn Rés. Vmf. ms. 88 c. 5r. 10. “Gelosa più
non sono”
(Attilia - I,
14)
S, b.c. 3/8 Do M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 3-4v. Anche I-PAc
11. “Gelosia la
vuoi con
me”
(Attilia - 1,8;
NA I, 7). S, b.c. Largo assai
C
Re m
Scarlatti
D-Dl, Mus.1-
J-2,3, cc. 101r-102r
E-Mn M/2246,
cc. 53v-54r. 12. “Io non so,
se nel mio
petto”
(Ariene - NA
I, 8). S, b.c. Minuet 3/8
Si m
Scarlatti
F-Pn, Rés. Vmf. ms 88,
c. 8v. 13. “Labro
dolce”
(Gubaldo -
1,9; NA 1,8). S, b.c. A tempo
giusto 12/8
Sol m
Scarlatti
E-Mn M/2246,
cc. 56v-59r. F-Pn, Rés. Vmf. ms. 35,
cc. 12r-15r. In F-Pn:
formato
simile a
quello delle
tre arie che
presentano 14. C, b.c. 12/8 Re M
Pollarolo
I-Rvat, Barb. Lat 4131 cc. 13-14v. 15. “Miei
pensieri
date a
l’armi”
(Attilia
- III,3). S, b.c. C Re M
Scarlatti
F-Pn, Rés. Vmf. ms. 88,
cc. 6r-6v. [Venezia:
Pensieri date
all’armi]
16. “No, che
non v’è
del mio
più crudo
affanno”
(Gubaldo -
NA 1,8). S, b.c. 3/8 La m
Scarlatti
F-Pn, Rés. Vmf. ms. 35,
cc. 5r-7r. Capolettera
ornato. Interprete
indicato in
F-Pn: S.r
Nicolino
[Grimaldi] 14. 201 Anna Tedesco Incipit
Personaggio,
atto, scena
Organico,
tempo
tonalità
Autore
Fonti musicali Note
17. “Non
palpitarmi”
(Attilia -
II,5; NA
II,4). S, b.c. 3/8 Mi m
Scarlatti
D-Dl, Mus.1-
J-2,3, cc. 104v-105v. F-Pn, Rés. Vmf. ms. 35, F. 7v-8. Appendice 18. S. b.c. 3/4 Do M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 17-18v. 19. “Sarà nel
volo un
folgore”
(Gelinda - I,
3; NA I, 2). S, b.c. C La M
Scarlatti
F-Pn, Rés. Vmf. ms. 35,
cc. 125v-126v. 20. S, b.c. C Re M
Pollarolo
I-Rvat, Barb. Lat 4131, cc. 1-2v. 21. “Su tre
sentieri
corra
veloce”
(La mente
umana, VE
I,1)
S, b.c. Re M 3/4
Pollarolo
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Institutionalism, Public Sphere, and Artistic Agency: A Conversation on 32° East Ugandan Art Trust
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Critical Interventions Critical Interventions Journal of African Art History and Visual Culture ISSN: 1930-1944 (Print) 2326-411X (Online) Journal homepage: http://www.tandfonline.com/loi/rcin20 Date: 12 October 2017, At: 02:02 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 In 2003, Koyo Kouoh (2013, p. 17) posed
the following question: “How is Africa, after fifty
years of independence, really determining its artis-
tic landscape?” This issue arises as a central con-
cern in order to confront some of the most
decisive elements in play in contemporary African
art. Kouoh’s question echoes a general concern
about the role of cultural policies and institutional
dynamics in producing sustainable engagement
within artistic and non-artistic communities and
effective interventions into the African public
space. At the core of her words lies an interest in
exploring the potentiality of organizational and
collective agency in shaping cultural agendas both
locally
and
transnationally. The
complexities
derived from operating simultaneously at different
levels, as well as from dealing with agencies and
agendas that in some cases have no previous expo-
sure to contemporary art, force us to reconsider
the terms under which infrastructural activist proj-
ects (Smith, 2012, p. 251) are critically framed. platforms and institutions has been fundamental
in complicating the limitations of a critique based
fundamentally on discursive and representational
achievements. If, as Mamdani (1996, p. 4) argued,
institutionalism is “that part of the colonial legacy
[...] which remains more or less intact,” this fully
applies to artistic production. Confronting this
legacy means paying attention to how economic
and cultural constraints often thwart African crea-
tive platforms and independent institutions. In the
same text, Kouoh (2013, p. 17) defined African
alternative art institutions as “power stations” that
“question hegemonic viewpoints, canons and nar-
ratives
of
art,
and
develop
and
manifest
approaches of knowledge production outside state
institutionalization.” My main objective in this
essay is to understand the potential and limitations
of those power stations and what kind of forces
they set into motion. The following interview with the current staff
of 32 East Ugandan Arts Trust (Teesa Bahana,
Fred
Batale,
Nikissi
Serumaga)
summarizes
the exchanges I held in Kampala during a
month-length research stay in November 2016
(Figure 1).1 Although Bahana and Serumaga
joined the institution fairly recently, they were
involved in previous activities in various ways. The conversation hinges intentionally on practice
and decision-making, attempting thus to chal-
lenge any clean and bureaucratic memory of the
initiatives organized by 32 East. By stressing
practical questions over intentionality and identi-
fication, it attempts to show how initiatives such Kouoh’s question also raises inevitable issues
of autonomy, social relevance, and sustainability. Critical Interventions 11, Issue 2 2017
© 2017 Critical Interventions: Journal of African Art History and Visual Culture
https://doi.org/10.1080/19301944.2017.1363501 Institutionalism, Public Sphere, and Artistic
Agency: A Conversation on 32° East Ugandan Art
Trust Carlos Garrido Castellano To cite this article: Carlos Garrido Castellano (2017) Institutionalism, Public Sphere, and Artistic
Agency: A Conversation on 32° East Ugandan Art Trust, Critical Interventions, 11:2, 116-131, DOI:
10.1080/19301944.2017.1363501 To link to this article: http://dx.doi.org/10.1080/19301944.2017.1363501 Published online: 11 Oct 2017. Submit your article to this journal
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A CONVERSATION ON 32 EAST UGANDAN ART TRUST Carlos Garrido Castellano, Universidade de Lisboa Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Although the conversations on institutional and
infrastructural
artistic
practices
have
gained
momentum recently (cf. Diouf & Fredericks,
2014; Harney, 2004; Kasfir, 2013; Makhubu &
Simbao, 2013; Okeke-Agulu, 2010; Okeke-Agulu
& Hassan, 2008; Pinther & Smooth, 2015), his-
tories of African art have traditionally focused
more on discourses and representations rather
than on institutional and instituent modes and
practices. In that sense, the activity of alternative Critical Interventions 11, Issue 2 2017
© 2017 Critical Interventions: Journal of African Art History and Visual Culture
https://doi.org/10.1080/19301944.2017.1363501 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 117 Figure 1. 32 East Ugandan Art Trust Space in
Kansanga,
Kampala. Copyright
Carlos
Garrido
Castellano. Alongside that archival purpose, 32 East has
attempted to transcend the elitism with which
contemporary art is still associated in Uganda,
despite the emergence of a concern for and inter-
est in socially driven collaborative practices and
public art.3 This has been done through two ini-
tiatives: the Artachat program and the KLA Art
Festival. The first consists of a series of talks tak-
ing place at 32 East’s space in the Kansanga dis-
trict, and focusing on a wide variety of topics,
among them public art, cultural industries, art
policies, entrepreneurship, and Kampala’s public
space as an artistic venue. Initially developed by
Gutteridge in Scotland before 32 East was
founded,
the
initiative
became
located
in
Kampala in 2013, where it began to address
pressing matters for the local community.4
Although the discussions were followed primarily
by people already interested in visual arts, the the-
matic scope of the debates has widened the reach
of
collaborators
in
order
to
include
other
“creatives” and publics not necessarily linked to
the academy or the gallery worlds. Limited as it
is, the experience has been valuable in bringing to
the fore key issues on urbanism and accessibility,
public culture, civic engagement, and resource
management, and it has therefore expanded con-
siderably the scope and focus of contemporary
art, while continuing a trajectory of politically
charged aesthetics.5 Whereas those debates are
connected with former debates on authenticity,
identity, and representation held above all at
Makerere University (Kyeyune, 2003), they also
attempt to enhance contemporary art’s social rel-
evance, addressing at the same time the contra-
dictions derived from the impact of neoliberalism
in post-dictatorial Uganda. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Against the backdrop
of collapsing national institutions and a gallery-
driven art panorama, the emphasis on processual
and collaborative practices over artistic produc-
tion we find in 32 East’s activities evidences a Alongside that archival purpose, 32 East has
attempted to transcend the elitism with which
contemporary art is still associated in Uganda,
despite the emergence of a concern for and inter-
est in socially driven collaborative practices and
public art.3 This has been done through two ini-
tiatives: the Artachat program and the KLA Art
Festival. The first consists of a series of talks tak-
ing place at 32 East’s space in the Kansanga dis-
trict, and focusing on a wide variety of topics,
among them public art, cultural industries, art
policies, entrepreneurship, and Kampala’s public
space as an artistic venue. Initially developed by
Gutteridge in Scotland before 32 East was
founded,
the
initiative
became
located
in
Kampala in 2013, where it began to address
pressing matters for the local community.4
Although the discussions were followed primarily
by people already interested in visual arts, the the-
matic scope of the debates has widened the reach
of
collaborators
in
order
to
include
other
“creatives” and publics not necessarily linked to
the academy or the gallery worlds. Limited as it
is, the experience has been valuable in bringing to
the fore key issues on urbanism and accessibility,
public culture, civic engagement, and resource
management, and it has therefore expanded con-
siderably the scope and focus of contemporary
art, while continuing a trajectory of politically
charged aesthetics.5 Whereas those debates are
connected with former debates on authenticity,
identity, and representation held above all at
Makerere University (Kyeyune, 2003), they also
attempt to enhance contemporary art’s social rel-
evance, addressing at the same time the contra-
dictions derived from the impact of neoliberalism
in post-dictatorial Uganda. Against the backdrop
of collapsing national institutions and a gallery-
driven art panorama, the emphasis on processual Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Figure 1. 32 East Ugandan Art Trust Space in
Kansanga,
Kampala. Copyright
Carlos
Garrido
Castellano. as 32 East couple theory and practice, aesthetic
and social interventions (Kester, 2015). Founded in 2012 by Rocca Gutteridge
and Nicola Elphinstone, 32 East has been
behind much of the development of contem-
porary Ugandan art taking place in the last
five years. More oriented toward processual
than to exhibitional activities, the Trust offers
a residency for artists and curators, a work-
place for the local artistic community, and an
open area for meetings and discussions. The
project’s space also functions somehow as an
archive, gathering catalogs, visual material, and
press releases of its own and other spaces’ ini-
tiatives. This is especially important in a con-
text with no tradition of documenting cultural
activities, and which seriously lacks archival
and written records concerning contemporary
art.2 In that sense, the Trust documents the
experiments produced in relation to it, includ-
ing those that took place in public locations. This is made evident both by the center’s
library, the only one in the city focusing on
contemporary African art, and by the 32 East
space
itself,
where
many
of
the
artworks
developed by artists in residency are kept and
integrated into the organization’s grounds. Interventions Interventions | Castellano 118 festival consisted of a public art exhibition called
12 Boxes Moving, which resulted from the joint
collaboration of eight local institutions, among
which 32 East was represented.7 After launching
an open call for artistic projects, 12 were distrib-
uted in an equal number of containers sparse
throughout the city.8 The choosing of the con-
tainers as artistic venue holds a strong symbolism
in the Ugandan context: containers evoke transi-
tiveness, mobility, and multipurpose usage; they
refer to the transit and exchange of goods, but certain malaise with Ugandan cultural policies. This leads to experimenting with forms of crea-
tivity bound to the contradictions of present-day
Kampala. 32 East addresses this issue by diversi-
fying their activities in order to reach multiple
audiences and serve as a nexus for contemporary
art in the city (Figures 2–5).6 oteca do conhecimento online UL] at 02:02 12 October 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 The second initiative fueled by 32 East is
the KLA ART Festival, whose third edition is
programmed for 2017. KLA ART was first pro-
duced in October 2012. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 In its first edition, the Figure 3. 32 East Library on African Art. Copyright
Carlos Garrido Castellano. Figure 2. 32 East Library and Media Center. Copyright Carlos Garrido Castellano. Figure 2. 32 East Library and Media Center. Copyright Carlos Garrido Castellano. Figure 3. 32 East Library on African Art. Copyright
Carlos Garrido Castellano. Downloaded by [b-on: Biblioteca do conhecimento online UL] a Figure 2. 32 East Library and Media Center. Copyright Carlos Garrido Castellano. Figure 3. 32 East Library on African Art. Copyright
Carlos Garrido Castellano. Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 119 Figure 4. Detail of a sculpture made by an artist in
residency in 32 East, now part of the regular display. Copyright Carlos Garrido Castellano. on art’s purposefulness, ownership, and rele-
vance. It also initiated a lasting dynamic of insti-
tutional collaboration binding together public
and private stakeholders. Katrin Pieters (2015, p. 65), who recently dedicated a long essay to public
art in Uganda, mentions that “KLA ART 012 was
a pilot, an experiment in many ways, from the
outset with the intention of following editions in
two or three-year intervals. The main aims were
to create new physical and mental spaces for
visual art projects and to interact with new and
different audiences. The festival was strictly non-
commercial to allow for ideas beyond a direct
saleability.” She added that “the festival had a
visionary, experimental aspect, attempting to
open up a space for new artistic but also curato-
rial approaches” opposed to the long tradition of
solo shows motivated mostly by marketable ends. oteca do conhecimento online UL] at 02:02 12 October 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Figure 4. Detail of a sculpture made by an artist in
residency in 32 East, now part of the regular display. Copyright Carlos Garrido Castellano. also to the informal economies within the city
landscape, where they are a common sight. The
idea behind this choice, furthermore, was to raise
public awareness of contemporary art, a phenom-
enon Ugandans usually identified with gallery
spaces and government-ruled museums. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 In that
sense, the accomplishment varied from project to
project: some aimed for interactivity and per-
formativity, while others were simply limited to
hanging the artworks inside the space.9 Whereas
some criticism focused on the fact that the con-
tainers were also “enclosing” art and containing it
in public spaces already exposed to “cultural for-
ms,” the festival did challenge the public’s views Downloaded by [b-on: Biblioteca do conhecimento online UL] a KLA ART 14, the second edition of the
event, was coordinated by 32 East and brought
interesting novelties to the Ugandan artistic
arena. Titled Unmapped, the festival attempted a
more intensive projection into the public space. In order to achieve that, the initiative was split
into three interrelated projects: a more conven-
tional exhibition that took place at the Uganda
Railways station in Central Kampala,10 a series of
studio visits highlighting the workplaces of local
artists,11 and a set of interventions called The
Boda Boda Project. The latter, which was not
intended to be the nucleus of the event nor the
depository of the major part of the funding, out-
numbered the regular gallery exhibition in terms
of visitors and critical response. All the local
newspapers and cultural journals dedicated a
space to the event, and the Contemporary& online
platform published a special focus on art in
Kampala.12 The initiative consisted of a series of
collaborations between Ugandan artists and the
boda boda drivers, whose vehicles were custom-
ized and then used regularly throughout the city. Figure 5. Detail of 32 East’s library, studios, and
workshop space. Copyright Carlos Garrido Castellano. Figure 5. Detail of 32 East’s library, studios, and
workshop space. Copyright Carlos Garrido Castellano. Interventions Interventions | Castellano 120 Figure
7. Detail
of
one
of
the
performances
integrating the Boda Boda Project. Image courtesy of
Teesa Bahana. The boda bodas, motorbikes providing taxi serv-
ices, are the most common mean of transport in
Uganda and East Africa. They also constitute a
cornerstone in Kampala’s popular culture and
informal trade network.13 Choosing them both
as artistic venue and as target community to col-
laborate with acknowledges their role in configur-
ing Kampala’s urban landscape and recognizes
their legitimacy (Figures 8–10). At the same
time, it raises questions about the capacity of
one-time artistic interventions for raising aware-
ness of regulatory and customary issues. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Partici-
pation
in
KLA
ART
was
framed
through
the customization of already existing boda bodas
(Figures 6 and 7). Figure
7. Detail
of
one
of
the
performances
integrating the Boda Boda Project. Image courtesy of
Teesa Bahana. In a recent article, Angelo Kakande (2016,
pp. 18–25) showed how Ugandan artists are
increasingly adopting partisan positions concern-
ing the influence of extralegal forms of violence
in the definition of the debates on Ugandan
public space. The projects belonging to The Boda
Boda Project make evident that concern. Among
the issues raised were everyday violence against
marginalized groups, for example, in the artistic
projects of Adonias Ocom Ekuwe and Xenson, Downloaded by [b-on: Biblioteca do conhecimento online UL] a The interventions, again, varied from project
to project, but in this case the terms of the dia-
logue were now more balanced and horizontal,
with each artist interacting with the drivers in
a sustained way. The Boda Boda Project also
functioned differently in terms of space, trading
the relatively controlled locations where the
containers were installed in KLA ART 2012 for
more daring routes patronized by boda boda
drivers. Figure 6. Workshop at 32 East space linked to the
preparation of the Boda Boda Project. Image courtesy
of Teesa Bahana. Figure 8. Detail of one of 32 East’s containers
during an exhibition opening. Image courtesy of
Teesa Bahana. Downloaded by [b-on: Bib Figure 8. Detail of one of 32 East’s containers
during an exhibition opening. Image courtesy of
Teesa Bahana. Figure 6. Workshop at 32 East space linked to the
preparation of the Boda Boda Project. Image courtesy
of Teesa Bahana. Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 121 Figure 9. Artachat flyer. Image courtesy of Teesa Bahana. by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] a Figure 9. Artachat flyer. Image courtesy of Teesa Bahana. the liability of passengers and drivers alike and
the lack of respect for passersby, which was the
case of the projects of Ronex Ahimbisibwe, Petro,
Babirye Leilah Burns, or the invisibility of boda
boda workers despite constituting a central sector
of Kampala’s economy, as Kino Musoke, Enock
Kalule Kagga, and Sandra Suubi did. Critical Interventions 11, Issue 2 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Other artis-
tic projects celebrated Ugandan popular culture
and vernacular creativity such as Stacey Gillian
Abe’s, Joshua Kagimu’s, Katumba Simon Peter’s,
Ogwang Jimmy John’s, or Kizito Mbuga’s. Finally, yet a third group of projects stressed the
choosing of the individual or collective with
whom the collaboration took place over the pro-
duction of symbolic value: Derrick Komakech Figure 10. Detail of one of the art conversation
sessions held at 32 East’s yard. Image courtesy of
Teesa Bahana. Figure 10. Detail of one of the art conversation
sessions held at 32 East’s yard. Image courtesy of
Teesa Bahana. Interventions Interventions | Castellano 122 chose to work with one of the few female boda
boda drivers, while the Disability Art Project
Uganda (DAPU) developed a wheelchair to be
attached to a motorbike, attempting at a time to
deal with social recognition and urban exclusion. In this last case, artists and audience join forces
from the conceptualization of the project to its
final development and materialization14 While
many of the interventions arose with a central
topic on mind that was supposed to develop into
a mobile artwork related to it,15 the format
allowed for more complex and interesting forms
of collaboration. The 2014 edition of KLA ART
generated mostly positive critical responses (see
Namakula, 2014; Serubiri, 2015), and it was
perceived by many of those interviewed as the
beginning of a “new mood” in contemporary art,
in terms of its projection into the public space
and deepening of the terms of collaborations set
into motion. addressing multiple and heterogeneous audien-
ces. Those elements are at the center of the fol-
lowing conversation. The interview has been
transcribed as is to preserve the voice and syntax
of the speakers. Castellano: Hello Teesa, Fred, Nikissi. All of you joined the 32 East Project
recently. What brought you here? Castellano: Hello Teesa, Fred, Nikissi. All of you joined the 32 East Project
recently. What brought you here? o conhecimento online UL] at 02:02 12 October 2017 Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Teesa Bahana: Fred has been around for
a longer time, I started in January and
Nikissi two months ago. Fred Batale: Good question. I had just
finished school, and I saw on the Internet
that 32 East was doing KLA ART, and
they wanted volunteers. So, I wanted to
get exposed to the art outside from the
school settings, because I had finished, I
wanted to explore what was happening,
so I applied to volunteer with KLA
ART. There were twelve containers, so
each volunteer was going to work with
one artist on one container, and then the
task of those volunteers was opening
those containers, which had artworks
inside, talking to people about the exhi-
bition, so you had to be knowledgeable
about the works and the artists. They
accepted me, Lillian Nabulime was par-
ticipating,18 and she wanted me to work
further with her. We worked at Kampala
International University, it was there
where our container was located, and
then later after the festival 32 East was
establishing, they started putting con-
tainers, studios, and so on, so I went
ahead to know which organization put
up this exhibition, and I came here to
know more about 32 East and the Besides operating at a local level, 32 East has
also attempted to redefine the geopolitics of
Ugandan art at a regional and transnational level
through several initiatives. To the already men-
tioned partnership that led into KLA ART 012
we can add its participation in the East African
artistic exchange program, a residency network
linking Uganda, Kenya, and Ethiopia, the collab-
oration in African and intercontinental platforms
such as PAN!C or ArtsCollaboratory,16 and the
elaboration of joint curatorial experiences such as
the Boda Boda Lounge project.17 32 East’s partic-
ipation in those platforms has brought new ques-
tions into the debates on art in Uganda, some of
which are addressed in the conversation that fol-
lows: the role of art residencies in the develop-
ment of regional solidarities, the possibilities and
limitations of external funding in determining
the artistic autonomy of local initiatives, and bet-
ter ways of producing a sustained engagement
with the local community while at the same time INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 123 opportunities they had. That’s how I
came and engage with them. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 When I
talked with Rocca [Gutteridge, one of
the project’s founders], she saw an
opportunity of me coming to work with
them, she was happy with the work I
did. I went ahead and applied, when she
showed me out, she mentioned there was
going to be a library, I asked who was
going to be the librarian, there was
nobody, so I saw that as an opportunity. I applied to be a librarian, and I started
covering some tasks, we collected inter-
esting books, we engaged the artists
asking them to suggest readings which
we could buy for the library. wanted to do, a sort of organizational/
business thinking, but in creative spaces,
that was perfect. But then I met Rocca
and she invited me to work at 32 East,
she originally said it was going to be
operational tasks, but in fact, she wanted
me to take over from her. I did not
know if I could do that, the project was
very big and I do not have a background
in art, which made it kind of intimidat-
ing, but then I thought about all the
other reasons why I wanted to do this
master’s degree, and I saw it as practical
application of that. It was giving me the
opportunity of putting into practice the
ideas that you have. I had been at 32
East a few times before, when I came
back to Uganda in my free time while I
was
living
outside
of
the
country. I started finding out what was going on
in the art community, about the differ-
ent galleries and people around. First of
all, I enjoyed the space, which is very
much a heaven in crazy Kampala, and I
also thought it as a base for a lot of things
to happen outside of it, a platform for
other people’s creativity. Castellano: Was it in 2012? Batale: Yes, it was KLA ART 012, it
happened in October, but I started
working here in February 2013. Bahana: I met Rocca from a friend, we
had some conversations, and she asked
me what are you interested in, what do
you like, and questions like that. That
was over a year, she was bringing these
up really often. I was working at that
time with a woman who ran an NGO
and a consulting, I was doing communi-
cation for her, and then on the side I was
working with music festivals, I was on
the team in charge of making it happen. Through that experience I enjoyed being
in touch with creative spaces, but I also
acknowledged that I am not that crea-
tive. I had been thinking about doing
my master’s [degree] after the job with
that woman, a program in creative cul-
tural entrepreneurship. It was what I Nikissi Serumaga: My background is in
film production and film festivals (I am
right now a bit out of work. . .). I have
been working for a festival, I discovered
that this position was available and I
wanted to apply, but I had also devel-
oped a connection with 32 East before,
because I really never lived far from the
space, so I used to come here quite often
to see what was going on, and then also
one of the projects I have been working
on came to 32 East, so there was a lot of
interaction with it beforehand. I also saw Interventions | Castellano 124 Castellano: How would you consider
the relation with the project’s funders? To what extent is external funding deter-
mining the program and the objectives? it as an opportunity on a personal level
to be able to move back to Kampala and
to stay here for a long period of time,
and really get to be an observer of the art
scene in a much long-term way. Bahana: The founders of 32 East did
not really put much of their own funds
into the organization, but had networks,
a lot of friends who were able to help,
they did a crowdfunding to get the stu-
dios opened and to create the library, so
that made it possible to set the project
up. Critical Interventions 11, Issue 2 2017 Castellano: Was it in 2012? However,
once
an
organization
moves from being funded in that sense
and becomes bigger, it is hard to find
economic support beyond personality
and family (Rocca’s mom did a lot, for
example). Some people stayed supportive
because they believed in the mission, and
others thought they did not know the
people anymore. But luckily the founders
did a lot to get long-term support, one of
our founders is based in the Netherlands,
that’s how we entered Arts Collaboratory,
which has been really helpful. Castellano: What differentiates 32 East
from other spaces in Kampala? What
does the “trust” structure bring to the
organizational scheme of the project? Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Bahana: I think the reason of it being a
trust is very random. When it was
founded it was a way of giving the proj-
ect this air of legitimacy, the fact that it
was founded by two British women who
were trying to say we are here for a while,
we are the Ugandan art trusts, we want
to play this role. . . The name at first was
a bit deceiving, but it has been helpful in
some ways, not so helpful in other ways. I think for being in Uganda, it’s particu-
larly useful when it comes to finding
local support, working with the Kampala
Capital City authorities. . . But then, for
Arts Collaboratory,19 which is the net-
work we are in and it is really about post-
colonialism, anti-structure, and so on,
being a trust is strange. The name of the
project thus sort of speaks to different
personalities and the way we have found
our place here. Batale: When they created this space, the
objective was to promote visual arts in
Uganda. They thought how best this can
be done, so they came with this idea of a
residence. You have many known artists
who are already promoted, so those ones
they do not need much support. The
point was how could we bring those
young artists who needed it, so we
attempted to make a mixture: you had
graduate artists and those just starting
working together with established artists
at the same time. That was the aim. Each
time you could have three artists. In our The other spaces in Kampala are either gal-
leries or studios, so for us it was really about the
artists, about thinking what does an artist need
most in terms of developing personal capacities,
and providing opportunities. Part of the project
is the studio, another is exhibition space, then
you have the library. . . It is a mix of things. Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 125 Castellano: Were the external funders
asking for any kind of conditions to
apply? first year, we did not get many applica-
tions. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Another aim was to bring art to a
broader public, that’s how we came with
KLA ART, and somehow artists could
connect with the artists to get feedback,
or to think about their practice, but also
for the public it made possible a contact
with art. Bahana: It was a very lengthy applica-
tion, about thirty pages. You had to
show what your organization is classified
as under the government, whom your
stakeholders are, and sort of prove that
you have an impact, but beyond that. . . I think part of what Arts Collaboratory
was looking for as well is that the found-
ers had an idea of how to run the organi-
zation
locally. They
wanted
local
leadership, whatever that looks like. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Castellano: What about arts collabo-
ratory? How would you describe that
partnership? What does it bring? In what
ways does it restrict 32 East’s Autonomy? Castellano: What about arts collabo-
ratory? How would you describe that
partnership? What does it bring? In what
ways does it restrict 32 East’s Autonomy? Bahana:
Arts
Collaboratory
brings
immense value to 32 East. It connects
us
to
twenty-three
different
spaces,
exposing our artists to residency oppor-
tunities
in
places
they
never
knew
existed. This is both in terms of general
application and more intentional efforts
to have some kind of artistic exchange. For example, Mas Arte Mas Accion in
Colombia worked with the Ministry of
Colombia on an open call that allowed a
32 East member and curator to travel to
Colombia for research. This year, we
welcomed a video artist from Colombia. These kinds of international exchanges
between countries with similar contexts
but weak direct connections would not
otherwise be possible. In addition, as an
organization we are directly connected to
our peers around the world facing similar
struggles or existing as models that we
can emulate. Arts Collaboratory is an
incredibly open network and we have
been able to learn so much from our
counterparts. I really cannot say that it’s
restricted our autonomy in any way. Castellano: How does the residency pro-
gram work? What kind of applications
do you receive? Bahana: I think it also varies over the
years, probably now since we are a bit
well known, the quantity has increased. Quality also varies a lot, some applica-
tions are very detailed, explaining what
they want to do, while others are not
defined at all. We meet, revise all the
applications, and then we decide. We
plan on a year-to-year basis. Batale: When we had just started in
2013 it was totally different. I think
people here are just starting to under-
stand the benefits of residencies. At
first only established artists understood
it, but they did not need our support. Young artists did not know how to
handle it. The first year we even filled
blanks with some friends. The follow-
ing year we had few applications as Interventions | | Castellano 126 Castellano: How does that support work
in terms of public art projects? well. Then the number increased and
we had to select carefully. well. Then the number increased and
we had to select carefully. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Castellano: How does that support work
in terms of public art projects? Castellano: How difficult was it to
maintain the programs you have? Batale: Being an art center working with
different artists, each of them brings a
different approach on how they want to
work. It depends on what each artist is
interested in doing. We try to facilitate
the process. Bahana: There are some programs that
started and have not been continued,
like the Tackling Texts initiative, a pro-
gram based on theoretical discussion. It
is easier to maintain those programs that
people follow more. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 During that time, we also
had an open studio with him. We also
had a Kenyan artist here at the same time,
and she had a very different experience,
and I think part of it has to do just with
personality. In Ethiopia, there is now no
art space such as 32 East, so when artists
were there they were working at a school,
so the experience was different as well,
but they had an exhibition there. In
Kenya, they have an exhibition at the Cir-
cle Art Gallery, which is the most success-
ful commercial art gallery in East Africa,
but we have not had anything that put
together artists from the three countries in
the same place, at the same time, although
the British Council wanted to do a joint
exhibition and to have the pictures of the
residencies online. Bahana: There have been different art
initiatives in sort of community spaces,
but not focusing on a permanent resi-
dency and contemporary art. Fas Fas, for
example, used to be like a restaurant
where everything was designed by artists,
and they had discussions there and held
exhibitions. But nothing had the objective
of supporting artists on a long-term base. Castellano:
Within
the
institution’s
space, there are many activities not
directly related to visual arts? Is that
common in other projects in Kampala? What does this variety bring to the
initiative? Bahana: We used to share this plot with
a dance NGO. Before you constantly
had dance classes, kids in the space,
which brought more activity, but now
those spaces are rented for many differ-
ent purposes. We coexist. If we have
resources to support other initiatives, we
support them, but our focus is on visual
art, because we are essentially the only
art organization not oriented to commer-
cial ends. Most spaces are single-issue, or
they are rented for everything. Castellano: What kind of comments
have you received about the fact of 32
East being a space for residencies and not
for selling? Batale: Some artists think this is good,
because it helps them to prepare them-
selves before they enter the commercial
world. For others, it would be nice to
have production and selling together. I think it is good to have spaces like this,
concentrated in developing and collabo-
rating. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Castellano: Now you mentioned the
artists, what proportion of Ugandan
artists do you usually have in your
residencies? Batale: But some of those programs
needed extra funding, which we could not
raise, but also the vision and the persons
working here changed. The Ekyoto pro-
gram, in which people used to share drinks
and conversation around a fire (a tradition
coming from our grandparents, who nor-
mally used those moments to talk about
history, how can we behave, how can we
develop, and so on), was also discontinued. Bahana: It varies. If you look at the pro-
gram and the funding we have, the
majority of residents are Ugandans. If
international artists have their own fund-
ing and want to apply, then we can sup-
port them with the application, but we
do not have a budget for that. The excep-
tion is the East Africa Exchange Program,
which is founded by the British Council. Through it we can send artists to Kenya
and Ethiopia, and they can come. Arts
Collaboratory also brings specific funding,
but it is something sporadic. The ratio
would be two international artists for
each five national ones. Castellano: How would you define your
audience? Is it the same as those in art
galleries or the Kampala Biennial? Batale: For me, there is a mixture of dif-
ferent audiences. When we did KLA
ART, we saw boda boda guys interacting
with the art pieces, we saw government
officials interacting as well, they came to
collaborate with us through the public
pieces in town. So, I think there is a mix-
ture. Each category works differently,
there are those who are interested in
what are we planning, those who attend
our activities, and then we have another
group which is happy just supporting the
art, coming onboard particular projects. Castellano: How does the East Africa
exchange program work? Is there any vis-
ibility in the home country of the proj-
ects developed through the residencies? Bahana: Again, it varies. We still have
some pieces, some of them because artists
cannot move them. Immy [Mali],20 for
example, who went to Ethiopia, worked
on a bed project and she could not bring Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 127 it back. But then, when she came back,
Eyob Kitaba from Ethiopia was still here
and then we had discussions about both
experiences. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 But for other people it should be
our work to drive them to the commer-
cial world. Serumaga:
As
somebody
who
has
arrived very recently to 32 East, I could
say that the fact that the project has so
many different kinds of activities going
on introduced me to the space. You find
many different catching points to see
what is going on. The Boda Boda Lounge
exhibition we had in November 2016,
for example, created a collaboration in
which some people continued interact-
ing with 32 East besides the exhibition, Castellano: Can you think about any
antecedent to 32 East in Uganda? Interventions | Castellano 128 from outside the project fit the objectives
of 32 East? Do they work differently
than those you conceive and develop? and made many people know about the
space. For me that gives a strong feeling
of community. Batale: We focus on visual art, but we
are also trying to move visual arts fur-
ther. We bring on board many people,
so they know what we are doing, and
that facilitates the engagement with
them. Serumaga: It depends. For example, the
Boda Boda Lounge was a collaboration
that came from Vansa,21 and was very
useful. We would never be able to cover
so many different countries and to pro-
cess so many different applications and
then be able to adjudicate them all. It is
a lot of work, so sharing it was positive. We were fifteen organizations sharing
the project. Castellano: Why did you choose the
Kansanga neighborhood? How do you
engage the people living around? Batale: Kansanga is close to town and
Nassa Road, where you find all the artis-
tic material, printing. . . It is a busy
place, we are connected to it, but we
offer a quiet space where artists can sit
and think. Batale: KLA ART showed how differ-
ently things can be produced and how
interesting they can be, what are they
bringing in terms of local impact. The
two editions show different ways of
approaching audiences. In 2014, the
event was fairly understandable and
communicated to many people, as it was
intending to do, whereas in the 2012
event, so many people did not under-
stand why the containers were placed on
those roads apart from the artists who
knew about it. NOTES Carlos Garrido Castellano (cgc@campus.ul.pt) is
FCT Post-Doctorate Researcher at the Centre for
Comparative Studies of the University of Lisbon. His
research focuses on socially engaged art, curatorship,
and postcolonial theory. Currently, he is the main
researcher of the “Comparing We’s: Collectivism,
Emancipation, Postcoloniality” research project. He
has done extensive fieldwork research in the Caribbean
area, the United States, and Africa, and he has collabo-
rated with journals such as Third Text, Social Identi-
ties, Travessia, Anthurium, and Cultural Dynamics. On that trajectory, see Kakande (2008). One of
Kakande’s most productive conclusions has to
do with asserting how Ugandan artists have
always tended to adapt their political criticism to
the weakness of civil society. The current interest
in the public can be seen as a decisive shift, one
that Kakande summarizes by saying that in the
21st century “the visual arts are making signifi-
cant inroads on the political scene” (p. 326). Downloaded by [b-on: Biblioteca do conhecimento online UL] a A strong precedent in that sense is the Ngoma
International Artists’ Workshop Uganda, a proj-
ect started in 1995 by Rose Kirumira Namubiru. See Namubiru (2008, 2014). A strong precedent in that sense is the Ngoma
International Artists’ Workshop Uganda, a proj-
ect started in 1995 by Rose Kirumira Namubiru. See Namubiru (2008, 2014). A strong precedent in that sense is the Ngoma
International Artists’ Workshop Uganda, a proj-
ect started in 1995 by Rose Kirumira Namubiru. See Namubiru (2008, 2014). Downloaded by [b-on: Biblioteca do conh The author thanks Fred Batale, Teesa Bahana, Nikissi
Serumaga, and the 32 East Ugandan Art Trust team
and artistic community for their contributions to this
interview. 7
Besides 32 East, the organizing institutions
included Makerere University, AKA Gallery,
Nommo
Gallery,
the
Ugandan
Museum,
Alliance Fran¸caise Kampala, and the Goethe-
Zentrum Kampala. The partnership worked
not only at the level of raising funding, but
also at the level of curating and decision-
making. Besides 32 East, the organizing institutions
included Makerere University, AKA Gallery,
Nommo
Gallery,
the
Ugandan
Museum,
Alliance Fran¸caise Kampala, and the Goethe-
Zentrum Kampala. The partnership worked
not only at the level of raising funding, but
also at the level of curating and decision-
making. Color versions of one or more of the figures in the arti-
cle can be found online at www.tandfonline.com/rcin. 1
The 32 East art initiative is often referred to as
“32” in Uganda. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 The insistence of public art and the public’s
interest
for
monuments,
festivals,
and
the
occupation of the public space in Kampala is
eloquent on that regard. Downloaded by [b-on: Biblioteca do conhecimento online UL] at 02:02 12 October 2017 Visitors started asking
why the containers, what were we trying
to do, but in 2014 it was easier, because
it related to things they interact with like
the boda bodas. The approach was also
interactive, and that moved many people
to participate. For example, Kajimo’s
piece, Boda Boda Theater, had drum-
ming, performances. . . and had a very
good impact. The people working at the
organization tried to connect with issues
of
interests. We
even
incorporated
people on bicycles who sharpen people’s
knives. Bahana: We do not get drop-ins very
often. There are some people curious
about the rentals, but people do not usu-
ally come to see what is inside. Some
people know about us outside in the
near area, those even collaborated with
us. Batale: We had a screening outside the
space that brought people, but not a lot. We have just started here. Whenever we
have an event, we use to spread it
through the social networks. . . Castellano: Let me go back to KLA
ART. The first edition was part of a big-
ger project coordinated from abroad by
Simon Njami and David Adjaye. To
what extent do the initiatives organized Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 129 Castellano:
How
was
the
festival
organized? Besides the initiatives I commented on in this
article, we should consider as important the crea-
tion of an art biennial, the functioning of Start
Journal, an online magazine devoting to critical
evaluation of contemporary Ugandan art, or
curatorial
projects
such
as
Simon
Njami’s
AtWork (2015). More information about the lat-
ter can be found at http://www.contemporar
yand.com/magazines/the-kampala-episode/. Batale: It was hectic. Too much work, it
needed team collaboration, and it was
actually because of working together that
KLA ART got funding. It was not easy,
there
were
disagreements
within
the
partnering organizations... as everywhere. NOTES g
2
The main sources on contemporary Ugandan art
are the unpublished Ph.D. dissertations of the
Art History Department at Makerere University. The need to produce a visual and critical mem-
ory of exhibitions and other artistic activities has
been addressed only recently, when some galler-
ies (AfriArt Gallery, Makerere Gallery, AKA
Gallery) began producing elaborate exhibition
brochures and commissioning critics to write
about those initiatives. 8
The chosen artists were Bwambala Ivan Allan,
Emma Wolukau-Wanambwa, Eria Nsubuga
“Sane,” Eric Mukalazi, Lilian Nabulime, Ronex,
Ruganzu Bruno, Sanaa Gateja, Stella Atal,
Waswad, Xenson, and Sue Crozier Thorburn
(a British artist living in Uganda and the only
foreigner in the show). The case of Lilian Nabulime could be a good
example of the first. Her project was a continua-
tion of the activity she developed since the early Interventions Interventions | Castellano 130 its first edition in 2014, it has itinerated across
several African countries, activating a number of
side events and initiatives in the different con-
texts where it lands. 2000s, when she started experimenting with
sculpture as a way of generating social awareness
about HIV/AIDS among illiterate communities
in Uganda. Having already a vast experience in
artistic collaboration, for her intervention in
2012 Nabulime stood in front of her container,
dialoguing with the audience about the meaning
of her sculpture and the social relevance of
AIDS. 18
See
https://cedat.mak.ac.ug/staff-profiles/lilian-
mary-nabulime. See
https://cedat.mak.ac.ug/staff-profiles/lilian-
mary-nabulime. y
9
See http://www.artscollaboratory.org/. 20
See https://immymali.wordpress.com/. 21 oteca do conhecimento online UL] at 02:02 12 October 2017 21
See http://vansa.co.za/. 10 10
Closed in 1992, with KLA ART the station was
recovered for the first time since then for public
usage. REFERENCES 11
This element was original in Ugandan art. Besides
“placing the artists into the map,” it served to
encourage a climate of dialogue among the com-
munity of creators and the festival’s audience. Diouf, M., & Fredericks, R. (Eds.). (2014). The arts of citi-
zenship in African cities. Infrastructures and spaces of
belonging. London, UK: Palgrave. Harney, E. (2004). In Senghor’s shadow: Art, politics, and the
avant-garde in Senegal, 1960–1995. Durham, NC:
Duke University Press. 12
See
http://www.contemporaryand.com/maga
zines/kla-art-puts-east-african-art-on-the-map/. 13
It is essential to remark here on the existence of a
strong tradition of customizing and diversifying
boda bodas, matatus, and other vehicles dedicated
to informal transport. KLA ART 014 seconds
this phenomenon, using art to channel some of
the—again—already
existing
aspirations
for
social recognition and improvements in security
conditions of the sector, while benefiting from
the vibrant visual inventiveness of Kampala’s
vernacular scene. It in no way initiated this con-
text of practice. 13
It is essential to remark here on the existence of a
strong tradition of customizing and diversifying
boda bodas, matatus, and other vehicles dedicated
to informal transport. KLA ART 014 seconds
this phenomenon, using art to channel some of
the—again—already
existing
aspirations
for
social recognition and improvements in security
conditions of the sector, while benefiting from
the vibrant visual inventiveness of Kampala’s
vernacular scene. It in no way initiated this con-
text of practice. Kakande, A. (2016). Art and the “ghost” of “military
dictatorship”: Expressions of dictatorship in post-1986
contemporary Ugandan art. Start: Journal of Arts and
Culture (Kampala), December 14, 1–27. Retrieved
from
http://startjournal.org/2016/12/art-and-the-
ghost-of-military-dictatorship-expressions-of-dictatorship-
in-post-1986-contemporary-ugandan-art/ Kakande, A. (2008). Contemporary art in Uganda: A nexus
between art and politics (Unpublished doctoral disserta-
tion). Johannesburg: University of the Witwatersrand. Kasfir, S. L. (2013). African art and agency in the workshop. Bloomington, IN: Indiana University Press. p
14
On DAPU, see http://www.freetocharities.org. uk/dapu/aboutus.html. p
14
On DAPU, see http://www.freetocharities.org. uk/dapu/aboutus.html. Kester, G. (2015). On the relationship between theory and
practice in socially-engaged art. Retrieved from http://
www.abladeofgrass.org/fertile-ground/on-the-relationship-
between-theory-and-practice-in-socially-engaged-art/ 15
The boda boda project was not exempt from a
surprising and flashy effect. For many, the artistic
customizations of boda bodas might have been
perceived
as
crazy
objects
amid
the
rows
of vehicles populating Kampala. While that gim-
micky dimension was present in the relations
between artists, drivers, and audiences, it cannot
account for the whole diversity of experiences
and exchanges engendered by the initiative. REFERENCES 16
See http://panicplatform net/; http://www arts 15
The boda boda project was not exempt from a
surprising and flashy effect. For many, the artistic
customizations of boda bodas might have been
perceived
as
crazy
objects
amid
the
rows
of vehicles populating Kampala. While that gim-
micky dimension was present in the relations
between artists, drivers, and audiences, it cannot
account for the whole diversity of experiences
and exchanges engendered by the initiative. 16 Kouoh, K. (Ed.). (2013). Condition report: Symposium on
building art institutions in Africa. Ostfindern: Hatje
Cantz Verlag. Kyeyune, G. (2003). Art in Uganda in the 20th century
(Unpublished doctoral dissertation). London, UK: SOAS. Makhubu, N., & Simbao, R. (2013). The art of change in
South Africa. Third Text Special Issue, 27, 299–302. Mamdani, M. (1996). Citizen and subject: Contemporary
Africa and the legacy of late colonialism. Princeton, NJ:
Princeton University Press. 17
The Boda Boda Lounge project is a biennial video
festival linked to African urban practices. Since Namakula, E. (2014). Mapping Kampala with KLA ART
014. Start: Journal of Arts and Culture (Kampala), Critical Interventions 11, Issue 2 2017 INSTITUTIONALISM, PUBLIC SPHERE, AND ARTISTIC AGENCY 131 November 20, 1–5. Retrieved from http://startjournal. org/2014/11/mapping-kampala-with-kla-art-014/ Contemporary African Art, 31, 46–111. doi:10.1215/
10757163-1586481 Pieters, K. (2015). Art in Kampala at work 012. In K. Pinther, U. Smooth, & C. Nzewi (Eds.), New spaces for
negotiating art and histories in Africa (pp. 52–72). Berlin:
LIT Verlag. Namubiru, R. K. (2008). The formation of contemporary
visual artists in Africa: Revisiting residency programmes
(Unpublished doctoral dissertation). Kampala: Maker-
ere University. Namubiru, R. K. (2014). Why artists’ studios? In M. Seru-
biri (Ed.), Unmapped. Kampala Contemporary Art Festi-
val (KLA ART 2014) (pp. 5–6). Retrieved from http://
klaart.org/2014/catalogue.html Pinther K., Smooth U., &Nzewi C. (Eds.) (2015). New
spaces for negotiating art and histories in Africa. Berlin:
LIT Verlag. oteca do conhecimento online UL] at 02:02 12 October 2017 Serubiri, M. (2015). A tree in public space. Start: Journal of
Arts and Culture (Kampala), June 1, 2016, 1–4. Retrieved from http://startjournal.org/2015/06/a-tree-
in-public-space/ Okeke-Agulu, C., & Hassan, S. (2008). The twenty-first cen-
tury and the mega shows: A curator’s roundtable. Nka:
Journal of Contemporary African Art, 22/23, 152–188. Okeke-Agulu, C. (2010). Nka roundtable III: Contempo-
rary African art and the museum. Nka: Journal of Smith, T. (2012). Thinking contemporary curating. New
York, NY: Independent Curators International (ICI). Interventions Interventions
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Major Insect Pests of Paddy in Guyana
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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in Thanjavur delta were done at 10-day
intervals. Ten varieties were planted
each year except 1977-78, when only 8
were planted. No crop protection was
provided.
Varieties evaluated were:
1975-76: AD11585, ADT31, IET1722,
IR20, IR26, TKM8, Pusa 33-18, Tainan 3(M), Bhavani, Ponni.
1976-77: AD54-1, AD5620, AD6380,
AS3821, ADT31, CRM10-5747,
IET2881, IR20, Pusa 4-1-11-1,
Bhavani.
1977-78: AD9186, ADT31, IET1722,
IR20, IR26, TM1251, Tiruveni,
Vaigai.
1978-79: AD5231, AD6120, AD6970,
AD7211, AD7481, AD13893,
ADT32, AS3704, IR26, IR34.
1979-80: AD6120, AD4481, AD8991,
ADT31, ADT32, ADT33, AS3704,
IR34, PL29, TKM9.
The average disease incidence was
recorded at the ninth stage of crop
growth using the standard evaluation
system. Higher incidence of BS and
NBLS was observed in late plantings
(see table). The diseases appear to cause
significant yield reductions only in the
second season crop.
Individuals organizations, and media
who wish additional details of
information presented in IRRN should
write directly to the authors.
Seasonal incidence of brown spot and narrow brown leaf spot in Thanjavur, India.
Date of planting
Disease incidence (%)
1975-76
1976-77
15/6
25/6
5/7
15/7
25/7
5/8
15/8
25/8
5/9
15/9
25/9
5/10
15/10
25/10
5/11
15/11
25/11
5/12
15/12
25/12
–
–
–
15.0
18.9
14.0
9.7
7.2
5.1
8.4
7.9
12.2
17.5
26.4
31.9
35.5
47.7
46.8
53.1
51.6
17.2
13.4
13.2
18.1
17.3
14.2
11.0
14.6
14.4
17.5
16.8
21.2
26.5
26.9
25.7
37.1
42.0
50.3
68.7
62.4
15/6
25/6
5/7
15/7
25/7
5/8
15/8
25/8
5/9
15/9
25/9
5/10
15/10
25/10
5/11
15/11
25/11
5/12
15/12
25/12
6.3
8.7
–
–
–
5.0
7.3
5.4
–
–
10.4
9.9
8.9
10.0
10.9
15.4
29.9
31.7
32.2
28.5
Pest management and control
Major insect pests of paddy in
Guyana
I. Rambajan, entomologist, Guyana Rice
Board, Research and Extension Division,
Guyana, South America
A double-cropping system of rice cultivation is recommended in Guyana,
although continuous cropping is practiced extensively. The autumn crop
(May-Jun) accounts for 90,000-110,000
ha and the spring crop (Dec-Jan) for
40,000-50,000 ha. The rice belt spans
16
IRRN 6:6 (December 1981)
1977-78
1978-79
1979-80
Mean
Brown spot
4.8
11.6
7.9
14.0
12.4
13.7
16.4
8.4
11.2
9.5
14.4
19.3
15.5
31.4
46.4
53.1
67.4
59.5
66.1
64.2
–
–
6.9
10.4
9.7
12.2
15.2
7.9
6.4
14.2
17.1
19.9
16.4
19.7
29.4
37.0
51.4
48.1
47.4
48.0
7.4
5.8
9.4
6.3
14.8
19.2
18.5
14.0
20.3
12.2
11.0
9.9
18.2
37.4
35.2
42.6
43.1
53.3
55.2
50.0
5.9
6.2
7.4
12.8
14.6
14.7
14.2
10.4
11.5
12.4
13.4
16.5
18.8
28.4
33.7
41.1
50.3
51.6
58.1
55.2
Narrow brown leaf spot
–
15.1
–
15.5
–
–
–
12.7
–
13.2
–
13.7
7.3
7.2
9.6
–
17.2
–
10.1
9.5
–
11.6
–
14.6
–
15.6
7.5
14.6
13.6
13.4
19.1
12.9
20.6
18.5
47.2
29.7
43.0
26.8
36.5
27.5
–
5.6
10.5
–
3.6
9.4
14.5
7.8
10.2
–
4.6
7.9
–
–
15.2
17.6
19.7
37.3
26.2
24.4
12.6
7.4
9.5
–
–
–
–
–
–
–
–
3.5
9.6
–
–
14.7
20.3
32.5
29.3
22.0
6.8
7.4
3.9
2.5
3.4
5.6
7.3
4.6
5.5
3.9
5.3
7.2
6.8
6.4
10.6
15.9
21.8
39.7
31.5
27.8
INSECTS
most of the Atlantic coastal lands from
Crabwood Creek in the east to the
Pomeroon in the west, including Berbice, Demerara, and Essequibo counties.
Farmholdings range from less than 1 ha
to more than 500 ha.
Rice, the staple cereal, also is an
invaluable foreign exchange earner and
provides more than 50,000 jobs. Land
preparation and harvesting are completely mechanized. Pregerminated seeds
are broadcast under wet cultivation
method and fertilizers and pesticides are
applied manually except in large hold-
ings where applications are by air.
Pest problems have increased with a
change from dry to wet cultivation,
introduction of double or continuous
cropping, increase in hectarage, and
development of pesticide resistance. The
cultivars used have no genes for insect
and disease resistance. The table shows
the major insect pests of wetland rice in
Guyana and their occurrence in relation
to growth stages. Two storage pests
have been included because recent studies show significant field infestation
and damage.
Major insect pests of wetland rice in Guyana, South America.
Growth stage
Emergence to maximum
tillering
Panicle initiation to
heading
Heading to hard dough
Hard dough to harvest,
storage
Pest
Scientific name
1. Helodytes foveolatus Duval
(Curculionidae: Coleoptera)
2. Hydrellia deonieri Rambajan
(Ephydridae: Diptera)
3. Spodoptera frugiperda (Smith)
(Noctuidae: Lepidoptera)
4. Mocis punctularis Hubner
(Noctuidae: Lepidoptera)
5. Neoconocephalus Spp.
Caulopsis Spp.
(Tettigoniidae: Orthoptera)
1. Caulopsis cupsidata Scudder
(Tettigoniidae: Orthoptera)
1. Oebalus poecilus Dallas
(Pentatomidae: Hemiptera)
Rice water weevil
1. Sitotroga cerealella (Olivier)
(Gelechiidae: Lepidoptera)
2. Rhyzopertha dominica (Fabricius)
(Bostrichidae: Coleoptera)
Angoumois grain
moth
Lesser grain borer
Rice gall midge incidence in the dry
season
C. Bhaskara Rao, rice breeder, All India
Coordinated Rice Improvement Project,
Regional Centre, Maruteru - 534122,
Andhra Pradesh, India
Damage
Common name
Larva feeds on roots. Adult feeds on eye of pregerminated seeds.
Larva feeds in growing shoot, causes deadheart.
Rice leafminer
Rice blackfly
Fall armyworm
Larva feeds on seedling leaves, causes burnt tip,
later defoliation.
Rice looper
Long-horned grasshopper
Feeds on leaves that appear shredded, cobweb.
Long-horned grasshopper
Stink bug or paddy
bug
Feeds on leaves and ball, causes whitehead.
Nymph and adult feed on grains at milk stage,
cause wind paddy or atrophied glumes; at
dough stage cause broken barrels, discolored
rice after milling.
Larva feeds on kernel.
Percent silver shoots in dry season, Maruteru, India.
Silver shoots (%)
Trial
1979
Mean
Uniform Variety Trial 2
Uniform Variety Trial 3
Uniform Variety Trial 4
11
17
16
1980
Maximum
20
24
28
Mean
Maximum
5
18
13
8
25
8
Rice gall midge (Orseolia oryzae), a
major insect pest in late-planted fields in
the wet season (May-November) in the
Andhra Pradesh Krishna and Godavari
deltas, has become a major problem in
the dry season (November-April) in the
Godavari delta since 1978. The percentage silver shoots or galls recorded in
coordinated variety trials show the high
pressure of gall midge at Maruteru center (see table).
It is likely that extensive use of pesticides to control insects has resulted in the
destruction of predators and parasites of
Effects of rice plant age on diopsid
oviposition and plant susceptibility
Diopsis thoracica eggs laid on rice plants of different ages and subsequent development of deadhearts in Sierra Leone. a
rice gall midge. Popular rabi season varieties Jaya, Prabhat, Prakash, Rasi, and
Tella Hamsa are susceptible to rice gall
midge. BPT1235 and IR36 are promising early-maturing, gall midge-resistant
varieties.
IR5
A. M. Alghali, research fellow, IITA, Rice
Research Station, Rokupr, Sierra Leone;
and E. O. Osisanya, Agricultural Biology
Department, University of Ibadan, Nigeria
Diopsis thoracica (West), a serious pest
limiting rice yields in West Africa, prefers plants of particular ages for oviposition. Confirmation of the specific age
range would be useful in planning pest
management programs involving host
plant resistance and other control
measures.
Pregerminated seeds of 2 rice varieties
DT
b
10
20
30
40
50
60
Eggs
(no.)
4 cd
11 ab
14 a
8 bc
2
d
1
d
Deadhearts
(no.)
6 b
13 a
14 a
6 b
2
c
0
c
GH106-76
Deadhearts/
egg
(no.)
1.5
1.2
1.0
0.8
1.0
0.0
Eggs
(no.)
2
2
5
3
1
0
ab
ab
a
ab
b
b
Deadhearts
(no.)
Deadhearts/
egg
(no.)
4 ab
3 b
6 a
3 b
0
0
2.0
1.5
1.2
1.0
0.0
0.0
aTotal
of 6 replications. In the same column, means followed by the same letter do not differ at the
1% level of probability. bDT = days after transplanting.
with similar tillering abilities, IR5 and
GH106-76, were sown at 10-day intervals. Seedlings 21 days after sowing
(DS) were transplanted separately at 2
seedlings/pot. Rice plants 60, 50, 40, 30,
20, and 10 days after transplanting (DT)
IRRN 6:6 (December 1981)
17
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https://openalex.org/W3135826316
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https://www.scielo.br/j/jaos/a/Jvz8ZS4bczTQSvdpqYqyYzp/?lang=en&format=pdf
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English
| null |
A prospective and randomized clinical trial evaluating the effectiveness of ART restorations with high-viscosity glass-ionomer cement versus conventional restorations with resin composite in Class II cavities of permanent teeth: two-year follow-up
|
Journal of Applied Oral Science
| 2,021
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cc-by
| 7,721
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Abstract Submitted: July 20, 2020
Modification: September 16, 2020
Accepted: October 19, 2020
Objective: To compare the effectiveness of ART restorations using High
Viscosity Glass-ionomer cement (HVGIC) with conventional restorations
using resin composite in Class II cavities of permanent teeth, in a 2-year
follow-up. Methodology: Seventy-seven restorations were made with each
restorative material, Equia Fil-GC Corporation (ART restorations) and
Z350-3M (conventional restoration), in 54 participants in this parallel and
randomized clinical trial. Restorations were evaluated at 6 months, 1 and 2
years using the ART and the modified United States Public Health Service
(USPHS) criteria. Chi-square test and Survival Analysis (p<0.05) were used
for statistical analysis. Results: The success rates for ART restorations were
98.7% (6 months) and 95.8% (1 year) for both criteria. At 2 years, success
rate was 92% and 90.3% when scored by the modified USPHS and ART criteria
(p=0.466), respectively. The success rates for conventional restorations were
100% (6 months), 98.7% (1 year) and 91.5% (2 years) for both assessment
criteria. ART restorations presented a lower survival rate by the criterion of
ART (83.7%) when compared to the modified USPHS criterion of (87.8%),
after 2 years (p=0.051). The survival of conventional restorations was 90.7%
for both evaluation criteria. Conclusion: At the 2-years follow-up evaluation,
no statistically significant difference was observed between the success rate
of ART restorations with HVGIC compared to conventional restorations with
resin composite in Class II cavities of permanent teeth. Keywords: Permanent dentition. Atraumatic restorative treatment. Glass-
ionomer cement. Resin composite. Clinical trial. Rafael MENEZES-SILVA1
Sofia R Maito VELASCO2
Eduardo BRESCIANi3
Roosevelt da Silva BASTOS¹
Maria Fidela de Lima NAVARRO¹
1Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Materiais
Dentários, Endodontia e Dentística, Bauru, SP, Brasil. 2Universidade de São Paulo, Faculdade de Saúde Pública, São Paulo, SP, Brasil. 3Universidade Estadual Paulista (UNESP), Instituto de Ciências e Tecnologia, São José dos Campos,
SP, Brasil. Corresponding address:
Rafael Menezes Silva
Alameda Dr. Otávio Pinheiro Brisola, 9-75 -
Vila Nova - Cidade Universitária -
Bauru - SP - 17012-901 - Brasil. Phone: +55 14 998146904
e-mail: rafa18ms@hotmail.com Objective: To compare the effectiveness of ART restorations using High
Viscosity Glass-ionomer cement (HVGIC) with conventional restorations
using resin composite in Class II cavities of permanent teeth, in a 2-year
follow-up. 1Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Materiais
Dentários, Endodontia e Dentística, Bauru, SP, Brasil.
2Universidade de São Paulo, Faculdade de Saúde Pública, São Paulo, SP, Brasil.
3Universidade Estadual Paulista (UNESP), Instituto de Ciências e Tecnologia, São José dos Campos,
SP, Brasil. Original Article
http://dx.doi.org/10.1590/1678-7757-2020-0609 Original Article
http://dx.doi.org/10.1590/1678-7757-2020-0609 Original Article
http://dx.doi.org/10.1590/1678-7757-2020-0609 Original Article
http://dx.doi.org/10.1590/1678-7757-2020-0609 A prospective and randomized clinical
trial evaluating the effectiveness of
ART restorations with high-viscosity
glass-ionomer cement versus
conventional restorations with resin
composite in Class II cavities of
permanent teeth: two-year follow-up A prospective and randomized clinical
trial evaluating the effectiveness of
ART restorations with high-viscosity
glass-ionomer cement versus
conventional restorations with resin
composite in Class II cavities of
permanent teeth: two-year follow-up Submitted: July 20, 2020
Modification: September 16, 2020
Accepted: October 19, 2020 Introduction have investigated the clinical performance of multiple-
surface restorations using GICs and resin composites
in permanent teeth.12-14 Evaluating restorations
performed with HVGICs under the ART approach would
provide important data considering three aspects:
testing a substitute for dental amalgam, the ART
approach being a more socially available technique
due to the non-use of electrical equipment, and ART
might be an important approach in COVID era as it
does not generate aerosols.1,8,15-17 Currently, the main alternatives of direct restorative
material to substitute dental amalgam are resin
composite and polyalcenoate-based materials, with
the glass-ionomer cements (GIC) being the most
biomimetic ones.1 In contrast to resin composite, the
GIC presents a coefficient of linear thermal expansion
similar to tooth structures and it releases fluoride,
characterizing its anticariogenic property.2 Therefore, the objective of the present study
was to compare ART restorations with HVGIC versus
conventional restorations with resin composite in
Class II cavities of permanent teeth over a period of
2 years. Previous follow-up data have been published
elsewhere.18 That study presented similar success
rates considering both approaches, granting longer
evaluation assessments. GICs emerged as the most suitable restorative
materials in early studies on the impact of ART
to oral health.3 Today, ART is widely accepted by
the international scientific community and used
worldwide.4 Although High Viscosity Glass-ionomer cement
(HVGIC) is the material of choice for ART restorations,
there is still room for improvements. Thus, some
authors have proposed and tested additional retention
in cavities restored with GICs to provide greater
longevity to restorations in permanent teeth.5,6 Further,
encapsulating HVGICs led to in vitro increased flexural
strength,7 with possible positive influences to the
restorative treatment. The tested null hypothesis assumes there is no
difference after 2 years on the effectiveness of ART
restorations with HVGIC compared to conventional
restorations with resin composite. Abstract Methodology: Seventy-seven restorations were made with each
restorative material, Equia Fil-GC Corporation (ART restorations) and
Z350-3M (conventional restoration), in 54 participants in this parallel and
randomized clinical trial. Restorations were evaluated at 6 months, 1 and 2
years using the ART and the modified United States Public Health Service
(USPHS) criteria. Chi-square test and Survival Analysis (p<0.05) were used
for statistical analysis. Results: The success rates for ART restorations were
98.7% (6 months) and 95.8% (1 year) for both criteria. At 2 years, success
rate was 92% and 90.3% when scored by the modified USPHS and ART criteria
(p=0.466), respectively. The success rates for conventional restorations were
100% (6 months), 98.7% (1 year) and 91.5% (2 years) for both assessment
criteria. ART restorations presented a lower survival rate by the criterion of
ART (83.7%) when compared to the modified USPHS criterion of (87.8%),
after 2 years (p=0.051). The survival of conventional restorations was 90.7%
for both evaluation criteria. Conclusion: At the 2-years follow-up evaluation,
no statistically significant difference was observed between the success rate
of ART restorations with HVGIC compared to conventional restorations with
resin composite in Class II cavities of permanent teeth. Rafael MENEZES-SILVA1
Sofia R Maito VELASCO2
Eduardo BRESCIANi3
Roosevelt da Silva BASTOS¹
Maria Fidela de Lima NAVARRO¹ Rafael MENEZES-SILVA1
Sofia R Maito VELASCO2
Eduardo BRESCIANi3
Roosevelt da Silva BASTOS¹
Maria Fidela de Lima NAVARRO¹ Keywords: Permanent dentition. Atraumatic restorative treatment. Glass-
ionomer cement. Resin composite. Clinical trial. Corresponding address:
Rafael Menezes Silva
Alameda Dr. Otávio Pinheiro Brisola, 9-75 -
Vila Nova - Cidade Universitária -
Bauru - SP - 17012-901 - Brasil. Phone: +55 14 998146904
e-mail: rafa18ms@hotmail.com J Appl Oral Sci. 2021;29:e20200609
1/10 A prospective and randomized clinical trial evaluating the effectiveness of ART restorations with high-viscosity glass-ionomer cement versus conventional restorations with resin
composite in Class II cavities of permanent teeth: two-year follow-up Methodology According to a systematic review,8 it cannot be
suggested that resin composite has higher failure
rates and risk for secondary caries than amalgam
restorations due to the weak scientific evidence. Thus,
with the Minamata Convention and the reduction in the
use of mercury in several fields, including dentistry,
resin composite restorations are considered viable
alternatives to amalgam restorations.9 Therefore, in
studies looking for new restorative alternatives, resin
composites must be considered control. This is a prospective and randomized clinical
trial study with a 2-year follow-up, approved by
Institutional Review Board of the Bauru School of
Dentistry (CAAE: 24012913.0.1001.5417). The
study protocol was registered over the Brazilian
Registry of Clinical Trials – REBEC website (#RBR-
2jmbvt) and written in accordance with the CONSORT
(Consolidated Standards of Reporting Trials) and the
SPIRIT (Standard Protocol Items: Recommendations
for Interventional Trials) guidelines. Mickenautsch10 (2016) investigated the state of
the art of direct restorations in posterior permanent
teeth applying HVGICs. The author concluded that
ART restorations showed similar clinical performance
to amalgam restorations. Kielbassa, et al.9 (2017)
proposed that the promising HVGIC Equia Fil could be
an option to dental amalgam in countries where health
services do not cover resin composites or in cases of
allergy to polymers. Training sessions Prior to the beginning of the study, a single operator
(RMS) was trained in the ART approach, including
the creation of additional retentive grooves, and
for the resin composite approach. Two experienced
dentists in ART and Restorative Dentistry (MFLN
and JEF) supervised the training sessions, which
involved theoretical, laboratory and clinical exercises. The training was performed in the Clinical Research
Center of the Bauru School of Dentistry, Brazil, where
the examiners (SRVM and RSB) and assistants were
trained for CAST and data recording. One month before
each evaluation, the evaluators were trained in using
the evaluation criteria by means of theoretical and
clinical training. The exclusion criteria comprised the following:
participants presenting mobile teeth, having
paranormal occlusion, more than two multiple-surface
cavities in permanent teeth and wearing orthodontic
appliances. The size of the cavity was classified as small,
medium or large.18 Only children or adolescents
whose parents or the participant signed the Informed
Consent form were included in the study. Stratified
randomization was performed by subdividing class
II cavities into two homogeneous groups. Cavity size
and caries experience (DMFT) were the stratification
variables, in that order. Inter- and intra-agreements (Kappa coefficient)
were performed for caries diagnosis, cavity size
classification, and for the evaluation of restorations at
6 months, 1 and 2 years of follow-up, in 10% of the
included subjects. For stratified randomization, due to the difficulty in
obtaining the calculated number of class II cavities, the
tooth was considered a sample unit. The participants
were initially screened and in an Excel spreadsheet
the screened teeth were listed with the indication of
the cavity size and the patient’s caries index. Teeth
were initially ordered in this Excel spreadsheet by
the caries index and divided into two conglomerates,
the first with the lower DMFT values and the second
with the higher values. After this division, in each of
these clusters, the teeth were ordered by the size of
the cavity and each half was divided into two parts,
totaling four clusters namely: low DMFT-scores and
small cavity size, low DMFT-scores and large cavity
size; high DMFT-scores and small cavity size; and high
DMFT-scores and large cavity size. The intra-agreement values for the evaluator 1 for
the classifications of caries and size of cavities were 1
and 0.86, respectively. The values for the evaluator 2
were 0.84 and 0.84, respectively. The intra-examiner
agreement was 0.85. Training sessions For the evaluation of restorations, intra-examiner
agreement for evaluator 1 was 0.8 for 6 months and 1
year, and 0.84 for the 2 years follow-up. The values for
the evaluator 2 was 0.84 at 6 months, and 0.8 at 1 and
2 years of follow-up. The inter-examiner agreement
values were 0.82 for 6 months and 2 years, and 0.80
at the 1-year follow-up. SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF cavity sizes of the two groups (5%). Figure 1 shows the flow diagram of patient
randomization indicated by CONSORT (Consolidated
Standards of Reporting Trials). the state of São Paulo, Brazil. The inclusion criteria
were as follows: children and adolescents with no
medical history; individuals that cooperated for
dental examination; one class II cavity in permanent
teeth without active periodontal or pulpal diseases
or toothache; presence of occluding tooth; good oral
hygiene. The included participants were examined
using the Caries Assessment Spectrum and Treatment
(CAST) instrument,19 from which a mean DMFT-score
was retrieved. Sample Size and Study Population The sample size was calculated through the formula
for comparing proportions, considering a power of
80% and a significance level of 5%. According to the
literature, the failure proportions were 18% for ART/
HVGIC multiple-surface restorations with Ketac Molar
and 3% for multiple-surface conventional restorations
with resin composite. An additional 20% increase was
considered to compensate dropouts, resulting in 77
restorations for each treatment.18 Considering the lack of randomized and parallel
clinical trials with high internal validity comparing
resin composites and HVGICs, both in deciduous
teeth and permanent teeth, it is difficult to indicate
the superiority of a material,11 especially considering
the substitution for dental amalgam. Few studies The participants of this parallel trial were selected
from 17 public primary schools in the countryside of J Appl Oral Sci. 2021;29:e20200609
2/10 Examination The clinical examinations were performed under
adequate lighting. Patients were laid on a table. The
examiners seated at a 12-o’clock position and recorders
sat at a 9 o’clock position. The dental instruments used
were mouth mirrors, wooden spatulas, and the CPI
(Community Periodontal Index) probe. After this division into four groups, teeth were
allocated to groups using the “random” function
on Excel to ensure impartiality in the process of
randomization and allocation. After this randomization,
statistical tests were carried out to ensure that
the factors used in the randomization were equally
divided among the experimental groups. A T test for
independent samples was used for the comparison
between the caries index of the two experimental
groups and a Mann-Whitney U test to compare the Restoration Placement As different techniques and materials were tested,
it was not possible to have blindness in this study since
the operator, the evaluator and the patient would know J Appl Oral Sci. 2021;29:e20200609
3/10 A prospective and randomized clinical trial evaluating the effectiveness of ART restorations with high-viscosity glass-ionomer cement versus conventional restorations with resin
composite in Class II cavities of permanent teeth: two-year follow-up acid. After the placement of a matrix band and
wooden wedge, Equia Fil capsules (GC Corporation,
Tokyo, Japan) were used to fill the cavity extending
slightly over the marginal ridge. Then, the restoration
material was held for 40 seconds under pressure. That
step resulted in a sealed restoration on the occlusal
surface. After 2.5 minutes, hand instruments were
used to remove material flashes. The matrix was
removed with buccal-lingual and occlusal movements,
5 minutes after the start of GIC mixing. A carbon
paper was used to check the occlusion and a dental
floss to check the presence of a contact point with
the neighboring tooth. Any debris and humidity were
removed from the restoration surface and Equia Coat
(GC Corporation, Tokyo, Japan) was applied and light
cured for 20 s. A schematic drawing of the restoration
and its border locations was performed on the clinical
form of patients. Patients were recommended not to
eat solid food for 1 hour. which material was being inserted or evaluated. Figure 1- CONSORT flowchart for clinical trials Examination The
operator restored by randomization as previously
defined and knew the restorative techniques that were
being performed; the evaluator was also able to easily
identify the two types of restoration and the patient
was informed about the materials and techniques
that could be used: one with the use of anesthesia
and rotating instruments and the other with manual
instruments. The filling materials were used according to the
manufacturer’s instructions. ART/HVGIC protocol was
described in detail in a previous study.18 In summary,
hand excavators were used to remove soft dentin
and retentive grooves were made in the gingival-
occlusal direction in the buccal and lingual walls of
the proximal boxes, approximately 0.5 mm from the
dentin-enamel junction. The tooth was isolated with
cotton rolls. The cavity was cleaned with cotton wool
pellets, and conditioned for 15 s with 20% polyacrylic Figure 1- CONSORT flowchart for clinical trials J Appl Oral Sci. 2021;29:e20200609
4/10 SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF to analyze the distribution of scores according to the
ART and the modified USPHS criteria, as well as the
percentage of success and failure for ART and resin
composite. In addition, Kaplan-Meier test was used to
evaluate the survival percentages of the restorations
and the difference between survival curves was
determined with the Log-Rank test. Conventional restorations/resin composite
protocol: The tooth to be restored was anesthetized
and the operative field was isolated with rubber
dam. The cavities were prepared using minimal
invasive dentistry with # 245 or # 330 carbide burs
at high speed. Carious dentin was removed with #
1, 2, or 3 round burs (KG Sorensen, Cotia, Brazil). A gingival marginal trimmer was used to finish the
enamel margin in the proximal box. In the case of
deep caries, calcium hydroxide cement was applied,
followed by the application of a resin-modified glass-
ionomer cement (Vitrebond - 3M, Saint Paul, USA). The
enamel was etched with 35% phosphoric acid (FGM,
Joinvile, Brazil) for 15 s, washed with air/water spray
for 20 s and dried with absorbent paper. Afterwards,
Scotchbond Universal Adhesive (In Brazil: Single Bond
Universal) (3M, Saint Paul, USA) was actively applied
for 20 s with a microbrush, air gently sprayed for 5
s and lightcured for 10 s. For restoring the proximal
contact and the marginal ridge, a metallic matrix
system and Palodent clamp (TDV, Pomarode, Brazil)
were used with wood wedge. Examination Oblique increments (up to
2 mm in thickness) of Filtek Z350 XT resin composite
(3M ESPE, Saint Paul, USA) were inserted in the
proximal box (es), followed by the occlusal box. Each
increment was lightcured for 40 seconds with a LED
device (Elipar Free Light 2 LED light 3M ESPE, Saint
Paul, USA). Excess removal and occlusal adjustment
were performed with 12-blade FG 7803F multilayer
drills (KG Sorensen, Cotia, Brazil) and T & F 7802 (Jet
Carbide Burs, Kyoto, Japan). Polishing was carried out
with a 12 and 30-blade multilayer drills (FF9904 from
Jet Carbide Burs) and felt discs with the Poligloss paste
(TDV, Pomerode, Santa Catarina, Brazil). The level of significance was set at p<0.05. Statistical analysis was performed with SPSS version
23.0 (Statistical Package for Social Sciences, IBM
Inc., USA). Results In this study, the overall recalls at 2 years was
87%. ART restorations presented success rates of 98.7%
(6 months), 95.8% (1 year) and 90.3% (2 years),
and the success rates for conventional restorations
were 100% (6-month), 98.7% (1 year) and 91.5% (2
years), according to ART criteria. Significant difference
was observed between the restorative approaches at
6 months (p=0.033) and 1 year (p=0.033) but not at
2 years (p=0.064) (Table 1). According to the modified USPHS criterion, the
success rates for ART restorations were 98.7% (6
months), 95.8% (1 year) and 92.0% (2 years), and
for conventional restoration were 100% (6 months),
98.7% (1 year) and 91.5% (2 years). There was a
significant difference between ART with HVGIC and
conventional restoration with resin composite at the
6 months’ evaluation (p=0.001) but not after 1 year
(p=0.310) and 2 years (p=0.830) (Table 2). At the end of 2 years, two ART restorations
received the score 9 according to ART criterion, being
excluded from the analysis. Those restorations were
also excluded from the evaluation with the modified
USPHS criterion. Those restorations with code 9 (ART
criterion) can be identified in Table 2. It is important to
note that in this same evaluation period, another ART
restoration classified with the score 6 according to the
ART criterion was considered satisfactory as stated in
the modified USPHS criterion (Tables 1 and 2). Evaluation Photographs were taken before and immediately
after treatment and at the 6 months, 1 and 2 years,
for registration. The restorations were evaluated by
two experienced dentists (SRVM and RSB) according
to ART19 and modified USPHS criteria20. For this, they
used mouth mirrors, wooden spatulas and the CPI
probe. At each evaluation point, participants received
new brushing kits and were guided in oral hygiene. Furthermore, supplementary treatments were offered
to participants. Regardless of the evaluation criteria used, the
success rates for conventional restoration with resin
composite were the same. However, considering the
success rates for ART restorations with HVGIC after 2
years, they were 90.3% when using the ART criterion Statistical Analysis Statistical Analysis *1 and 2 = success; 3, 4, 6, 7 and 10 = failure; 9 = excluded. **Chi-square test with linear trend. Table 2- Distribution of scores according to the modified USPHS criterion for ART restorations with HVGIC and conven
with resin composite scores according to the modified USPHS criterion for ART restorations with HVGIC and conventional restorations Clinical
parameters
Rating
6
months
1 year
2 years
ART
restorations
n(%)
Conventional
restorations
n (%)
p
ART
restorations
n(%)
Conventional
restorations
n (%)
p
ART
restorations
n(%)
Conventional
restorations
n (%)
p
Color
Alpha
33 (43.4)
55 (71.4)
< 0.001
32 (45.0)
38 (51.4)
0.609
31 (50.0)
35 (53.0)
0.368
Bravo
43 (56.6)
22 (28.6)
39 (55.0)
36 (48.6)
31 (50.0)
31 (47.0)
Marginal
discoloration
Alpha
75 (98.7)
74 (96.1)
0.315
65 (91.5)
70 (94.6)
0.785
56 (90.3)
62 (94.0)
0.294
Bravo
1 (1.3)
3 (3.9)
6 (8.5)
4 (5.4)
6 (9.7)
4 (6.0)
Relapse of
caries
Alpha
76 (100.0)
77 (100.0)
-
69 (97.2)
74 (100.0)
0.235
59 (95.2)
64 (97.0)
0.221
Charlie
0 (0.0)
0 (0.0)
2 (2.8)
0 (0.0)
3 (4.8)
2 (3.0)
Anatomical
shape
Alpha
43 (56.6)
70 (90.9)
<0.001*
45 (63.4)
66 (89.2)
< 0.001
*
32 (51.6)
54 (81.8)
0.001*
Bravo
33 (43.4)
7 (9.1)
23 (32.4)
8 (10.8)
29 (46,.8)
10 (15.2)
Charlie
0 (0.0)
0 (0.0)
3 (4.2)
0 (0.0)
1 (1.6)
2 (3.0)
Marginal
integrity
Alpha
70 (90.9)
72 (93.5)
0.471*
56 (78.9)
69 (92.0)
0.072 *
50 (80.6)
56 (80.0)
0.361*
Bravo
6 (7.8)
5 (6.5)
12 (16.9)
5 (6.7)
10 (16.1)
8 (11.4)
Charlie
0 (0.0)
0 (0.0)
2 (2.8)
0 (0.0)
0 (0.0)
2 (2.9)
Delta
1 (1.3)
0 (0.0)
1 (1.4)
1 (1.3)
2 (3.3)
4 (5.7)
Surface
texture
Alpha
42 (55.3)
63 (81.8)
<0.001
32 (45.1)
56 (75.7)
< 0.001
32 (51.6)
48 (72.7)
0.022
Bravo
34 (44.7)
14 (18.2)
39 (54.9)
18 (24.3)
30 (48.4)
18 (27.3)
Restoration
quality**
Ideal
25 (32.5)
47 (61.0)
0.001*
27 (38.0)
36 (48.0)
0.310*
21 (33.9)
27 (38.6)
0.830*
Satisfactory
51 (66.2)
30 (39.0)
41 (57.8)
38 (50.7)
36 (58.1)
37 (52.9)
Unsatisfactory
1 (1.3)
0 (0.0)
3 (4.2)
1 (1.3)
5 (8.0)
6 (8.5)
*Chi-square test with linear trend. **The ideal and satisfactory scores = success; unsatisfactory = fail. Statistical Analysis The chi-square test with linear trend was applied J Appl Oral Sci. 2021;29:e20200609
5/10 A prospective and randomized clinical trial evaluating the effectiveness of ART restorations with high-viscosity glass-ionomer cement versus conventional restorations with resin
composite in Class II cavities of permanent teeth: two-year follow-up the conventional approach only within the clinically
acceptable scores, on color, anatomical form, and
surface texture (p<0.001). After 1- and 2-year recalls, and 92.0% when using the modified USPHS criterion,
with no difference between them (p<0.466). During the 6-month recall, ART differed from During the 6-month recall, ART differed from Scores*
6 months
1 year
2 years
ART
restorations
n(%)
Conventional
restorations
n (%)
p**
ART
restorations
n(%)
Conventional
restorations
n (%)
p**
ART
restorations
n(%)
Conventional
restorations
n (%)
p**
(1) Restoration present and
correct
65 (84.4)
74 (96.1)
0.033
56 (78.9)
73 (97.4)
0.003
43 (67.2)
59 (84.3)
0.064
(2) Small marginal defect and/
or wear with less than 0.5 mm;
without need of repair
11 (14.3)
3 (3.9)
12 (16.9)
1 (1.3)
13 (20.3)
5 (7.2)
(3) Marginal Defect exceeding 0.5
mm. Need of repair
-
-
1 (1.4)
0 (0.0)
-
-
(4) Wear exceeding 0.5 mm. Need of repair
-
-
1 (1.4)
0 (0.0)
-
-
(6) Restore and/or fracture tooth. Need of repair
1 (1.3)
0 (0.0)
1 (1.4)
1 (1.3)
2 (3.1)
4 (5.7)
(7) Restoration has completely
disappeared. Treatment is needed
-
-
-
-
1 (1.6)
1 (1.4)
(9) Tooth has been extracted
-
-
-
-
2 (3.1)
0 (0.0)
(10) Sensitivity or pulpal
involvement
-
-
-
-
3 (4.7)
1 (1.4)
*1 and 2 = success; 3, 4, 6, 7 and 10 = failure; 9 = excluded. **Chi-square test with linear trend. Table 1- Distribution of scores according to the ART criterion for ART restorations with HVGIC and conventional restorations with resin
composite Table 1- Distribution of scores according to the ART criterion for ART restorations with HVGIC and conventional restorations with resin
composite bution of scores according to the ART criterion for ART restorations with HVGIC and conventional restorations with res ble 1- Distribution of scores according to the ART criterion for ART restorations with HVGIC and conventional restorat
mposite *1 and 2 = success; 3, 4, 6, 7 and 10 = failure; 9 = excluded. **Chi-square test with linear trend. SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF Figure 2- Graphs for survival analysis of ART restorations with HVGIC and conventional restorations with resin composite, at the 2-year
follow-up. Graphs represent data assessed with the ART or the modified USPHS criteria
Figure 3- Class II (DO) ART restoration with HVGIC in the upper left second premolar: A) Baseline; B) 6 months; C) 1 year and D) 2 years
and Conventional Class II (MO) restoration with resin composite in mandibular right first molar: E) Baseline; F) 6 months; G) 1 year and
H) 2 years
SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF gure 2- Graphs for survival analysis of ART restorations with HVGIC and conventional restorations with resin composite, at the 2-year
llow-up. Graphs represent data assessed with the ART or the modified USPHS criteria Figure 2- Graphs for survival analysis of ART restorations with HVGIC and conventional restorations with resin composite, at the 2-year
follow-up. Graphs represent data assessed with the ART or the modified USPHS criteria Figure 3- Class II (DO) ART restoration with HVGIC in the upper left second premolar: A) Baseline; B) 6 months; C) 1 year and D) 2 years
and Conventional Class II (MO) restoration with resin composite in mandibular right first molar: E) Baseline; F) 6 months; G) 1 year and
H) 2 years Figure 3- Class II (DO) ART restoration with HVGIC in the upper left second premolar: A) Baseline; B) 6 months; C) 1 year and D) 2 years
and Conventional Class II (MO) restoration with resin composite in mandibular right first molar: E) Baseline; F) 6 months; G) 1 year and
H) 2 years Figure 3 illustrates the two types of restorative
treatment in this study. Figure 3 illustrates the two types of restorative
treatment in this study. differences were detected for anatomical form and
surface texture (p<0.022) (Table 2). The survival percentages for ART restorations
assessed by the ART criteria were 98.7% (6 months),
94.8% (1 year) and 83.7% (2 years), and for the
conventional restorations the percentages were 100
% (6 months), 98.7% (1 year) and 90.7% (2 years). There was no difference in the survival curves of ART
restorations with HVGIC and conventional restorations
with resin composite after 2 years (p = 0.181) (Figure
2). Statistical Analysis Table 2- Distribution of scores according to the modified USPHS criterion for ART restorations with HVGIC and conventional restorations
with resin composite J Appl Oral Sci. 2021;29:e20200609
6/10 Discussion After 1 and 2 years, differences in anatomical
shape and surface texture were detected between
the restorative approaches (Table 2). Differences in
color were lost after the 6-month recall. Although
there were differences between the restorations
and their anatomical forms and superficial textures,
during the 2 years of follow up, the quality of the
restorations was not compromised. Since the anatomy
of ART restorations is achieved by digital pressure,
their anatomical shape would be a disadvantage
over conventional restorations with resin composite. Besides the resin composite being nanoparticulated,
which ensures a high surface smoothness, the
polishing of restorations performed also collaborate
to a smoother surface texture, possibly explaining
the differences found in surface texture between
treatments. Other very important highlighted aspects for the
present study are the time lapsed from the beginning
of mixing and the initial removal of material excess
(2.5 minutes) and matrix removal (5 minutes). Those
are important to allow the initial material setting and
hardening.24 On the other hand, longer waiting periods
are not desired due to difficult excess removal with
possible occlusal interferences being left, a fact that
would lead to early restoration fracture. The operator
was aware and took into consideration bonding
stability of HVGICs to dentin is not so strong in the
early periods.25 Moreover, a step that may also increase
the longevity of multi-surface HVGIC restorations was
the creation of retention grooves in proximal boxes,
close to the amelodentinal junction, as noted by
Barata, et al.5 (2008) and Fernandes, et al.6 (2019). The main reasons for failure in this study according
to the ART criterion were: fracture of the restoration
and/or tooth (9 restorations), painful symptomatology
(5 restorations), one restoration failed due to excessive
wear and one restoration failed due to a marginal
defect of more than 0.5 mm. Only after 2 years one
restoration fail due to secondary caries. Discussion Both HVGIC and resin composite presented high
success rates after 2 years (>90%). Although clinical
success was similar within the assessed period of
time, one might consider performing ART restorations
since it has some advantages, among them the
use of inexpensive hand instruments, only infected
tooth tissue being removed, the employed material
presenting chemical adhesion to the tooth substrates,
and fluoride release.19 Considering modified USPHS criterion, survival
percentages of ART restorations were 98.7% (6
months), 94.8% (1 year) and 87.8% (2 years), and
for the conventional restoration 100% (6 months),
98.7% (1 year) and 90.7% (2 years). There was no
difference in the survival curves of ART restorations
with HVGIC and conventional restorations with resin
composite after 2 years (p = 0.552) (Figure 2). In the present study, regarding ART restorations,
the average annual failure rate was 6.3%, presenting
a longevity of restorations higher than a metanalysis J Appl Oral Sci. 2021;29:e20200609
7/10 A prospective and randomized clinical trial evaluating the effectiveness of ART restorations with high-viscosity glass-ionomer cement versus conventional restorations with resin
composite in Class II cavities of permanent teeth: two-year follow-up that reported 78.2%21. The difference can in part
be attributed to the material used, which was an
encapsulated GIC, presenting improved mechanical
properties compared with hand-mixed GICs commonly
used in ART.7 The present material also contains
improved liquid and powder and the restoration surface
is coated with nanofilled resin. The encapsulated
GIC removes the negative effects of proportioning
the powder/liquid ratio and diminishes the number
of porous produced by hand-mixing. In addition to
encapsulation, Equia Fil combines the main advantages
of HVGIC, with a nanofilled, light cured coat, which
protects the material in the initial setting phase and
occludes surface cracks and porosity, increasing its
wear resistance and toughness.12,14,22,23 and there was no statistical difference between them
according to the findings in the literature.29-31 This was
likely because the only two restorations with scores
3 and 4 (ART criteria) were also considered failures
according to the USPHS criteria. This study considered the use of the modified
USPHS criteria adequate and comparable to the
ART criteria. These criteria are relevant since they
can assess marginal discoloration, color and surface
texture, which are not measured by the ART criteria.32 At the 6-month evaluation, differences were
detected within the clinical acceptable parameters for
both treatments (color, anatomical shape and surface
texture). Authors' contributions Menezes-Silva, Rafael: Conceptualization
(Supporting); Data curation (Lead); Formal
analysis (Lead); Investigation (Lead); Methodology
(Supporting). Velasco, Sofia Rafaela Maito: Data
curation (Supporting); Formal analysis (Supporting);
Methodology (Supporting). Bastos, Roosevelt da
Silva: Data curation (Supporting); Formal analysis
(Supporting). Investigation (Supporting). Bresciani,
Eduardo: Formal analysis (Supporting); Methodology
(Supporting). Navarro, Maria Fidela de Lima:
Conceptualization (Lead); Data curation (Supporting);
Formal analysis (Supporting); Investigation
(Supporting); Methodology (Lead). The present results do not follow the main reasons
for the substitution of conventional restorations with
resin composite observed in the literature, which are
the development of secondary caries related to the
adhesive interface and fracture of the restoration that
is related to the mechanical properties of the material,
as well as to the quantity and quality of the remaining
dental structure.35,36 Poor oral hygiene along with high rates of DMFT
negatively impact the success of restorations in
general.37 Most publications do not report the oral
health status of subjects included in the studies nor
did they report whether a dental health program was
implemented along with clinical treatment.37 This may
also have been one of the reasons for the observed
restorative success of this study, because in addition
to explaining how to clean their mouth and reinforcing
the importance of preventive measures in each follow-
up, the participants received a new kit for oral hygiene
at each evaluation point. Acknowledgments One study reported34 that after a three-year follow-
up, the annual failure rates for resin composites and
resin-modified GICs in deep cavities were 14.6% and
26.7%, respectively. Now, regarding the management
of carious lesions, the annual failure rates for the
restorations were 17.3% when the selective removal
was performed and 13.1% when the total removal
of the carious lesion was performed. Thus, in the
present study, failure rates, especially for painful
symptomatology due to pulp involvement, were
considered low. The authors thank the São Paulo Research
Foundation (FAPESP, grant 2014/01626–3) and the
National Council for Scientific and Technological
Development (CNPq, grant 312060/2017-3) for their
support. Discussion The other
fractures in the study probably occurred due to
different intrinsic reasons of restorative materials, the
technique employed or patient habits.18,33 Regarding conventional restoration with resin
composite, the average annual failure rate was 4.3%,
being within the reported mean, which varies from 2
to 10% depending on the adhesive strategy used.26-28 Generally, ART restorations are evaluated by
ART criteria in most studies whilst the longevity of
restorations are assessed by USPHS criterion.15,29 It
has been suggested that the ART criteria are more
stringent than the USPHS criteria, since the marginal
defect or wear exceeding 0.5 mm is considered to be
a failure in the ART criteria and not for the USPHS
criteria, which will consider failure only if dentin is
exposed.30,31 Moreover, the ART criteria of dental
fracture considers failure even if the restoration
remains intact, opposing the USPHS criterion that
considers this scenario as success. Our results showed
that regardless of the evaluation criteria used for
restorations, the success rates were identical or similar, The results of the present study showed that
the preventive effect of caries was similar for both
materials. After 2 years, abscess and/or fistula were
present in three ART restorations and two conventional
restorations, suggesting a high level of efficacy after
2 years, regardless of the high caries experience of
the participants (DMFT=4.72). It is worth noting that
for those restorations, the protection of the dentin-
pulp complex with calcium hydroxide solution and J Appl Oral Sci. 2021;29:e20200609
8/10 SILVA RM, VELASCO SR, BRESCIANI E, BASTOS RS, NAVARRO MF the real effectiveness of restorations in non-inferiority
studies.38-40 cement had been applied for the ART restorations,
while calcium hydroxide solution and cement and GIC
base were applied when necessary for conventional
restorations with composite resin. Six other teeth
with deep carious lesions such as these responded
favorably to the protection of the dentin-pulp complex,
maintaining pulp vitality and restorative success after
24 months. The results of the present study showed that the
null hypothesis could not rejected, and there is no
difference after 2 years on the effectiveness of these
ART restorations with HVGIC compared to conventional
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7. doi: 10.1007/s00784-016-1832-1 36- Ferracane JL. Resin composite-state of the art. Dent Mater. 2011;27:29-38. doi: 10.1016/j.dental.2010.10.020 36- Ferracane JL. Resin composite-state of the art. Dent Mater. 2011;27:29-38. doi: 10.1016/j.dental.2010.10.020 19- Frencken JE, Makoni F, Sithole WD. Atraumatic restorative treatment
and glass-ionomer sealants in a school oral health programme in
Zimbabwe: evaluation after 1 year. Caries Res. 1996;30(6):428-33. References Clin Oral Investig. 2014;18:753-9. doi: 10.1007/s00784-013-1026-z 12- Diem VT, Tyas MJ, Ngo HC, Phuong LH, Khanh ND. The effect of
a nano-filled resin coating on the 3-year clinical performance of a
conventional high-viscosity glass-ionomer cement. Clin Oral Investig. 2014;18:753-9. doi: 10.1007/s00784-013-1026-z 30- Lo EC, Luo Y, Fan MW, Wei SH. Clinical investigation of two glass-
ionomer restoratives used with the atraumatic restorative treatment
approach in China: two-year results. Caries Res. 2001;35:458-63. doi: 10.1159/000047490 30- Lo EC, Luo Y, Fan MW, Wei SH. Clinical investigation of two glass-
ionomer restoratives used with the atraumatic restorative treatment
approach in China: two-year results. Caries Res. 2001;35:458-63. doi: 10.1159/000047490 13- Molina GF, Faulks D, Mulder J, Frencken JE. High-viscosity glass-
ionomer vs. composite resin restorations in persons with disability:
five-year follow-up of clinical trial. Braz Oral Res. 2019;33:e099. doi:
10.1590/1807-3107bor-2019.vol33.0099 13- Molina GF, Faulks D, Mulder J, Frencken JE. High-viscosity glass-
ionomer vs. composite resin restorations in persons with disability:
five-year follow-up of clinical trial. Braz Oral Res. 2019;33:e099. doi:
10.1590/1807-3107bor-2019.vol33.0099 31- Lo ECM, Holmgren CJ, Hu D, van Palenstein, Helderman W. Six-
year follow up of atraumatic restorative treatment restorations placed
in Chinese school children. Community Dent Oral Epidemiol. 2007;
35:387-92. doi: 10.1111/j.1600-0528.2006.00342.x 31- Lo ECM, Holmgren CJ, Hu D, van Palenstein, Helderman W. Six-
year follow up of atraumatic restorative treatment restorations placed
in Chinese school children. Community Dent Oral Epidemiol. 2007;
35:387-92. doi: 10.1111/j.1600-0528.2006.00342.x 14- Gurgan S, Kutuk ZB, Yalcin Cakir F, Ergin E. A randomized controlled
10 years follow up of a glass ionomer restorative material in class
I and class II cavities. J Dent. 2020;94:103175. doi: 10.1016/j. jdent.2019.07.013 32- Hickel R, Roulet JF, Bayne S, Heintze SD, Mjör IA, Peters M, et al. Recommendations for conducting controlled clinical studies of dental
restorative materials. Clin Oral Investig 2007;11:5-33. doi: 10.1007/
s00784-006-0095-7 15- Frencken JE, Holmgren CJ. ART: a minimal intervention approach
to manage dental caries. Dent Update. 2004;31:295-8, 301. doi:
10.12968/denu.2004.31.5.295 15- Frencken JE, Holmgren CJ. ART: a minimal intervention approach
to manage dental caries. Dent Update. 2004;31:295-8, 301. doi:
10.12968/denu.2004.31.5.295 33- Kemoli AM, Opinya GN, van Amerongen WE, Mwalili SM. Two-year
survival rates of proximal atraumatic restorative treatment restorations
in relation to glass ionomer cements and postrestoration meals
consumed. Pediatr Dent. 2011;33:246-51. 16- American Dental Association (ADA). Covid-19 Resources for
dentists [internet]. Chicago: ADA; 2020 [cited 2020 Apr 9]. Available
in: https:// www.ada.org/en/member-center/coronavirus-resource-
toolkitfor-ada members. References doi: 10.1159/000262355 37- Amorim RG, Frencken JE, Raggio DP, Chen X, Hu X, Leal SC. Survival
percentages of atraumatic restorative treatment (ART) restorations
and sealants in posterior teeth: an updated systematic review and
meta-analysis. Clin Oral Investig. 2018;22:2703-25. doi: 10.1007/
s00784-018-2625-5 37- Amorim RG, Frencken JE, Raggio DP, Chen X, Hu X, Leal SC. Survival
percentages of atraumatic restorative treatment (ART) restorations
and sealants in posterior teeth: an updated systematic review and
meta-analysis. Clin Oral Investig. 2018;22:2703-25. doi: 10.1007/
s00784-018-2625-5 20- Zanata RL, Fagundes TC, Freitas MC, Lauris JR, Navarro MF. Ten-
year survival of ART restorations in permanent posterior teeth. Clin
Oral Investig. 2011;15(2):265-71. doi: 10.1007/s00784-009-0378-x
21- Amorim RG, Leal SC, Frencken JE. Survival of atraumatic restorative
treatment (ART) sealants and restorations: a meta-analysis. Clin Oral
Investig. 2012;16(2):429-41. doi: 10.1007/s00784-011-0513-3
22- Ilie N, Hickel R, Valceanu AS, Huth KC. Fracture toughness of
dental restorative materials. Clin Oral Investig. 2012;16(2):489-98. doi: 10.1007/s00784-011-0525-z 38- Altman DG. Practical statistics for medical research. Chapman &
Hall, London; 2003. 38- Altman DG. Practical statistics for medical research. Chapman &
Hall, London; 2003. 39- Clark TG, Bradburn MJ, Love SB, Altman DG. Survival analysis part
I: basic concepts and first analyses. Br J Cancer. 2003;89:232-8. doi:
10.1038/sj.bjc.6601118 10.1038/sj.bjc.6601118 40- Botelho F, Silva C, Cruz F. Epidemiologia explicada-análise de
sobrevivência. Acta Urol. 2009;26:33-8. 40- Botelho F, Silva C, Cruz F. Epidemiologia explicada-análise de
sobrevivência. Acta Urol. 2009;26:33-8. 23- Friedl K, Hiller KA, Friedl KH. Clinical performance of a new glass
ionomer based restoration system: a retrospective cohort study. Dent
Mater. 2011;27:1031-7. doi: 10.1016/j.dental.2011.07.004 23- Friedl K, Hiller KA, Friedl KH. Clinical performance of a new glass
ionomer based restoration system: a retrospective cohort study. Dent
Mater. 2011;27:1031-7. doi: 10.1016/j.dental.2011.07.004 J Appl Oral Sci. 2021;29:e20200609
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Metode Istinbat Hukum Jaringan Islam Liberal (JIL)
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Al-Manahij : jurnal kajian hukum Islam/Al-Manahij
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* Mahasiswa STAIN PurwokertoJurusafl Syariah Program Studi Ahwalus Syakhsiyy.h
^'Igk^t^n 2@3. Abstract Tlte netbods oJ interpreting al-Qtr'an often claborate tpon
?bihtEb, brt, arrd ftali, and nse g1u rcu pmpctiw taw*
some m m,aties in Islanit lays- Tbis clupter expbt: abo* one
nethod y'hicb is practiced b1 lilanic Liberal N*work $Q. Connon! JIL nntrib*es lh rdional ilntqlbl and it is based ot
tlx na:lahah pincipbs. Kaa kunci metode istinbit mas. lahah, pemikiran rasional METODE IS TIATEAT HUKUM JARINGAN
ISI.AM LIBERAL OIL) Aif Suandi* A. Pendahuluan Jaringan Islam Libetal SIL) secara tesmi didirikan pada tanggal
8 Maret 2001 di Jakarta. Pada mulanya ia adalah sebuah forum
diskusi melalui milis (islamlibetal@yahoo.com) yang telah ada sejak
tahun 1999 (setqlah lengsemya Soeharto). Enam aktivis mudanya
yang telah bertemu Gunawan Muhammad pada tanggal 4 lmuart
2001 mulai metekrut para mahasiswa dan intelektual untuk
bergabung di milis tersebut. Meteka memutuskan untuk
menggunakan ianngan, bukannya organisasi, asosiasi, ataupun partai
politik. Sebagai contoh Hamid Basyaib beratgumen bahwa JIL adalah 17 Al-Manlhii, Vol. 3 No l Jaouati -Juni 2009 SyuFaat SyuFaat gerakan intelektud den kultural, oleh karena itu ia bukanlah partai
politik, oryanisasi" atau sekte ^g rn yarg mempunyai ritual yang
ketat dengan sanksi dan hukuman. Oleh karena fokusnya adalah
melawan fundamenalisme,Jll memilih untuk mewadahi diti mereka
&l"m bentuk iaring'an, bukannya sebaga.i organisasi yang ketat aau
melibatkan diti &lam partai politik. Dengan demikianJll merupakan
aliansi yang teduka bagi siepapun yang mempunyai keinginan untuk
mempublikesikan ide-ide Islam libetal. Tulisan ini akan menielaskan
sedikit mengenai metode isti it htkum yang merupakan poros
petdebatan para ahli hukum Islam. I Satria liffendi, Uhl Fiqrb Q^lrta Prenada Media 2005), hd. 79.
Yusuf Rahman, "Pluralitas Penafsinn al-Qucao", dalam Iy'ilrul lrbt Lbcml,haL 12.
'Penafsiran scperti ioi dapat dil.ihat pad. tafsi Jalibh. ,
q
Q lrta
),
Yusuf Rahman, "Pluralitas Penafsinn al-Qucao", dalam Iy'ilrul lrbt Lbcml,haL 12.
'Penafsiran scperti ioi dapat dil.ihat pad. tafsi Jalibh. I Satria liffendi, Uhl Fiqrb Q^lrta Prenada Media 2005), hd. 79. ,
q
Q lrta
),
Yusuf Rahman, "Pluralitas Penafsinn al-Qucao", dalam Iy'ilrul lrbt Lbcml,haL 12 usu
a
a ,
u a tas Penafsinn al Qucao , dalam Iy ilrul lrbt Lbcml,haL 12
'Penafsiran scperti ioi dapat dil.ihat pad. tafsi Jalibh. aZllhziri Mtsrew:- Alprat Ktae ToLtati, flakarta Fitrah, 2m4, hel. 64.
5R brD2r, "Plur.lit s...", hal. 13.
6Kusmaaa, "Iafsir d-Qulan lnl rdrf', mVV-nt6thdr, did@'oload pad. tanggal
12 November 2008. Di .ot r. pegar-p.gar yang dikcmbaogkao e,ddrah a$a-b al-nV-|,
peodas.nn terhad+ pedielasao alQulaa yeog disampaikao geaerasi pcrume (Nabi d:o
Sahzl!,,lt), tdi'bt t&i' al-tilih. Petdzsztzo a mcnurut Kusmaoa adalah prinsip dasrr dzri
metode t fsir yzog dikeoal detrga$ al-t$& bi ol-nall*, Laidah kebrhasaaa abrogasi (ptinsip
penyeleseian kootradilsi tntzt tyt ddzm asprk huLum aau redaksinye), saopai pade
pers,,arrt.n iotegtitas moral dzo otoritas l(eilJtrurn peoafsir. Dalan peogeobaogan pzgar-
pagar pemfsino ters€but teLh terr.di ortodoksi tafsir al-Qur'rn bahkao sampei pada
kesirupuleo hahva tafsir yang bener edalah tafsir yang sejeuh ruuagkio bta aemagari
keterlibaua subye\ akel ideologi dan oe&heb penafsir. Artioya, afsir yrng seLmr ini
dianggap "beoa1' adalah peoafsiran yang berpusar Vad^ tey (la<t cnkt d ,nlalnbiot).
lMtsnwt, dpn m..., hd. 1 O. 1. AI-Qut'an. Al-Qulan dalam kaiieo usul 6kih merupaken obyek utama dan
Pertama kaiian d^lam memecahLan suatu hukum. Al-Qur'an
menrrnrt bahasa berarti "ba,cztt" dan menurut istilah usul 6kih
ia berarti kahn (petkatazn) Allah yang diturunkan-Nya dengan
perantaraan Malaikat Jibril kepa& Nabi Muhammad dengan
bahasa Arab dan membacanya adalah ibadah.l Pandangan
Jaringan Islam Libetal tentang al-Qur'.n tercpresentzsikan dalam
dua tema besar sebagai berikut: a. Al-Qur'an sebagai teks terbuka
Memahami al-Qur'an deogan membiatken ia berbicara seodiri,
menurut Yusuf Rahman,2 disebut dengan penafsirao yang
berpusat pada teks (xxt ccntcnd inkrprctatiott). Metode ini
dilakukan dengan menganalisa atti bahasanya, gaya
bahasanya, m.aupun struktur bahasa.3 Tafsir al-Qur'an hanya
bersandat pada teks dan teks dianggap sebagai benda yang
diam. Zuhaid Misrawi yakin bahwa al-Qur'an adalah teks
yang terb"ka. Maksudnya al-Qut'an bukaolah barang antik a. Al-Qur an sebagai teks terbuka
Memahami al-Qur'an deogan membiatken ia berbicara seodiri,
menurut Yusuf Rahman,2 disebut dengan penafsirao yang
berpusat pada teks (xxt ccntcnd inkrprctatiott). Metode ini
dilakukan dengan menganalisa atti bahasanya, gaya
bahasanya, m.aupun struktur bahasa.3 Tafsir al-Qur'an hanya
bersandat pada teks dan teks dianggap sebagai benda yang
diam. Zuhaid Misrawi yakin bahwa al-Qur'an adalah teks
yang terb"ka. Maksudnya al-Qut'an bukaolah barang antik 18 Al-Manihii, Vol. 3 No. 1 Januad - Juni 2009 Konseptualisasi Qat'i dan Zannidalam Diskursus Usul Fikih yang tidak dapat drjamah sama sekali. Dalam bahasa yang
lebih sederhana, dibutuhkan ke{a-keria penafsitan yang
mempunyai televansi dengan konteks masyarakat pembaca.a
Pembacaan tidak lagl hanya terpusat pada teks akan tetapi
hatus menghargai peran pembaca atau penafsir, nafur ce nd
iiterprctation, meminiam istilah Yusuf Rahman,5 yakni
menekankan pentingnya petan pembaca di dalam mencad
dan membedkan makna suatu teks. Pare pemethati kaiian
al-Qut'an sebenarnya menyadari potensi problem dalam
menafsitkan Kitab gusi. Hf ini ssrlihet dengan upaya mereka
unruk mengembangkan apa yang kita kenzl sebzgi ' ttn al-
Qtr'an, di dabmnya dibaogun p^gr-p^glt a,g;rl produk tafsir
itu "benal'.6 Sebagai teks, al-Qulan mempunyai karaktet yang
terbuka, bahkan progtesif, yaitu membangkitkan lahimya
betbagai macam pen&pat, bahkan ia telah mendotong umat
uotuk menghasilkrn perubahan progtesif.T b. Al-Qw'an sebagai produk budaya
Al-Qut'an sel^in 5slagai "teks wahyu" iuga sebagai "teks
budaya" yaitu teks yang sangat erat hubuogaffiya denga.n
budaya. Dalam posisinya sebagai teks wahyu umat Islem
meyakini bahwa al-Qut'an adaleh wahyu yang dituru''k,n
oleh Allah melalui malaikat Jibril kepada Nabi Muhammad
SAW Kemudian al-Qu/an dnl"m Posisinya sebagai teks yang b. Al-Qw'an sebagai produk budaya
Al-Qut'an sel^in 5slagai "teks wahyu" iuga sebagai "teks
budaya" yaitu teks yang sangat erat hubuogaffiya denga.n
budaya. q
r0 Lihat Nasr tlamid Abu Z^id, T.krrldlitdr alp1rbr, teri. Khoiron Nahdlilyin,
fiogyakarta: LKIS, 2005), ha1.29-61. Pemahaman Jaringao Islam Liberal mengeoai d-
Qulen memang sangat dipengaruhi oleh pemikiran Abu Zaid. Al-Qur'an edalah produk
budaya schingga bersifat historis datr lokal ( rab). Al-Qur'an sama halnya dengen teks-teks
lain yang tidak bisa legras dari kitik. Al-Qur'en schagei sebuah tcks, pada dasemyr merupalan
produk budaya. Anhya tcks terbentuk dalam realias dan budaya mdalui rentang waktu
lebih dari duapuluh tahun. ,,
'Nlengenai pandango kritis JIL terhedap kodiElasi d-Quran dapat dilih.t pada Luth6
Assfirkanie, "Mereouogkan Sejarah a.lQur'an",delzm ljtibd kbt bbcral, ha.l. 1. Assyaukani
mempertanyakan dafl mengukan apakah d-Qu lar mulai dti srat al-Fdtibalt sempai a/-N,4r
ada.lah kzlam Alleh yang diturudktu keprda Nebi Muhmmad baik kaa maupun malnanya?
Menurutoya al-Qur'eo adalah kelen Allah yang di,*ahyukan kepada Nabi Muhammad
tetapi kemudian meogalami berbagai p.oses "aP) dirirl' oleh pera sahabat, ra&ii, ehli
taczzn, qmi', rxogn6, mesin cetat den kekuasaan. Ilid,, heL 66. Ilid,, heL 66.
'Nlengenai pandango kritis JIL terhedap kodiElasi d-Quran dapat dilih.t pada Luth6
Assfirkanie, "Mereouogkan Sejarah a.lQur'an",delzm ljtibd kbt bbcral, ha.l. 1. Assyaukani
mempertanyakan dafl mengukan apakah d-Qu lar mulai dti srat al-Fdtibalt sempai a/-N,4r
ada.lah kzlam Alleh yang diturudktu keprda Nebi Muhmmad baik kaa maupun malnanya?
Menurutoya al-Qur'eo adalah kelen Allah yang di,*ahyukan kepada Nabi Muhammad
tetapi kemudian meogalami berbagai p.oses "aP) dirirl' oleh pera sahabat, ra&ii, ehli
taczzn, qmi', rxogn6, mesin cetat den kekuasaan.
r0 Lihat Nasr tlamid Abu Z^id, T.krrldlitdr alp1rbr, teri. Khoiron Nahdlilyin,
fiogyakarta: LKIS, 2005), ha1.29-61. Pemahaman Jaringao Islam Liberal mengeoai d-
Qulen memang sangat dipengaruhi oleh pemikiran Abu Zaid. Al-Qur'an edalah produk
budaya schingga bersifat historis datr lokal ( rab). Al-Qur'an sama halnya dengen teks-teks
lain yang tidak bisa legras dari kitik. Al-Qur'en schagei sebuah tcks, pada dasemyr merupalan
produk budaya. Anhya tcks terbentuk dalam realias dan budaya mdalui rentang waktu
lebih dari duapuluh tahun. 1. AI-Qut'an. Dalam posisinya sebagai teks wahyu umat Islem
meyakini bahwa al-Qut'an adaleh wahyu yang dituru''k,n
oleh Allah melalui malaikat Jibril kepada Nabi Muhammad
SAW Kemudian al-Qu/an dnl"m Posisinya sebagai teks yang 79 Al-Manihii, Vol. 3 No. 1 Jaouad -Juni 2009 Arif Susandi erat hubungannye dengan bu&ya belum mendapat perhatian
yang semestinya khususnya perihal dimensi linguistik dan
historisitas kodifikasi al-Qur'an.6 Faktanya al-Qur'an pada
masa awal tutunnye adalah berbentuk verbal dan baru
dikodifikasikan pa'dt ztmzr Usman ibn 'Affan. Kodifikasi
al-Qur'an yang dil"kuk,q oleh Usman memang tidak berialen
dengan mulus da'' banyak tedadi kontroversi di anara pata
sahabat meskipun pada akhirnya Usman berhasil
menyelesaikannya dan kemudian memusnahkao seluruh
mushaf lainnya yang tidak disepakati.e erat hubungannye dengan bu&ya belum mendapat perhatian
yang semestinya khususnya perihal dimensi linguistik dan
historisitas kodifikasi al-Qur'an.6 Faktanya al-Qur'an pada
masa awal tutunnye adalah berbentuk verbal dan baru
dikodifikasikan pa'dt ztmzr Usman ibn 'Affan. Kodifikasi
al-Qur'an yang dil"kuk,q oleh Usman memang tidak berialen
dengan mulus da'' banyak tedadi kontroversi di anara pata
sahabat meskipun pada akhirnya Usman berhasil
menyelesaikannya dan kemudian memusnahkao seluruh
mushaf lainnya yang tidak disepakati.e Apa yang dikemukaken oleh Jadngan Islam Libetal kongruen
dengan tesis dari Nasr Hamid Abu Zud yxrg menyatakan
bahwa budaya Isl,"' adqlah bu&ya tekstual kebu&yaan yang
selalu bedandaskan kepada teks, yakni al-Qur'an dan al-
Hadis. Al-Qut'an, meskipun ia adalah wahyu dari Allah SWT
akan tetapi tidak &pat dilepaskan dari konteks historis pada
waktu al-Qw'an diturunkan. Al-Qur'an adalah iawaban atas
tantaogen umat Muhammad SAW waktu itu, sehingga al-
Qur'an metupakan produk budaya, menyeiarah, dan dapat
.likritit layaknya teks-teks lain.l0 Menurut Ulil, seiak menit pertama proses gradual
penuruneonya, al-Qut'an telah meniadi pokok petsoalan di 20 Al-Man;hii, \rol. 3 No. l Januari -Juni 2009 Al-Man;hii, \rol. 3 No. l Januari -Juni 2009 l i Metode Istinbal Hukum laingan Islam Liberal QIL) kal"ngan orang Arab yang menyaksikan turunnya wahyu. Al-
Qur'an dengao cukup baik metekam fenomgla ifl dqlam Q.S. al-Ftrqit: 32:
"{
i'l*' tt
i
i kal"ngan orang Arab yang menyaksikan turunnya wahyu. Al-
Qur'an dengao cukup baik metekam fenomgla ifl dqlam Q.S. al-Ftrqit: 32:
"{
i'l*'
i "{+,i'l*'ctt;pi *b Jj Si it; u-itr',lsS
" g5'.;S'r3L a 5t i --. 4-,!))-' i-L "{+,i'l*'ctt;pi *b Jj Si it; u-itr',lsS
" g5'.;S'r3L a 5t i --. 4-,!))-' i-L Menurut A.lirn Husaioi pemahaman seperti ini betbahaya
karena penafsiran tethadap al-Qut'an dianggap banyak
mengandung ploblem Iantemn rentang waktu dan situasi yang
sangat iauh berbeda aotata dulu dan sekarang. ItPemyataao terebut timbul d.i'kdeogan oreng-orang Areb karena a&nya asumsi yeng
tidak seluruhaye tepat bahua yrh1ar-c.hp yrng tumn pada Nzbi-Nabi sebelum Mubemmad
bc,nld.tjdnbb Aifut.'I^*^l^ omng-oaog Anb memp€rtaryaloo mengapa tidak diturunk2n
sekzligus? Jawabm yeng diberilen ,{Dh seog.tlah merar:Jl ydtra b4iitlz t dzbbita Uh
fu'Aah ,DtdlrahAt ulliUll r.l,e,,d;qeazhkan ayit ters€but dengan "pcncicilaa itu Aku
(Anah) hkukan agar Aku membuat hrtimu rozng dao kukuh, (dan sekdipuo ayat-a,zt itu
turuo secera bcrahap, tet p0 ALu susuo kerlbali begitu rupa s€hingga menjadi beruruten
daa teni6 (artil)." l ^t
AbdDlla Mrq*6*a Knbat, h . 180.
DAdian Husaini" "Kritik HermeaeutiL dQut'an ," ddzm V/V4Y.blantb.aa, didolthad
pada t-.ggd 12 November 2008.
'r Historisios teks dQu1m menumtJ"'i.fn lslem Libeol lebih masuk akal kerena
untuk mendukung budaya oespfllat Arzb yrog masih bersifat od bukennya budaya nrlis.
Peocicilan al-Qulan meodutuog dan rueoudahkan para Sahahat Nabi untuk mengahfal.
Dengan roemahami konsep historisitas teLs dQur'aa Jaringa Islarn Liberal, bisa diarik
kesimpulan bahwa teks dQur'm adalzh rcsloo terhadap feromcoa sosid yeog tedidi pad.
ma.syar2k2t Areh Oleh L2rcrll itu, wrhfr s.og2t terpengaruh oleh determioasi sosid histor\
ertinye $rehyu ditentukm, dikondisikeo, d:n dipengaruhi oleh keadaan sekitar. 1. AI-Qut'an. Dengan
demikian pedu adanya upaya tafsir ulang, tetmasuk tafsir
tethedap ^y^t-^y^t mrbaamit sekalipun di mana hal ini
dianggap sebegai dinamisasi ayat-ayat al-Quran dan
merupakan ptestasi akrl yang brilian.t'z Keyakinan Jaingan
Islam Libetal eken historisitas teks al-Qut'an semakin
menemukan pettguetrn ketika ditemukan adanya metode
pernzhaman al-Qdr'an klasik yang mempertimbangkn arlaT
al ttg-l dzr riikb nauikh.n tr!-l{mitii, Vol. 3 No 1 Jmueti - Jrmi fi09 ItPemyataao terebut timbul d.i'kdeogan oreng-orang Areb karena a&nya asumsi yeng
tidak seluruhaye tepat bahua yrh1ar-c.hp yrng tumn pada Nzbi-Nabi sebelum Mubemmad
bc,nld.tjdnbb Aifut.'I^*^l^ omng-oaog Anb memp€rtaryaloo mengapa tidak diturunk2n
sekzligus? Jawabm yeng diberilen ,{Dh seog.tlah merar:Jl ydtra b4iitlz t dzbbita Uh
fu'Aah ,DtdlrahAt ulliUll r.l,e,,d;qeazhkan ayit ters€but dengan "pcncicilaa itu Aku
(Anah) hkukan agar Aku membuat hrtimu rozng dao kukuh, (dan sekdipuo ayat-a,zt itu
turuo secera bcrahap, tet p0 ALu susuo kerlbali begitu rupa s€hingga menjadi beruruten
daa teni6 (artil)." l ^t
AbdDlla Mrq*6*a Knbat, h . 180. " Lihat Abi Diwud Suleimaa ibn d- Ash'eth d-Silis6ni, Jm AE DAd @iR t
D;r rt-Hadd, 1999), I: 494. 16 Menurut Husseien Nesr sebageimaaa ditutip dch Kmnrddin tlitayaq Hermes
tidak lein adalah Nabi I&is. Heraeoeutita, yrog diemtrfl &ri peren Hemes, rdebh sebueh
ilrnu dao seni oeogioterpreasi}ao sebuah tels Drgi Hffi Fsel2r! fog Frt rm. dih.d.pi
adalah bagaimanr rncnafsirkao pcsao Tuhao pog bertn:re deagen b2h2s. 'l2ogit" agrr
dapat dipahami oleh manusia Fng berbirn deogm b.h-- 'hrmi". Seiek eud hetmeoeutila
berunrsan deogan tugzs meoerzogkeo kata-Laa dao eh yrogdiasat n asiog ohh nasyrolrt
(alid tp*tb),baik it dt"^g dari Tuhea rnaupun yrog terblzn deogzo bohesr bngit mruprm
yeng &ang dari generasi tedahulu yeng hiclup dalam todisi rLn iuga bahesa y.og'.sing".
Komaruddin lljdtytt, Mcaabai Bzhu dQr'o* Schd Aia Hamutih, {zLznz:
Paramadina, -1996), h.L 125-126. fsUlil Abshar Abdalla, 'Scl Kcdudulen ll2dis", d.Lm anEbTa@,afugn4.*,
didowoload pada tmggal t2 Novernber 2008. Pcoi:hs tcnteng kitcria srhih, hasan, drn
dalf dtpzt dillhx dalam Muhamoed llashin Kzodi, Di.4 dd Tari-t&.i Ht*n lrb,
(Yogyak rt Pustaka Pclaiar, 1996), hd. 10t- 17 Mochamad Nur Ichwrn , Mtntat K*djd@ Kritr olQn'a Q*zrtz: "let$t, NO3),
hel. 89. 2. Al-Hadis 15 bertentangan dengan semang^t l; ikiba f dl-din. Uhl dd*
menedma hadis tersebut .l-ti segi matao atau sekutang-kurangnya,
harus ditafsh ulang. 15 2. Al-Hadis Mengenai kehuii"h". hadis sebagei sumbet hukum l5lam &pat 21 tr!-l{mitii, Vol. 3 No 1 Jmueti - Jrmi fi09 Arff Susandi Arff Susandi .lilihat peda rekeman peristiwa diutusnya Mu'az ibn Jabal ke
Negeri Yaman oleh Rasul sebagei berikutra
il tJrJtl5t , e-,:r 6irrri[ .1."f il Jy1 ol
."a;i :Ju tel-ii iJJ ,f p tttL.a;;:-;"{ :)t;
:."+ :J6 3r.,t56s li.,li:Ju.irr .,t(,
ir J,
z,',-" J G I dli :Ju .l .,-p,lr J4
.rJI Yr.1ir-rxr*i:Ju.,6rt.+t.5 J )l .f ..r
6.iJr .6 -tJr :JUJ o.s.t-,a . p. uc i,tt ) r-, -trai
irr Jr; ,-f -c J..lr J4 Jrs ds ,
Posisi hadis sebagai sumbet hukum Islam yang kedua diakui oleh
Jaringan Islam Libetal. Hadis kedudukannya adalah sebagai
penjelas terhadap al-Qu'an. Pandangan Jaringn Islam Libetal
mengenai kedudukan hadis ti&k jauh beda dengan mayoritas
ulama. Akan tetapi kendati mengakui hadis sebagai sumbet
hukum Islam namun ia tetap harus diteliti kembali secara kritis. Dzi segS sanad,hadis telah diseleksi secata ketat pada masa ta&tin
untuk mencad hadis yang benar-benar sahih. Tatapi Ulil Abshar
Abdalla beqpendapat bahwa untuk menyatakao hadis bemilai
sahih ataupun sebaliknya, tidak cukup hanya dilihat dari segi
sanadnya saia akan tetapi hatus iuga kdtisi .l"ri segi mataonya. Kritik matan hadis menutut Ulil adalah bidang yang sangat
tertinggal perkembangannyr daJtm ilmu hadis. Banyak hadis
yang tidak sesuai dengan semangat al-Qu'an, misalnya hadis
tentang perinteh membunuh otang murtad. Bagi Ulil, ini Posisi hadis sebagai sumbet hukum Islam yang kedua diakui oleh
Jaringan Islam Libetal. Hadis kedudukannya adalah sebagai
penjelas terhadap al-Qu'an. Pandangan Jaringn Islam Libetal
mengenai kedudukan hadis ti&k jauh beda dengan mayoritas
ulama. Akan tetapi kendati mengakui hadis sebagai sumbet
hukum Islam namun ia tetap harus diteliti kembali secara kritis. Dzi segS sanad,hadis telah diseleksi secata ketat pada masa ta&tin
untuk mencad hadis yang benar-benar sahih. Tatapi Ulil Abshar
Abdalla beqpendapat bahwa untuk menyatakao hadis bemilai
sahih ataupun sebaliknya, tidak cukup hanya dilihat dari segi
sanadnya saia akan tetapi hatus iuga kdtisi .l"ri segi mataonya. Kritik matan hadis menutut Ulil adalah bidang yang sangat
tertinggal perkembangannyr daJtm ilmu hadis. Banyak hadis
yang tidak sesuai dengan semangat al-Qu'an, misalnya hadis
tentang perinteh membunuh otang murtad. Bagi Ulil, ini 22 A-Meoihii, vol. 3 No I Jrouad - Juai 2009 A-Meoihii, vol. 3 No I Jrouad - Juai 2009 Metode Istinbat Hukum laringan Islam Liberal (IL) bertentangan dengan semang^t l; ikiba f dl-din. Uhl dd*
menedma hadis tersebut .l-ti segi matao atau sekutang-kurangnya,
harus ditafsh ulang. fsUlil Abshar Abdalla, 'Scl Kcdudulen ll2dis", d.Lm anEbTa@,afugn4.*,
didowoload pada tmggal t2 Novernber 2008. Pcoi:hs tcnteng kitcria srhih, hasan, drn
dalf dtpzt dillhx dalam Muhamoed llashin Kzodi, Di.4 dd Tari-t&.i Ht*n lrb,
(Yogyak rt Pustaka Pclaiar, 1996), hd. 10t-
16 Menurut Husseien Nesr sebageimaaa ditutip dch Kmnrddin tlitayaq Hermes
tidak lein adalah Nabi I&is. Heraeoeutita, yrog diemtrfl &ri peren Hemes, rdebh sebueh
ilrnu dao seni oeogioterpreasi}ao sebuah tels Drgi Hffi Fsel2r! fog Frt rm. dih.d.pi
adalah bagaimanr rncnafsirkao pcsao Tuhao pog bertn:re deagen b2h2s. 'l2ogit" agrr
dapat dipahami oleh manusia Fng berbirn deogm b.h-- 'hrmi". Seiek eud hetmeoeutila
berunrsan deogan tugzs meoerzogkeo kata-Laa dao eh yrogdiasat n asiog ohh nasyrolrt
(alid tp*tb),baik it dt"^g dari Tuhea rnaupun yrog terblzn deogzo bohesr bngit mruprm
yeng &ang dari generasi tedahulu yeng hiclup dalam todisi rLn iuga bahesa y.og'.sing".
Komaruddin lljdtytt, Mcaabai Bzhu dQr'o* Schd Aia Hamutih, {zLznz:
Paramadina, -1996), h.L 125-126.
17 Mochamad Nur Ichwrn , Mtntat K*djd@ Kritr olQn'a Q*zrtz: "let$t, NO3),
hel 89 It Moch. Nur lchran, "Al-Qurho sebagai teks, teori tets dalam hermeneutik al-
Qu-1an Nasr Hernid At.l, Zoid', ddattt Stuli alQn'a, Ktrt npv, el,. AlxJd Musaqim dan
Syahiron Syamsudin, (Y%d.rta: Tiara !(|cara 2002), h2l. 158-159.
r Komaruddin Hidayat,Mcaabai Bahta alQrr'oq Schab l:ata Ha*t*iN {ahxtt.
Parrmadina, 1996), hal. 130. C. Pendekatan fletmeneutik daLrn [f66rherni Nas Syarak Istilah hetmeneutik ateu hermeoegtik- meruiuk pe;da nzru
dewa Yunani kuno yang bername Hermes yaog tugesnya
menyampaikan berita dari Saog Mehe Dewa (Zeus) kepada
manusia.l6 Sebenarnya banyak teori hetmeneutika yang telah
betkembang, akao tetapi di sini penulis henya akan memapetkeo
teori hermeneutika Nasr Hamid Abu Zaid. Hd ini penulis lah:kan
karena teori hetmeneutika Abu Zaid seiak permulaan dipetsiepken
untuk membedah al-Qur'an dan hadis Pemahaman Abu Zaid tentrng m'ko' dnn sign i6lrosi rcqera
umum didetivasi dati Hfusch. "Makna" adalah makna yang
dipresentasikan oleh teks &n signifikansi qd^lqh 7p7 yxog muncul
rlalam hubungan antar makna d-n pembeca- Dengan 4"615a11t"akna
adalah "makna kontekstual original" yang hrtnpir-hampir m-pan
(fixe$ dtsebtbkan historisitasnya" sedangkan signifikansi "bisa
berubah" (hangabb).l1 Abu Zaid membedakan antera makna yang
dimaksud penulis dan makna yang dipetgrmakan oleh teks. Dia lebih
menekankan pa& makna yaog teralhir, epebila .natnq bergantung hel. 89. 23 Al-Maoihif, vol. 3 No. 1 Jaouari - Juni 2009 Arif Susandi Arif Susandi pade meksud penulis, yaitu Allah SWT, hd ini akan menutup makna
d"; sigoiEkansi apapun. Kata-kata hter (nantiq) teks al-Qur'an betsifat ilahiyah,
namun ia meniadi sebuah "konsep" @oJhn ) yang relatif dan bisa
betubah ketika ia dilihat d'.i perspektif manusiawi. Menurut Abu
Zid te ttzs adalah dasar. Dad realias dibentuklah teks (al-Qur'an)
dan dari bahasa dan budayanya tetbentuklah konsepsi-konsepsi
(naJihin). Di tengah-tengah gerak intetaksi dengan manusia
terbentuklah mzkm (dalilah).\8 Wilayah teori Abu Zid renlcaLng
^1-
Qut'an adalah wilayah pemahaman terhadap al-Qur'an secata
sosiologis bukan teologis. Yang tetpenting dati konsep yang
ditawarkan oleh Abu Zild adzlah dalam memahami teks al-Qut'an
seseorang harus paham tktn tigz level makna pesan dalam level
teks, secara ringkas konsep level teks dapat diielaskan sebagai
berikut: 1. Teks level pertama, yaitu makna yxlg halnyt menuniuk kepa&
bukti atau fakta historis yang tidak dapat diintelptetasikan secata
metafotis. Misalnya ayet tentang tentata gajah. 2. Teks pada level kedua, yaitu makna yang menunjuk kepada bukti
atau fakta historis &n &pat diidentifik.sikan secata metaforis,
misaftya
^y^t tentang penggambaran surga. y t
3. Teks pada level ketiga, yaitu makna yang dapat diperluas
berdasarkan aas signifik^nsi yang diungkap dari konteks sosio
kultural dalam teks itu berada. Misalnya ayat teotang waris
maupun poligami. Hubungan antara pikiran, bahasa, dan wacana adalah
metupakan salah satu agenda pokok kaiian hetmeneutik. Yaitu
mengkaji pikiran &n petasaan otang y,ng telah tedembagakan dalam
bahasa n:lis, sementala pembicaranya su&h tidak ada.1e Artinya 24 Al. -M.n,hii, Vot. 3 No. n IU{ haJ^. 134. Di da.lam berhadapan deogan suatu teks, proses penjaralan tidak
hanya terjadi anara si pengarang dan pembac^ @ntunriihdftorn lb. a ln ), atan tetapi iuga
dengan konteks. Jika di delam suatu dialog lisan, masing-masing pihak (pembicara darr
pendengar) meogetahui kooteks pembicaraan dan bila terjadi kesalah-pahaman di antara
keduanya mereke dapat merujuk secara langsung kepada konteks apa yang sedang dibicarakan.
Sebaliknya ketika membaca suatu teks kita sering kali mengalami kesuhran di dalam
mengetahui apa konteks awal dari pembicaraan, walaupun kadang-kadang si penprang
telah mencoba menyelipkan konteks tersebut. Oleh karena itu adelah sangat mungkin
terjadinya kesalahpahaman di dalam membaca suatu karye tulis kalau kita tidak mengetahui
konteks pembicaraan (Attarciat dton ,h rorrt x4. Lihat Suramo, "Pluralismc Agama dalam
Hermenentika Paul Richev", V4VlV.Itb ib.an, di&*nlad ptdo, trulgal 12 Agustus 2008.
" Abd. Muqsith Ghazati, "Meenceng Keidah Usul Fikih Baru," makalah diseminarkao
pada acara Wotkshop JIL III, Jakaru: Wahid Iostitute, 2008, hd. 12. g
" Abd. Muqsith Ghazati, "Meenceng Keidah Usul Fikih Baru," makalah diseminarkao
pada acara Wotkshop JIL III, Jakaru: Wahid Iostitute, 2008, hd. 12. 2 Pare ulama sa.laf telah menerapL^ t orbE (metode) med.fsirkao Al -Qttrzo. Patana,
untuk rDeoafsirken sebuah ayat harus tedebih dulu dicari tafsimya dcngao ayat yaog lain,
misalola teotang hzri ki.rn * (al.qii'ab),ryatah lcDn* int (D; ohA'ot), d$ eyat seterusnya.
Kedu,hila idak ada, ryat yang meoafsikan ay.t ters€but, meka hanrs dicad drlam Sunnah
(tradis) Nabi SAti[ &a!a, bila tidak juga ditemukeo Sunnah yaog menerangkan ayat tersebut,
maka langkah selanjumya dicarikan perkataao dai sahabat. IGoPar, bila tidak ada perkaaen
sehabat mengeoai tafsL sebuah ayat, maka dilacak dad pe*atran pan tabi'in, scpeni Hasao
Das{ Qatdah, Muiahid, dm lain-lah. Iirln4 setelah perketarn geoerasi tabi'in pun tidai
ada, baru dicarilrun peodapat par. imam, s€perti SpEi Malili, Heobdi, dar. Haoaft- Ktatoa,
bila semua sandaran di eas juge tidak ditemukao, mata haru ryat tersebut diafsirkan secara
&3lan @ehasa). Menurut Adian, selain metode di etas diengap sebagai metode tafsir & /-
raJi (deag ^
akal semata) rtau dengan kaa lein, mengguoalao metode apa yang disebut
herneneutila. Lihat Adien Husaioi, "Kritik Hermeneutika".
a Ghazali "Meraocang Kaidah", hal. 11. C. Pendekatan fletmeneutik daLrn [f66rherni Nas Syarak 1 Januati - Juoi 2009 Metode Istinbaf Hukum )aringan Islam Liberal (IIL) ketika pembaca meflghadapi teks ia harus sadar bahwa sebenarnya
ia menghadapi "dunia lain", yaitu dunia teks dan dunia konteks. Teks tidak bisa dilepaskan dari konteks yang melingkupinya kenka
s ng aribor melembagakan pemikitainy^.l^l^hi d^; pemikiran yang
saling betbenturan dengan konteks sosial masyarakat. Hal yang ingin
dijembatani oleh hermeneutila adalah kesen)angan ^ntata pembaca
dan penulis, y^ig m^n keduanya dihubungkan oleh teks. Petsoalan
lain yang hendak diselesaikan oleh hermeneutika adalah persoalan
mengenai zdanya, ia.nk dan petbedaan bahasa, ttadisi, dz/J cztz
berpikir antara teks dan pembaca karena bahasa dan muatannya tidak
bisa dilepaskan dari kultur.a Tedepas dati hal di atas, menurut Jaringan Islam Libetal
penyelesai2n terhadap paradoks yang betsifat isu dan gagasan belum
banyak dilakukan. Gagasatr tentang pluralisme ^galm dalam al-
Qur'an, misalnya. Di d.lam satu spektrum, plwalisme ala Qur'ao
diungkapkan melalui ianji penyelamatan terhadzp orang-oraflg yang
bengama selain Islam (QS. al-Baqatah: 62). Sementara pada spektrum
yang lain, absolutisme Islrrn iuga terpampang dengan tegas dalam
al-Qur'an. Hal ini harus diatasi untuk memungkinkan tegaknva
sebuah tata kehidupan berdampingan secara damai dengan umat
^gltrr
l^;n-21 Dengan metode tafsir hetmeneutika Jadngan Islam
Liberal hendak membuat produk tafsir yang lebih tasional dan
hendak lepas dai pa,gx-prgar metode tafsir klasik yang kurang
mementingkan kondisi sosio historis. Singkat kata, metode yang 25 Al-Maaihii, vol. 3 No. 1 Jaouati - Juoi 2009 Arif Susandi kemudian muncul meniadi sangat betbe& dengan kebanyakan ulama
s tf-u Suatu tindakan yang sangat betani manrkrlo Jaringen Islam
Liberal menawarkan suatu cara pan&ng yang be6eda ketika melihat
hukum Islam. Seperti apa yang di looarkan oleh Muqsith Ghazdir
tentang hiratki sumbet hukum lslan. Maqi;id al-sltai'ab merupakan
sumber hukum pertama dalam Islam, batu kemudian diikuti secata
be'iri.gan al-Qut'an dan al-Sunnah. Maq;id al-sba;'alr merupakan
inti da'i totalitas ajaran Islrm. Ia meoempati posisi lebih ti"gg d"ri
ketentuan-ketentuan spesifik al-Qur'an. MaqiSid al-shai'ab rnl
merupakan sumber inspirasi tatkala al-Qur'an hendak menanam
ketentuan-ketentuan legal spesifrk di lapangan. Moqiid al-sbai'ab
adalah sumber dari segala sumbet hukum dalem Islam, tetmasuk
sumber dari al-Qur'an sendid. Oleh ka'ena itu, sekiranya ada suatu
ketentuan baik di dalam al-Qut'an maupun al-Hadis yang
bertentangan secara substansif dengan naqi;id al-shai'ab meka
ketentuan tersebut mesti &efotmesi. Ketentuan terebut harus total
atau dibatalkan demi logika ilaq;id al sba;'ab. Secara garis besar penafsiran Jaringan Islam Liberal lebih
mengutamakan realitas dari pada bunyi teks, meskipun lebih
menoniolkan pertimbangan pa& aspek reaftas tetapi masih teap
menggunakan teks sebagai piiakan. 2r VirgrnA M. Hooker, 'Developing Idarnic Argumetrt fot Chznge Through libem.l
lslzm" , WV\Y*yuhzni,aa, dibuld oadt teng l 2 November 2008.
5 Ghezdi, "Metode daa Kaidah Peoefsiren al-Qur'ao", dalan Pcaikirar I an
Ktttr;Poar di hbnuia, (ogyakzra: Pusaka Pelaiar, 2005), hal. 138.
, Ghazali, "Menilik Metode Qiyas Sytfi"', VIVIY.I a b.raz, didownlord pada
tangal 12 Novernber 2008. J,;ng.q Istam Lberal juga menuduh kitlb-kitab usul frkih
H*rk sqem al-Rtilat karya Imam Sfafrl diduge kerzs meogi&p seiumleh pers<-ralen yang
kronis. Pnrztz, usul 6kih Syaf i beraroma Arebsentris. Arabisme meruPa[aa ideologi yang
lek t dalam ternbok usul E\jh,leml Krds,ktfrah png banyak dilansir oleh kitab usul fikih
Syai'irz.h ythrn " al-'ibab bi 't rot dl-14<l; ti klu& al-raW' tetkes n tetuln memberhalakan
teks dao tneogabaik n kooteks. Usul fikih konverrsiooal lebih menitikberatkan pada @trX
Q<aa) dao buLan pada nqind $del morzl). C. Pendekatan fletmeneutik daLrn [f66rherni Nas Syarak jarlngan Islam Liberal
mengkombinasik^n a;ntat^ teks dan konteks, pembahesan mengenai 26 Al-Maaihii, Vol. 3 No. I Januati - Juoi 2009 Metode Istinbat Hukum laringan Islam Liberal (JIL) hal ini akan diielask,n lebih laniut pa& b"gi,n selaniumya. Satu hal
yang perlu penrrlis lskirnk"n di sini, bahwa iitihad yang dilakukan
olehJadngan Islam Liberal dengan merunggalkan bunF teks al-Qur'an
telah dicontohkan oleh Khalifah Umar ibn Khattab.2a Al-M.6;hii, VoL 3 No. 1 Jaouati - Juoi mO9 ' Ghazali "Usul 6kih Alternetif", rryz.tbttb.oa, dithuhad pada anggat 14 Juni
2008. Iihat juga ddam tulisan Muqsith lain oeagenei usul 6kih. D. Dekonstruksi UsuI Fikih Menuju UsuI Fikih Altematif Batawal dati ketidakpuasan kalangan Islam libeml tethadap
model metodologi Islam klasik yang dianggap rapuh, mereka
berupaya metumuskan sebuah model batu metode penggalian
hukum Islam. Muqsith Ghazali menjelaskan mengenai kerapuhan
te6ebut. Palana, metodologi lam" 16da1u m.mandang sebelah mata
terhadap kemamp,'"n rknl publik di dalam menyrlih dan menganulir
ketentuan legal fotmalistik di rlalarn Islam yang tid2[ l"gi relevan. Kcdta, metodologi klasik kutang peduli terhadap kemampuan
manusia di dalam metumuskan konsep kemaslahatan walaupun
untuk umat manusia sendiri, maousia tak memiliki reputasi dan
kedudukan apa pun dalam ruang usul fikih klasit kecuah meniadi
sasamn hukum yang tak betdayt (n*allaJ). IGtiga, pemberh azn
teks dan pengabaian rcalitas meruprk.n cid umum metodolo g1l^rn .'?s Mengenai korsep qiyas (analogi) yeng terutam.a diusung oleh
Syaf i di mana ia fidsfinisik-n 5g$agei menganaloEkan sesuatu yang
belum ielas ketentuao hukumnya fw)
deogan yang sudah ielas
hukumnya dalam al-Qur'an dan al-Sunnah (ay) karena ada kesamaan
'illat, mereka menilni qiyas seperti ini betmasalah seudaknya katcna
dua hal berikut e 27 Arif Susandi 1 . Bahwa trdak ada dua peristiwa yang petsis sama, sehingga hukum
keduanya bisa dipatalelkan. Petsamaan ilht yxrg rnetizdt dzsar,r
pengoperasian qiyas sesungguhnya metupakan tindakan
srmphfikasi. 2. Menyangkut hal-hal yang betsifat sosial, qiyas Syaf i tampak
mengabailan konteks yang melandasi kehadiran hukum arl. Setelah membetikan kdtik tethadap metode usul 6kih Hasilq
J-i"S"" Islam Libetal menawarkan kaidah usul fikih alternatif. Dati
kaidah ini drharapkan dapat meniadi jembatan bagi kebuntuan usul
fikih klasik dalam memecahkan kasus-kzsus kontemporer. Adapun
kaidah usul 6kih tetsebut adalah sebagai berikut: 1. Al-'Ibrah bi al-Maqalid la bi al-AlJ-a7 Kaidah ini berarti bahwa yang mesti menjadi pethatian seorang
mujtahid di dalzm metg-istinbal-kan hukum dari al-Qur'an &n
al-Sunnah bukan huruf dan aksaranya melainkan dri naqa;id
yang dikandungnya. Yang dipethatikan adalah cia-cita etik moral
dari sebuah ayat dan bukan legislasi spesifik atau fotmulasi
litetzktya.2T Kaidah ini dimaksudkan untuk menguak makna di
balik makna litetal teks. Tidak lagi betkutat pada bunyi teks tapi
hendak membuat suatu hukum yeng bisa iadi sangat berbeda
dengan bunyi teks. Menutut penulis, meskipun Jaringan Islam
Liberal meninggalkan bunyi teks akan tetapi meteka tidak &pat
lepas dad teks, hanya saia mereka menggunakan teks al-Qur'an
sebagai piiakkan awal dalam menentukan hukum. Dengan
menguak prinsip atau semangat al-Qur'an .l^d bulyi teks tercebut
kemudian dari ptinsip yang didapatkan dicoba untuk
dikontekstualisasikan dengafl zamao 66i da1 di sini (bukan AraQ. '' Ibid. hal. 212. Menurut Ibn Qudamah sebagaimana yang dikutip oleh Hafidz
Abdvtahman, na.slzlnb dibagi meniedi tiga yaitu na.slafiab rulabarab, yaki na'lalab yang
diterima dan dan diakui oleh syara', yaitu hukum yaog diwik dari rasiona.litas al-Qudan
(aa'q al ru1) dal, jjaa'. Kemuditn naialab n gbifab (nallalab yxrg ditolak) yaitu
aa'lahab tltsif yangkebatilannya telah dinyatakan oleh syara', kerena bertcntangan deogan
al-Qulan dan hadis dao jika diubah maka akan b€rakibat pada diubahnya hukum syara'; dao
nalltlab nrralab (naiahah yarrg ttdak dkeime dan tidak ditolak). Art\oya na;labab tutt
tidak dinyatakan maupun ditolak dalam al-Qur'ao maupun Hadis. Lihat ,{bdurrahman,
U:zl fkib, he.l. 113-114. ' Pandangan al-Thufr tmtang pertefltangao antara ms dan na. alah ii dapat dilihat
dalam nrlisan Suraji, "U ryensi leoi Ma'labal Najmuddin al-Thufi dalam Pernbaruan Hukum
Islam Kontemporer", dalam Jrnal Petelitiar Agm4 Vol. 5, no 2, Juli-Desember, 20o4, hal.
210. Menurut Suraji" al-Thufi tidak pemah mencontohkan adanya pertc'ntangafl artara flas
da aayblah. Al-Manihii, VoL 3 No. 1 Januati - Juni 2009 s Muqsith jugr meoemb.hk2tr b.hu,r.yrt y.og mengandung konsrp tolerensi rdalah
ayat pokok (4rQ sedznglen apt kekensan dao pcaag memplkan apt cebang (nf.l,rhat
Adien Huseini, "Kdtik Hermeneutikr-"
n Gh2zrlf 'Usul 6kih". Jrriog.n lslzm l,ftcr.l tilak menielasken secara rinci apa yang
di'razksrd Ql d-njtata: Atzn tet pi menurut peoulis seteleh melecrk di berbagai surnber,
ia t€rlait iugl deog2n koos€p kemaslahaun publil Dalanr merreotuk .o kemxslehatan suatu
hukum pedu diidentifikesi meoggunah berb.gai ilmu, baik ilmu agerna oaupun ilmu
soeial laio bahkan ilrnu eksat- Kemudian diblukzn perurnusan-perumusan yaog melibatkan
onng banyak @ublik).
r? Abdurrahmen, Ufll lbb, M.111. ). Tanqil1 alNryu; bi al-Aql al-Mtrjtana'. Kaidah ini menyatakan bahwa akal publik memitki kewenangan
untuk menyulih dan mengamandemen seiumlah ketentuan
"dogmatik" egama yang menyangkut petkata-perkara publi\ baik
dalam al-Qur'an m2upun dalam al-Sunnah. Sehingga ketika
teriadi pertent^"f n antara akal publik denf " bunF harfah teks
aiaran, maka akal publik memiliki otoritas untuk mengedit,
menyempumakan, &n memodifikasikannya. Modifi}asi ini terasa
sangat dibutuhkan ketika berhadapan d,eng l ^y^t-^y^t
partikulrr, seperti ayat hdid (sepem potong tangen, taianr, dan
sebagainye), waris, dan sebagainya. Ay^t-^ytt tersebut dalam
konteks sekarang, alih-alih bisa menyelesaikan masalah-masalah
kemanusian, yang teriadi iusteru merupakan bagian da'i masalah
yang herus dipec,hL^q melalui prosedut tanqTlyngbentpt taqid
bi al-'aql, takh;i; bi al- aql, dzn ta$in bi al-'aql.3l 2. Jann\ Na:kh al Nryz,; bi al-Maskbab Al-Jifi menyatakan bahwa tidak mungkin tetiadi pertentangan 28 Al-Manihif, Vol. 3 No. 1 Jaouad - Jrmi 2009 Metode Istinbat Hukum )aringan Islam Liberal (JIL) Metode Istinbat Hukum )aringan Islam Liberal (JIL) antara nas d*r na;lalah, karena apa yang diuiarkan oleh nas
adalah kemaslahatan itu sendiri.26 Sedng disinyalir bahwa
kemaslahatan yang diandailan oleh manusia adalah kemaslahatan
semu dan telati( sementara kemaslahatan yang drtetapkan Tuhan
melalui bunyi harfiah nas adalah kemaslahatan hakiki dan
obyektif.'ze Menurut Muqsith Gha,z i teks baru betmakna sekiranya
menyertakan cita kemaslahtttn ba.gi umet manusia. Kemaslahatan merupakan aiararr a.grma. y^ng tba,y;bit (tld^k
betubah, pokolq dan univetsal), sementara wuiud pelaksanaan
cita kemaslahatan inr metupakan perfu a;gama ya;ng nilaghayir
(berubah-betubah mengikuti petubahan alut sejatah dan
peradaban). Konsekuensinyz uskh uda'k dapat dilakukan
terhadap teks al-Qut'an yang menganduflg prinsip-prinsip ajaran
yang universal, ,;lzit,rt ma,l; telah melintasi ruang dan waktu,
mengatasi pelbagai etnis dan keyakinan. Ay*-ayar ini sebagar
zyzt detgrn kedudukan p"ling tingg (al-i1ah al-ali qinatan) *at
al alab al ryinyah ^tav
,q,t7 alp- r'arr. Misatrnya seperti ry* '1aa
idqa bakantm baya al-ris an tahkzmi bi al'adl", 'I'dili hwa aqrub
li al-taqual', dan sebagainya. Sementata ^ylt-ay^t mu'amalah
dalam al-Qut'an yang bersifat teknis operasional, menurut
Muqsith disebut dengan al-a1ab al adni qinatdr ^tz,u
al @ah al
fui foat *zu fqh alprrbn, seperti ayat yang berbicara tentang 29 Al-Manihii, VoL 3 No. 1 Januati - Juni 2009 Arif Susandi bentuk-bentuk hukuma r ('ryibil) , sanksi bagi para pelaku pidana
(biliQ, bntr.gzn waris dan sebagainya, make tetep terbuka
kemungkinan untuk dinasakh den difalsifikasi, sekiranya ayat
tersebut ti&k efektif lagi sebagai sanna untuk mewuiu.lkrn cita
kemalahatan.30 bentuk-bentuk hukuma r ('ryibil) , sanksi bagi para pelaku pidana
(biliQ, bntr.gzn waris dan sebagainya, make tetep terbuka
kemungkinan untuk dinasakh den difalsifikasi, sekiranya ayat
tersebut ti&k efektif lagi sebagai sanna untuk mewuiu.lkrn cita
kemalahatan.30 bentuk-bentuk hukuma r ('ryibil) , sanksi bagi para pelaku pidana
(biliQ, bntr.gzn waris dan sebagainya, make tetep terbuka
kemungkinan untuk dinasakh den difalsifikasi, sekiranya ayat
tersebut ti&k efektif lagi sebagai sanna untuk mewuiu.lkrn cita
kemalahatan.30 @
)
r? Abdurrahmen, Ufll lbb, M.111. s Muqsith jugr meoemb.hk2tr b.hu,r.yrt y.og mengandung konsrp tolerensi rdalah
ayat pokok (4rQ sedznglen apt kekensan dao pcaag memplkan apt cebang (nf.l,rhat
Adien Huseini, "Kdtik Hermeneutikr-" E. Penekanan Terhadap Prirsip Maglallah dalam Istinbip
Hukum Ma;lallah berkonotasi jalb al-nana.fi' (mengusahakan
kemanfaatao) du, dof' al-nafind (mencegah kerusakkan).32 Dalam 30 Al-Meaihii, Vol. 3 No. 1 Januad - Jrmi 2009 Al-Meaihii, Vol. 3 No. 1 Januad - Jrmi 2009 Metode Istinbat Hukum laringan Islam Liberal (lIL) khazanah usul fikih, naqi;id al-shari'ah itu adalah keadilan,
kemaslahatan, kesetaraan, hikmah-kebiiaksanaan, dan cinta kasih. Maqaltd inilah yang sejatinya menjadi sumber inspirasi latkqlq xf,-
Qut'an hendak melabuhkan ketentuan-ketentuan legal-spesi6k di
lapangan. Deogan petkataan bn, naqi;id al-shaibl adalah sumber
dati 5sgaln sumbet hukum dalam Islarrr, termasuk sumber d-ri el-
Qur'an seodid.3l Dalam syari'at tetdapat beberap a, vziala yzrng mesti dipahami
secata utuh, antera lain hukum, tuiuan hukun, .f^lil, dqn iitihxd. Hal ini menuniukkan bahwa syari'at tidak hanya hukum belaka,
karena ada varian lain yang sangat penting yaltu tuiuan-tuiuan ut"mq
diturunkannya syxi'* (naqa;id al-thai'ab).v Adapun kemeslahatan
umum seceta rinci ditui"kan untuk memelihar^
^ganrE, diri (iwa),
akal keturunan, dan hata. Petlindungan terhadap kelima hal ini
harus menjadi landasan utame dalam mefletapkan produk hukum. Dalam upaya melindungi eksistensi kelima hal tersebut pam ulama
merrbagl dalam trga tingkaan yakni: s Ghazali, "Kaidah Usul Filih."
r {
,
tt Danusi4 "Episternologsyat{',d zfiEl*ta*ologi OwaI ed. Anang Hrris Himaumn
(Yog,%k rta: Pustaka Pelajer, 20m), hd. 47. s Ghazali, "Kaidah Usul Filih."
r Nucholis M.dN. dW Fnib Undr A2aa {abxt,- Pafloadina 2ma) hd. 10.
tt Danusi4 "Episternologsyat{',d zfiEl*ta*ologi OwaI ed. Anang Hrris Himaumn
(Yog,%k rta: Pustaka Pelajer, 20m), hd. 47. r Nucholis M.dN. dW Fnib Undr A2aa {abxt,- Pafloadina 2ma) hd. 10.
tt s Ghazali, "Kaidah Usul Filih."
r Nucholis M.dN. dW Fnib Undr A2aa {abxt,- Pafloadina 2ma) hd. 10.
tt Danusi4 "Episternologsyat{',d zfiEl*ta*ologi OwaI ed. Anang Hrris Himaumn
(Yog,%k rta: Pustaka Pelajer, 20m), hd. 47.
Al-Meaihii, vol. 3 No. 1 Jaouati -Jlrni 20og
31 b Mad1d, Fiqib Dnta Agaa4 h . 11. Medurut kalangan Fuodameoulis pendapat
dari Jaringan Islam Liberal di ata.s sanget iauh berseberangan dengan usul 6kih. Mcnurut
mereka Jariogan Islarn Uberal telah menyelewengkan epa yrng telah term2ktub dalam
risalah-risa.lah tedahulu, bahwa mmurut Usul 6kih penjagaan terhadap agama adalah dalam
bentuk kctmtuao hukum s,,ara' dengan membuouh orang kafir yang menyesatkan dan
hukuman atas pembrnt hdbb yang mengaiek kepada bid'at. Hd, ri dilaLsanal.n untuk
menjaga egame Meni.g. erau melinduogi 4ama adalah menjaga Islam zgat tidak rusak,
maka perusak egama dihukum bunuh. Dalun cootoh di atas orang keft yeng menyesatkan
hukumannya adalah dibunuh. Akaa tetapi perlunya mdindungi agama (ffial-&r) mertat
kalaogeo fundamcntal telah dipelintir oleh Jaringan Islam Libcral meoiadi "setiap manusia
mesti menghargai keberagamaan orang lain." Iihat Hartono Ahmad Jaiz, 'Nafsu Besar
Tenega Kurang ', dabm V,lYlY.Sloanuln,on ,
t IUd, hil. 11 . Ddzm hal lt$foat dm talitfod pettetangm anao fundarneotalis 1. Qam-iya1 Pada tingkatan /ainlyit (primer) kelima hal tetsebut hatus
diupayakan sebegai agenda utama. Apabila satana itu ti&k ada
akan meogancam hilangnya petlindungan tethadap kelima ha1
tersebut. Sesuatu yang mutlak harus ada untuk menoPang
kehidupan manusia baik dalam urusan agema maupun kedunraan. Pada tingkatan /ainlyit (primer) kelima hal tetsebut hatus
diupayakan sebegai agenda utama. Apabila satana itu ti&k ada
akan meogancam hilangnya petlindungan tethadap kelima ha1
tersebut. Sesuatu yang mutlak harus ada untuk menoPang
kehidupan manusia baik dalam urusan agema maupun kedunraan. Seandainya sesuanr itu tid^k ada, maka rusaklah tata kehidupan
ini.l5 Menurut Jeringan Islam Liberal, yang dimaksud kemas-
Iahatan primet yaitu perlunya melindungi agama, iiwa, akal,
ketutunan, dan harta. Setiap otang hatus menghotmati
kebetagamaan otaog lain, menghargai iiwa, menghargai
kebebasan berpikit dan berpen&pat, meniaga ketutunen (rak
teproduksi) setta menghatgai kepemilikan harta setiap otang. Seandainya sesuanr itu tid^k ada, maka rusaklah tata kehidupan
ini.l5 Menurut Jeringan Islam Liberal, yang dimaksud kemas-
Iahatan primet yaitu perlunya melindungi agama, iiwa, akal,
ketutunan, dan harta. Setiap otang hatus menghotmati
kebetagamaan otaog lain, menghargai iiwa, menghargai
kebebasan berpikit dan berpen&pat, meniaga ketutunen (rak
teproduksi) setta menghatgai kepemilikan harta setiap otang. 31 Al-Meaihii, vol. 3 No. 1 Jaouati -Jlrni 20og Arif Susandi Kemaslahatan yang betsifat primet tersebut metupakan inti
semna agarna dtn \*tn.x 2
H4ryo1
H oJ-,yit adalah kemaslahatan yang bersifat sekunder, yaitu
kemaslahatan vang tidak menyebabken ambruknya aanan sosial
dan hukum. Misalnya.l^l"t hal ibe&t a& dispensa si *a't mkltab
atau kednganan apabila dalam pelaksanaannya tet&pet kesulit n. Bagi mereka yang melakukan perjalanan iauh, sakit rlrn sraog
tua renta dibedkan ketinganan dalam menialakan ibadah. Menurut Islam Libetal kemaslahatan ltajfoit nt menuoiukkatr
adanya keringat^n y^ng bertuiuan untuk mewujudkan
kemaslahatan dan kenyamanan bagi pemeluk agama. Dengan
demikian beragama dan beribadah tidak akan ada rasa keberatan
dan keterpaksan. Mengamalkan ^garn secata tulus ikhlas
metupakan inti dari ibadah sehingga unsur keterpaksaan
merupakan lawan yang sengat bertolak belakang.rT Tenega Kurang ', dabm V,lYlY.Sloanuln,on
n Maditd5 Fiqib L,ir!a!. . . , h2l. 1 'l .
t IUd, hil. 11 . Ddzm hal lt$foat dm talitfod pettetangm anao fundarneotalis Maditd5 Fiqib L,ir!a!. . . , h2l. 1
.
t IUd, hil. 11 . Ddzm hal lt$foat dm talitfod pettetangm anao fundarneotalis Metode Istinbat Hukum laringan Islam Liberal (lIL) Maslahat betsumber dad konteks sosial. Jika da.hl a.garna. bertent2ngan dengan konteks sosial, maka konteks harus
didahululan di atas teks agama. "Men&hulukan" dalam pandangan
al-Tu6, bukan bem11i msrn[at^lkan dan menganulir sama sekali dalil
agrrna. Sebaliknya, konteks sosial dianggap sebagi "pet+a*.h;i!'
atau spesiEkasi dat"bgif'atau menerangkan teks atau dalil agama
yatg zda. Dalam hukum fikih fakta sosial ielas bisa menjadi dasat
pefletapan hukum. Katena itulah ada kaidah terkenal 'taghayn al
altkan bi tagltayr al arytat ya al-amkinab." 3e TaIiny1ir
3 Kemaslahatan talskjlit adalah kemaslahatan yang betsifat
komplementet, yaitu kemaslahatan yang memberikan perhatian
pada masalah etika dan estetika. Misalnya zizrzrr tentzng
kebetsihan, berhias, sadaqah, dan bantuan kemanusiaan. kemaslahatan ini menyempurnakan kemaslahatan primer dan
sekunder.16 Kemaslahatan talskjlit adalah kemaslahatan yang betsifat
komplementet, yaitu kemaslahatan yang memberikan perhatian
pada masalah etika dan estetika. Misalnya zizrzrr tentzng
kebetsihan, berhias, sadaqah, dan bantuan kemanusiaan. kemaslahatan ini menyempurnakan kemaslahatan primer dan
sekunder.16 32 Al-Menihii, Vol. 3 No. 1 Januari - Jr:ni 2009 Metode Istinbat Hukum laringan Islam Liberal (lIL) dao liberalis kuaflg b€gnu meogemul4 perdehaan serius dan a6, teriedi pada maslahet
dant$ut, karena ii dalah yangkel en fibir dn ,abi"foA dao liberalis kuaflg b€gnu meogemul4 perdehaan serius dan a6, teriedi pada maslahet
dant$ut, karena ii dalah yangkel en fibir dn ,abi"foA
" Ulil Abshar Abdella, "Kritik atas Argumea Aktivis Hizbut Tahrir,"
lYlYV ltlaatb coa
ddoa rload pad^ tangpl 10 November 2008 y
g
fibir
" Ulil Abshar Abdella, "Kritik atas Argumea Aktivis Hizbut Tahrir,"
lYlYV.ltlaatb.coa, ddoa,rload pad^ tangpl 10 November 2008. F. Kesimpulan Kendati selalu menekanlan dekonstruksi terhadap usul fikih
klasik dan menyerukan pemberuan di dalamnya, penalaran hukum
Jaringan Islam Liberal pd dasamya masih tetap berpijak pada kaidah
usul fikih lama. Indikasinya adalah bahwa penalatan hukum mereka
masih berpiiak pada al-Qur'an dan Hadis. Di samping itu mereka
iuga serhg mengadopsi metode ldasik lainnya misalnya 't$ istibsan,
dan juga kotsep na;laf;ah yang digagas oleh pata ulama klasik,
meskipun pada beberapa sisi terdapat petbedaan. Atau lebih tepatnya
adalah penekanen berbe&. Sumbet hukum Islam menurut Jaringan Islam Liberal adalah
al-Qur'an &n Hadis. Akan tetapi pemahaman tethadap keduanya,
menurut mereka, pedu menggunakan berbagai macam pendekatan,
misalnya dengan hetmeneutika. Je ringan Islarn Liberal meniembatani
ayat yang kontradiktif dengan konsep ^y^t t!7 d^n
^y^t jrsil. Korsep
ini menuut penulis edalah hasil turunan dari gagasan besar tentang
naqa;id al:bai'ah. Ay* ry ul"l"h aya:t y^ng mengisyata&an secara
ielas akan gagasat naqalid al-shai'ah, seperti ayat tentang pendngnya
menegakkan keadilan, toletansi, dan saling menghargai kebera-
g ma 1. Sedangkan
^y^t f,tr;l z.drbh zy* yang mengisyaratkan JJ d-Manihil, Vol. 3 No. 1 Januari - Juni 2009 Al-Maaihii, Vol. 3 No. I Januari -Juai 2009 Arif Susandi rdarryz naqi;id al- ai'ah a.karl tetapi telah menielma dalam konsep
yang lokal (Atab). Misalnya ayat tentang perang tethadap orang kafu,
pembagian waris, &n poligami. Metode-metode yang digunakan oleh Jeringan Islatn Liberal
pada umumnya mengarah kepe& m"kna di balik bunyi hetfiah teks. Meninggalkan bunyi teks bukan merupaken kesalahan, asalkan masih
dalam semangat al-Qut'an. Metode istihin, 'wl, istislib, dan yang
semacamnya telah dimakoai secata lebih longgar oleh Jaringan Islam
Liberal. Menurut penulis yang meniadi prioritas pcrtama adala,h
rco,litzs, Acdta adalah tealitas, dzn lectiga adalah realitas. Attinya
kedudukan teks meniadi "kutang penting", ia hanya digunakan sebgai
pelengkap dalam menyulih kebeneran. Jika terdapat dalil yang
bettentzngan dengan semangat toleransi misalnya, dalil itu harus
diafsir ulang atau bahkan ditolak. Jika ada .{^lil yang mendukung
dengan gagasan itu maka ekan diambil mentah-menteh tanpa
inteq)retasi, meskipuo dalil tetsebut metupakan wilzyth fugil
(partikular) . 34 Al-Maaihii, Vol. 3 No. I Januari -Juai 2009 Al-Maaihii, Vol. 3 No. I Januari -Juai 2009 Al-Man-hii, Vol. 3 No I Januari - Juai 2009 DAFTAR PUSTAKA Abd,ll,, UIil Abshar Mc4lcgarkm knbali Pcmikiran lthn. Jrkarta:
Nalar, 2007. "Soal Kedudukan Hadts;' *lanlibcral@aahoogrotp.eom "Kritik atas Argumen Aktivis Hizbut Tahrir."
lVlVlV.lslamlibrom. Abu Zaid, Nasr Hamid. Tcksnalilrc al-par'an, terj. Khoiron
Nahdliyyin. Yogyrka'6; LKIS, 2005. Assyaukanie, Luthfi. "Metenungkan Seiatah al-Quran" dalzm Ijtiltad
Islan Liberat. Danusid. "Epistemologi Syar:t"' ddzn Epistenobgi Slara', ed,. Ararrg Haris Himawan. Yogyakata: Pustaka Pelaiar, 2000. Effendi, Satria. Unl fkib. Ja,keta: Plrnzda Media, 2005. Ghazali, Abd. Muqsith. "Metode dan Kaidah Penafstan al-Qur'an",
dalam Pcmikiran Islan Ko enponr di Indoncia, ed. Abd. Muqsith Ghazali. Yogyakarta: Pustaka Pebix, 2005. "Menilik Metode Qiyas Sya,f i;' V/V47.lslanlib.con, y
"Usul fi kih Altematif." VlVlY.Islanlib.con, "Metancang Kaidah Usul fikih Baru," (makalah
diseminarkan ptdt acalt Wotkshop JIL III, Wahid
Institute, J a,krttz, 2O08). Hidayat, Komaruddin. Mcmabami Bahasa al-ptr'a4 Sebmh Kajian
Hctmennlik Jakarta: Pataoadina, 1 996. Hoket, Virginia M. "Developing Islamic Argument fot Change
Through Liberal Islam." IYV\V-4sgaakanic.nm. 11g5rini, Adian. "Kdtik Hermeneutik el-Qut'ao." lVlYlV.i anlb.an. Ichwan, MochemadNur. Mcrhtis lQsalianaan Kritis al-pnbn. Jekarta:
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al-Qut'an Nast Hamid Abu Zoit', d^l^rt Sttdi al-prrbr
KorrtcmPorrr, ed. Abdul Mustaqim dan Syahiron
Syemsgdil. Yogyakerta: Tizrt Vlsc na.,2002. 85 A!-Mq.;hii, VoL 3 No 1 Jaoueri -Juai 2009 Arif Susandi Jaiz, Hattono Ahmad. "Nafsu Besat Tenaga Kurang",
IV|VII/. S va ra m rc li m. co m. Kamali, Muhammad Hashim. Pitip dan Teoi-hoi Htkm lslan. Yogyakatta: Pustaka Pelaiar, 1996. Kusmana. "Tafsir al-Qur'an Inklusif." lVVlV.islanlib.nn. Madiid, Nurcholis, dld<. Fiqih L)ntas Agama. Ja,kzt:o: Par.amrd)lt't,
2004. Misnwi Zrhtin Al-ptrbn Kitab Tohrani. Jakarta: Fitrah, 2007. Rahman, Yusuf. '?luralites Penafsiran al-Quran", ddzm ljtibad Islan
bbcral. Al-Siiistani Abu Davrud Sulaiman al-Ash'ath. Sttan Abi Danttd, 4
iilid. Betut Dat al-Hadis, 1999. Sutaii. "Urgensi Teori Ma"lahab Naimuddin al-Thufi dalam
Pembaruan Hukum Islam Kontemporet", dalant Janal
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Suramo. "Plurdisme Agama dalam Hermeneutika Paul fuchew."
\N4I4V-Islamlib.com. \N4I4V-Islamlib.com. Syarifuddin, Amn Usil fkib. Jakarta: Kencana,2008. 36 Al-Man-hii, Vol. 3 No I Januari - Juai 2009
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Bosman: A proto-postcolonial author?
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Dates: Keywords: Herman Charles Bosman; postcolonial theory; proto-postcolonial author; English
colonialism; South African War; apartheid; cultural hybridity; multiculturalism; linguistic
hybridity; parody. How to cite this article:
Loonate, F., 2022, ‘Bosman:
A proto-postcolonial
author?’, Literator 43(1),
a1868. https://doi. org/10.4102/lit.v43i1.1868 How to cite this article:
Loonate, F., 2022, ‘Bosman:
A proto-postcolonial
author?’, Literator 43(1),
a1868. https://doi. org/10.4102/lit.v43i1.1868 Note: This article has been republished to correct specific details in the first paragraph of the Introduction, including changing the pronoun
‘their’ to ‘his’ and updating ‘proto-colonial’ to ‘proto-postcolonial.’ Additionally, the indented quote from Bosman (1977:80) on page 7 has
been revised to include previously omitted wording. The publisher apologises for any inconvenience these corrections may have caused. Literator - Journal of Literary Criticism, Comparative Linguistics and Literary Studies
ISSN: (Online) 2219-8237, (Print) 0258-2279 Literator - Journal of Literary Criticism, Comparative Linguistics and Literary Studies
ISSN: (Online) 2219-8237, (Print) 0258-2279
Page 1 of 11
Original Research Literator - Journal of Literary Criticism, Comparative Linguistics and Literary Studies
ISSN: (Online) 2219-8237, (Print) 0258-2279
Page 1 of 11
Original Research Literator - Journal of Literary Criticism, Comparative Linguistics and Literary Studies
ISSN: (Online) 2219-8237, (Print) 0258-2279 Page 1 of 11 Introduction Copyright:
© 2022. The Authors. Licensee: AOSIS. This work
is licensed under the
Creative Commons
Attribution License. Bosman scholars cover a wide spectrum of concerns. Some earlier scholars analysed his linguistic
talents, especially his use of irony and satire, to create humorous depictions of the bushveld
Afrikaner who have attracted and entertained readers. Meihuizen (1991:35) contends that Bosman’s
‘principal concern, is to entertain’, as illustrated by the quirky narrator in most of his short stories,
Oom Schalk Lourens. Other scholars have read between the lines, disclosing political themes and
concerns through his use of this same narrator as a parodic tool, embodying the writer’s critique of
Afrikaner racial ideology. They have noted that Bosman conveys his most compelling, subversive
messages and serious political statements through parody, amusing his readers as he works with
themes later exemplified in postcolonial literature. The present study explores Bosman as a satirical
political writer who worked in a historical context of colonialism, but whose work also anticipated
key characteristics of a movement that had not yet found its voice. In other words, this study assesses
the extent to which Bosman can be classified as a ‘proto-postcolonial’ author. Bosman’s political intentions have frequently been overlooked. In a 1986 collection of critical
essays on Bosman (Gray 1986:147), Dickson sees Bosman as appearing ‘much too involved with
his personal life to show any of that obsession with racial conflicts which have been so marked in
some of his contemporaries’. Similarly, Davis (2006:8) questions the presence of any political
beliefs in Bosman’s writing, indicating that ‘The reader who seeks evidence of firmly-held political
beliefs of whatever kind in Bosman’s work will, inevitably, be disappointed’. By contrast, Hayden
(2002:1) contends that Bosman’s ‘bushveld stories concern themselves directly with race, and are
devoted to a meticulously constructed recording of Afrikaner ideology’. She sees in Bosman’s
complex use of metaphor, irony and symbolism, the delivery of a highly politicised message. Bosman, at times, has been called a racist because he used the ‘k-word’1 so often that the reader ‘is
left to wonder where Bosman stands in relation to the views of his character-narrator’ (Chapman
2006:155–156). Author Phaswane Mpe admits to being ‘a little shocked’ at the racist terminology
when first reading Makapan’s Caves but recognises Bosman’s satirical intention: ‘I don’t think
words in themselves are bad. I’m more interested in how those words get used. Bosman: A proto-postcolonial author? Bosman scholars tend either to have focused on the humour and entertainment value of his
works or to have leaned towards appreciation for the satirical quality of his writing and the
serious political commentary that accompanies and underpins it. Building on these insights, the
present study investigates Bosman’s preoccupation with South Africa’s politics in order to
determine whether he could be classified a ‘proto-postcolonial author’. It discusses key features
of postcolonial theory and writing and elucidates the term ‘proto-postcolonial’. It then analyses
selected texts in terms of their political themes – five short stories from the collections Ramoutsa
Road (1987), Unto Dust (1991) and the novel Willemsdorp (written in 1951, first published in 1977). The focus is on Bosman’s form of subtle protest against contemporary inequalities and injustices
through his use of satire and techniques such as parody, irony and other linguistic and stylistic
devices. Political themes that emerge from this analysis – including the detrimental effects of
colonisation, racism, displacement, subjugation, repression and hybridity – are echoed and
developed further in discussion of other, subsequent postcolonial writing. This study, therefore,
reveals Bosman as a precursor of this later important body of literature and as a writer ahead of
his times who has earned his place as a ‘proto-postcolonial’ author. Corresponding author:
Farzanah Loonate,
farzanah.loonate@nwu.ac.za Introduction We need to 1.This is a reference to the derogatory and offensive term ‘kaffir,’ which was used to refer to black South Africans. Open Access http://www.literator.org.za http://www.literator.org.za distinguish between insults and ironies
Cuthbertson (2006) similarly defen
accusations of racism:
[N]aming is such a controversial matter
it is difficult to see beyond the offen
commonly used word ‘kafir’ in Bosman
the extent to which he systematically l
behind the prejudiced ascriptions of his
was actually trying to subvert the conten
always acknowledged in a society which
correctness and unsophisticated abou
contextuality. (p. 159)
Contextualising Bosman’s narratives
shows how the socio-economic and
South Africa during Bosman’s time are r
He later notes that as Bosman wrote:
[I]n an era of increasing Afrikaner con
propaganda apparatus, he sought to
developing Afrikaner nationalist ideolo
stories that drew attention to the darke
less welcome secrets. (Mackenzie 2003:3
Although critical of Afrikaner racist id
also sympathetic to their plight under
Cuthbertson (2006:151), for example, po
of the volksmoeder (mother of the nation
the suffering, yet emotionally strong
concentration camps during the S
Nevertheless, Cuthbertson (2006) also n
most of Bosman’s literary historians:
[R]evere his irony, see his satire as a w
war, and admire his conscious antipat
read in his work a dislike of white rac
criticism of Boer wars against Africans. (
Bosman may not have openly conde
narratives, but what Hayden (200
‘remarkable’ is the ‘unique, unflinching
documents racial discrimination in Sou
Various studies have drawn attention to
intent in Bosman’s writing. The pur
investigation is to explore these claims
his work for themes that characteri
literature by writers who, from a dec
portray the legacy that colonialism left
guiding this study was whether Bosma
about the colonial and apartheid
sufficiently reflect the themes of sub
writers to warrant considering him
writer’, that is, a writer whose work Page 2 of 11 Original Research Page 2 of 11 distinguish between insults and ironies’ (in Miller 2006:n.p.). Cuthbertson (2006) similarly defends Bosman against
accusations of racism: distinguish between insults and ironies’ (in Miller 2006:n.p.). Cuthbertson (2006) similarly defends Bosman against
accusations of racism: colonialism, racism and attempts by colonial authorities to
suppress cultural hybridity. 2.Formerly known as the second Anglo-Boer War (1899–1902), this new term is
considered more appropriate as it suggests that all South Africans were affected Postcolonial studies and theory Postcolonialism or postcolonial studies refer to the academic
study of the cultural legacy of colonialism and imperialism,
focusing on the human consequences of the control and
exploitation of colonised people and their lands. Loomba
(1998:2) observed that when colonisers formed a community
in the colonised land, it meant ‘unforming or re-forming
the communities that existed there already’ and involved
adverse effects such as ‘warfare, plunder, genocide and
enslavement’. Loomba (1998:12) therefore reflected that the
term ‘postcolonialism’ should not be considered literally,
as a period after colonialism, but that it should signify the
‘contestation of colonial domination and the legacies of
colonialism’. Contextualising Bosman’s narratives, Mackenzie (1999)
shows how the socio-economic and political changes in
South Africa during Bosman’s time are reflected in his stories. He later notes that as Bosman wrote: [I]n an era of increasing Afrikaner control of the State and its
propaganda apparatus, he sought to expose the flaws in
developing Afrikaner nationalist ideology. He, therefore, wrote
stories that drew attention to the darker, little-known and still
less welcome secrets. (Mackenzie 2003:3) Although critical of Afrikaner racist ideology, Bosman was
also sympathetic to their plight under British colonial rule. Cuthbertson (2006:151), for example, points to Bosman’s use
of the volksmoeder (mother of the nation) image in portraying
the suffering, yet emotionally strong Afrikaner women in
concentration camps during the South African War.2
Nevertheless, Cuthbertson (2006) also notes that even though
most of Bosman’s literary historians: Postcolonial theory, on the authority of Ashcroft, Griffiths
and Tiffan (2003): Postcolonial theory, on the authority of Ashcroft, Griffiths
and Tiffan (2003): involves discussion about experience of various kinds: migration,
slavery, suppression, resistance, representation, difference, race,
gender, place and responses to the influential master discourses
of imperial Europe. (p. 2) [R]evere his irony, see his satire as a way of writing ‘another’
war, and admire his conscious antipathy to stereotypes, they
read in his work a dislike of white racism, particularly in his
criticism of Boer wars against Africans. (p. 157) Driving developments in postcolonial theory, according to
Mambrol (2016), is a concern at the heart of postcolonial
studies, which analyse: [T]he metaphysical, ethical and political concerns about cultural
identity, gender, nationality, race, ethnicity, subjectivity, language
and power. Once colonised peoples had cause to reflect on and
express the tension which ensued from this problematic and
contested … mixture of imperial language and local experience,
post-colonial ‘theory’ came into being. (n.p.) Bosman may not have openly condemned racism in his
narratives, but what Hayden (2002:n.p.) observes as
‘remarkable’ is the ‘unique, unflinching manner in which he
documents racial discrimination in South Africa’. Bosman may not have openly condemned racism in his
narratives, but what Hayden (2002:n.p.) observes as
‘remarkable’ is the ‘unique, unflinching manner in which he
documents racial discrimination in South Africa’. Various studies have drawn attention to evidence of political
intent in Bosman’s writing. The purpose of the present
investigation is to explore these claims further by examining
his work for themes that characterise later postcolonial
literature by writers who, from a decolonised perspective,
portray the legacy that colonialism left behind. The question
guiding this study was whether Bosman’s political concerns
about the colonial and apartheid world around him
sufficiently reflect the themes of subsequent postcolonial
writers to warrant considering him a ‘proto-postcolonial
writer’, that is, a writer whose work, although produced
during a colonial period and decades before the development
of postcolonial literature and theory, shares and prefigures its
key themes and concerns. This study, therefore, focuses in
particular on his portrayal of the psycho-social impact of This view implies that the detrimental effects of colonialism
triggered the creation of postcolonial theory and writing. Introduction The analysis focuses on five
short stories and one novel, each of which relates to the socio-
political effects of colonialism, in the context, first, of the
aftermath of the South African War (1899–1902) and, second,
of the rise to power of the National Party in 1948 and its
implementation of the system of apartheid. [N]aming is such a controversial matter that for many Africans,
it is difficult to see beyond the offensive designation of the
commonly used word ‘kafir’ in Bosman’s writing, to appreciate
the extent to which he systematically lampoons the meanings
behind the prejudiced ascriptions of his own time. That Bosman
was actually trying to subvert the content of racist epithets is not
always acknowledged in a society which is literal about political
correctness and unsophisticated about, and insensitive to,
contextuality. (p. 159) The ‘proto-postcolonial author’ … that is the enemy. I can see them under those trees. There’s
that man with the long beard eating out of a pot with his hands. Why doesn’t he use a knife and fork? I don’t think he can be a
gentleman. Bring out the maps and we’ll attack them. (Bosman
1987:25) Although not concretely theorised, the term has been used
specifically to describe authors who anticipated postcolonial
views even whilst writing within their colonial context. Rao
(2007), for example, views James Joyce and Rabindranath
Tagore as writers who, within their own colonial context,
were critical of nationalism and its ‘stifling of the individual
spirit’ and calls them ‘proto-postcolonial thinkers’ (p. 182)
who articulate their thoughts ‘beyond the temporal context
in which they are writing’ (p. 184). Bosman’s strong irony in this extract resides in the genuinely
ungentlemanly behaviour of the English who savagely ‘attack’
the Boers. The Boers’ resistance to the English army is a
typical response of people who experience colonial
domination and thus portrays one of the key postcolonial
themes as explained by Ashcroft et al. (2003). Similarly, Clarke (2015:127) refers to English novelist Anthony
Burgess’ ‘proto-postcolonial perspective’ on the fall of the
British Empire, which, written during the high noon of English
colonial rule, demonstrated his thinking beyond his own
context. Kimber (2018) examines five short stories by author
Katherine Mansfield published between 1912 and 1913 that
‘display a conspicuous leaning towards an exposé of the harsh
realities of colonial life’ and that, therefore, ‘can be considered
in the context of proto-postcolonialism’ (p. 104). Mansfield’s
depictions of life in colonial New Zealand ‘with a postcolonial
mind-set’ mean, according to Kimber (p. 121), that they form ‘a
subconscious story cycle in Mansfield’s work at a specific
moment in her writing career: a proto-postcolonial cycle’
(Kimber 2018:109). Loomba’s (1998) inclusion of ‘warfare and plunder’ as
characteristically postcolonial themes is aptly reflected in
Bosman’s presentation of the harshness endured by
Afrikaners under British rule. His realistic depiction of
the physical, emotional and psychological effects on the
Afrikaners during the South African War is embodied in the
character Ben Myburg in ‘Peaches Ripening in the Sun’. Whilst fighting in the war, his farmhouse and peach orchard
were burnt down by the English, and his wife had taken
comfort in the arms of an English soldier. The ‘proto-postcolonial author’ Upon his return,
the devastation and trauma he experiences on seeing the
charred peach farm plunge him into a state of psychological
shock and his memory is so severely affected that he is
unaware that the farm belongs to him (Bosman 1991:59). This
scene highlights the plight of many farmers affected by the
implementation of the British scorched-earth policy that
included the burning of Boer houses and farms during the
war. Postcolonial writing, as pointed out by Loomba (1998),
highlights and contests such destructive legacies of
colonialism, and in this story, Bosman sympathetically
presents the Boers as oppressed victims of war at the hands
of the colonising power. Drawing on these uses of the term, I have defined a ‘proto-
postcolonial author’ as a writer whose works are constructed
during a colonial period, but in using a postcolonial viewpoint
when writing about the effects and anticipated legacies of
colonialism, prefigure and pioneer postcolonial discourse. Original Research Original Research Page 3 of 11 South African War, was first published in 1931, the year in
which South Africa became legislatively independent from
Britain with the passing of the Statute of Westminster, which
abolished the remaining powers of the UK Parliament to
legislate on the country. The story reflects this significant
moment in history as shaping the attitudes of Afrikaners and
of Bosman, towards stronger resistance to colonial rule. Bosman satirises the English general using the wrong map to
plan his attack on the Boers: Postcolonial literature Postcolonial literature, by people from countries that were
formerly colonised, often addresses the problems and
consequences of a country’s decolonisation. For Katrak
(1989:157), such writers characteristically ‘respond seriously
to the many urgent issues of their societies’ such as divisions
of race and culture. Typical postcolonial themes include a
special concern with the political and cultural independence
of formerly subjugated people. [A]nyway, they would work out the plans of our position for half
an hour, and at the end of that time, they would find out that
they had got it all wrong, because they had been using a map of
the Rustenburg District and actually they were half-way into the
Marico. So by the time they had everything ready to attack us,
we had already moved off and were making coffee under some
other trees. (Bosman 1987:26) Bosman’s life (1905–1951) witnessed two forms of colonisation
in South Africa: first, the British colonisation of the country,
which included the Union period (1910–1961) and, second,
the apartheid form created by the Boers in 1948. This study
analyses the grave concerns expressed in Bosman’s writing
in this socio-historical context and examines them in terms of
concerns that appear later in postcolonial writing. Nevertheless, the English general is pompous, disdainful
and snobbish in describing the ‘lowly’ Boers: Page 3 of 11 Original Research This view implies that the detrimental effects of colonialism
triggered the creation of postcolonial theory and writing. Three decades earlier, Ngugi wa Thiong’o (1986) already saw
the purpose of postcolonial studies as assisting in the
decolonisation of societies, both psychologically and
politically, intending to re-assess and sometimes reject the
master discourses of imperial Europe and thus revive pre-
colonial cultures. Stam and Spence (1983:4) had understood
that racism was always an ‘ally and product of the colonisation
process’, and that race, therefore, remained a relevant and
significant aspect of postcolonial theory, being central to the
power of imperial discourse. More recently, Overbey
(2012:146) continued to see, amongst the fundamental
concerns of postcolonial studies, the key issues of race,
conquest and socio-political representation. Open Access http://www.literator.org.za http://www.literator.org.za Page 3 of 11 The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War He ensures that the reader’s
sympathetic response towards the Afrikaner does not entirely
obscure their own shortcomings. Through parody, the
eponymous character, Karel Flysman, is portrayed as
behaving in cowardly fashion by running away and hiding
during several attacks in the war. This story was Bosman’s
first experiment with the socio-political hensopper3 theme,
repeated subsequently in his writings. Mackenzie’s (2003:3)
analysis of Bosman’s ‘A Boer Rip Van Winkel’ elucidates
Bosman’s aim to ‘de-bunk the over-glamorised and
embellished tales that he heard about the war’ by exposing
the cowardice of Afrikaners who betray their comrades by
joining the English troops in the South African War. This racist comment refers to the National Convention in
1908 (just before South Africa became a Union in 1910), which
permitted former Boer republics to exclude African people
from their electoral rolls, whilst the Cape was allowed to
retain its own franchise qualifications. This compromise
secured the franchise rights of coloured people in the Cape
Province (Dugard 1978:26–27). Bosman’s representation of
Oom Schalk’s and Stoffel Oosthuizen’s racially derogatory
attitudes towards non-white7 people in this short story is in
keeping with his critique of the Afrikaners’ (as colonisers)
sense of superiority and echoes the fundamental postcolonial
concerns of socio-political representation and racism as
expressed by Overbey (2012). In ‘Peaches Ripening in the Sun’, the heart-rending depiction
of Ben Myburg also includes the narrator’s earlier recollection
of Ben’s arrogance and racism in his younger days in
boasting, after his engagement to his fiancé, Mimi: ‘I was so
happy that I just kicked the first three kafirs I saw’ (Bosman
1991:58). This recollection of the disregard for human dignity
renders a negative impression of the character and his racial
ideology. Bosman’s critical view of the Afrikaner in these four short
stories comes mainly from his experiences of their racial
ideology. Whilst it is evident that the critique of the Afrikaner
features in Bosman’s writings, his stinging critique of the
English during the South African War is relentless and
features equally strongly in his short stories and novels. The
experience of suppression, which is highlighted by Ashcroft
et al. (2003) as one of the focal points in postcolonial theory,
features strongly in Bosman’s writing through conveying the
experiences of Boers under British rule, as well as black
people before and after apartheid. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War Recalling Overbey (2012:146), the issue of conquest is central
to postcolonial discourse, and Bosman’s critique of English
conquest is conducted mainly through his parody of the
English army. The short story, ‘Karel Flysman’, set during the Resistance, as a further significant postcolonial theme,
appears in ‘The Traitor’s Wife’, which depicts the guerrilla
tactics used by the Boer troops against the English. The http://www.literator.org.za Open Access Page 4 of 11 Original Research Page 4 of 11 characters Jan Vermeulen and Kobus Ferreira are wearing
clothing unbecoming of their soldierly status, during an
attack by the English: they portray themselves to be but who represent the religious
institution at the heart of the Afrikaner community,
controlling its values, mindset and political views.5 Bosman
highlights religious hypocrisy as the foundation of the
Afrikaner community’s racial attitudes and criticises the use
of religion to sanctify discriminatory practices. [A]nd instead of a jacket, he was now wearing a mealie sack with
holes cut in it for his head and arms […] Kobus Ferreira was
wearing a missionary’s frock-coat that he had found outside
Kroendal, where it had been hung on a clothes-line to air. (Bosman 1991:78) Scathing critique of the Afrikaner is also evident in ‘Unto
Dust’, written in 1949, 1 year after apartheid was instituted
by the newly elected National Party government. This story
depicts the racial ideology of the Afrikaner and is one ‘in
which apartheid’s obsessions are made to bite the common
earth’ (Meihuizen 1991:37). Oom Schalk approvingly shares
Stoffel Oosthuizen’s opposition to inclusively liberal racial
policies of the British government: The imagery may superficially ridicule the Afrikaners, but
their pitiful sight also evokes empathy. These two short stories, with their postcolonial themes of
suppression and resistance, take on poignant human
dimensions in portraying the destructive colonial power of
the English ‘plundering warlord’ robbing the Afrikaners of
their land, livelihood, dignity and women. [A]fter all, that was one of the reasons why the Boers trekked
away into the Transvaal and the Free State […] because the
British Government wanted to give the vote to any Cape
Coloured person walking about with a kroes6 head and big cracks
in his feet. (Bosman 1991:16) Whilst these short stories reveal Bosman’s serious political
objection to the English colonisation of South Africa, his
critique of the Afrikaner is also skilfully and subtly woven
into the same narratives. 5.See Oliver’s (2010) view of the church and politics: ‘The church shared the
government’s power and control by ensuring that laws were passed to shape and
discipline society to conform to Christian values and laws …’ (Oliver 2010:6). 7.A term devised by the apartheid authorities to refer to black, coloured or Indian
people in South Africa and could be considered to be offensive or problematic. 3.An Afrikaans word, originally used as a derogatory term for Boers who surrendered
to the British during the Boer War, ultimately derived from the English phrase –
‘hands up!’ 4.The Afrikaans term, used by Bosman, refers to members of the Reformed Church. 6.The term ‘kroes’ is an offensive reference to tightly curled/frizzy African hair. https://
dsae.co.za/entry/kroes/e04120 The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War (Bosman 1977:70)
The violence and destruction of colo
power are also evident in Willems
remarks of Jack Brummer, the minin
[I]f they did not burn down all our f
not put our women and children in
English would never have won the B
Similar human consequences of B
embodied in other characters in the
represents the impoverished white
Johannes Erasmus, through his con
Cyril Stein, can secure a job for Krisja
[T]o be a lorry driver was to be a k
about it, after the poverty and the mis
his life as a bywoner [poor-white tenant
This basic form of employment be
starving bywoner and reflects th
experienced by the Afrikaner po
colonial rule. Lena Cordier, who leaves Willemsdo
Johannesburg, relays a tragic story
white’ Afrikaner schoolchildren:
[T]hey came from very poor familie
their parents tried to keep them de
seemed so awful about it, because
whites trying to keep up a show of
and my people are there in the slum
got no idea how awful it is. (Bosman
8.The ‘Poor White Problem’ was a social phenome
Carnegie Commission. Page 5 of 11 Original Research Page 5 of 11 Such examples show Bosman portraying Afrikaners suffering
at the hands of the powerful colonial government. Postcolonial
writing similarly concerns itself with the powerlessness of the
colonised. Such examples show Bosman portraying Afrikaners suffering
at the hands of the powerful colonial government. Postcolonial
writing similarly concerns itself with the powerlessness of the
colonised. empire could not reach them’ (Bosman 1977:7). Reasons
include the cruelty of the British concentration camps, where
Afrikaner women and children were interned as a means of
coercing the Boer guerrilla soldiers to surrender: ‘And in
each small town there is a Boer War Cemetery: women and
children of the concentration camps lie there. Time does not
heal all wounds’ (Bosman 1977:8). The implication of such
cruelty is that generations of future Afrikaner men and
women would carry the pain and bitterness of this British
colonial policy. Postcolonial writing correspondingly focuses
on the legacies of colonialism through the scars that remain
after decolonisation. Cultural identity, a topic associated with postcolonial
discourse, also appears in Bosman’s portrayal of Johannes
Erasmus who, when he sees the ‘half-castes’ (children of
mixed race) playing in the Willemsdorp streets, is reluctant to
admit that the ‘pure white identity’ of the town does not
exist. 8.The ‘Poor White Problem’ was a social phenomenon officially named by the 1932
Carnegie Commission. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War Snyman (2009) associates this reluctance with Erasmus’s
own sense of inferiority as an after-effect of the colonial
experience and his need to make up for it by denigrating
others: Postcolonial discourse centres on the severe consequences of
control and exploitation of the colonised people and their
land. In Chapter 5 of Willemsdorp, Bosman portrays the
Afrikaner as a ‘refugee’, fleeing from such control and
exploitation: [H]is inferiority complex – which he tries to hide behind a facade
of self-assuredness – haunts him and causes him to suffer spells
of depression and melancholy. At another level, Erasmus’s state
of mind is emblematic of the demoralised Afrikaner psyche after
the Boer War. It appears that racially superior attitudes were, for
many Afrikaners, a means of compensating for the humiliation
suffered during the war and the rebellion. (p. 129) And in the end, the thousand miles turned out not to have been
enough. A thousand miles by ox-wagon was very far. And that
was the only form of transport that the Voortrekkers knew. And
they thought that a distance of a thousand miles between
themselves and the English – an ox-wagon’s thousand miles –
would be enough. They could not foresee the day of steam and
the petrol engine. (Bosman 1977:70) Opperman-Lewis (2016) supports the view that the
psychological trauma and humiliation inflicted on Afrikaners
under British colonial rule resulted in ‘narcissistic rage’
against black South Africans and that Afrikaners reflexively
undertook to oppress black people in markedly similar ways
to which they themselves had experienced oppression
(Opperman-Lewis 2016:15). She examines the psychological
impact that this historical scar left not only on the Afrikaners
who suffered under harsh British rule, but also on future
generations of Afrikaners. The violence and destruction of colonisers seeking to retain
power are also evident in Willemsdorp through the bitter
remarks of Jack Brummer, the mining commissioner: [I]f they did not burn down all our farm houses and if they did
not put our women and children in concentration camps, the
English would never have won the Boer War. (Bosman 1977:95) Bosman already notes this legacy of colonialism in his short
story ‘Funeral Earth’. He highlights the irony of the
Afrikaners’ attempt to ‘teach Sijefu’s tribe of Mtosas to
become civilized’ (Bosman 1991:160). Oom Schalk’s sarcastic
tone brings this critique to the fore: Similar human consequences of British colonisation are
embodied in other characters in the novel. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War Krisjan Erasmus
represents the impoverished white Afrikaner.8 His brother,
Johannes Erasmus, through his connection to the character
Cyril Stein, can secure a job for Krisjan as a lorry driver: [E]ven after we had set fire to their huts in a long row round the
slopes of Abjaterskop, so that you could see the smoke almost as
far as Nietverdiend, the Mtosas remained just about as
unenlightened as ever. (Bosman 1991:160) [T]o be a lorry driver was to be a king. That was how he felt
about it, after the poverty and the misery and the degradation of
his life as a bywoner [poor-white tenant farmer]. (Bosman 1977:97) This basic form of employment becomes a lifeline for the
starving bywoner and reflects the extent of poverty
experienced by the Afrikaner population under British
colonial rule. This basic form of employment becomes a lifeline for the
starving bywoner and reflects the extent of poverty
experienced by the Afrikaner population under British
colonial rule. Just as the English had burnt down the Afrikaners’ farms, so
do the Afrikaners repeat similar atrocities against black
people. Postcolonial writers, such as Fanon (1963:29–30),
expressed the view that colonialism is characterised by
‘pervasive violence against the marginalised natives’, by the
State and the settlers. This behaviour is clearly evident in
Bosman’s texts as he presents the destruction and devastation
experienced by Afrikaners under English colonialism and,
later, by Africans under Afrikaner rule. De Kiewiet (1957:48)
interpreted Black-Boer conflicts as fed more by their
similarities than by their differences. Both struggled for
control over the same natural resources such as water and
land. By the end of this story, Bosman presents the Afrikaners’
awareness of the irony when they realise that black people,
too, have a love for and an attachment to their land: Just as the English had burnt down the Afrikaners’ farms, so
do the Afrikaners repeat similar atrocities against black
people. Postcolonial writers, such as Fanon (1963:29–30),
expressed the view that colonialism is characterised by
‘pervasive violence against the marginalised natives’, by the
State and the settlers. This behaviour is clearly evident in
Bosman’s texts as he presents the destruction and devastation
experienced by Afrikaners under English colonialism and,
later, by Africans under Afrikaner rule. De Kiewiet (1957:48)
interpreted Black-Boer conflicts as fed more by their
similarities than by their differences. Both struggled for
control over the same natural resources such as water and
land. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War Kobus Ferreira in ‘The Traitor’s Wife’, ironically adorned in
missionary garb, not only spits out violent instructions to his
troops but also hints at having ‘unholy’ thoughts about
Serfina, the traitor’s wife. Oom Schalk, the story’s narrator,
hints at hypocrisy in concluding that ‘in a strange way, it
seemed as though his violent language was not out of place
in a missionary’s frock coat’ (Bosman 1991:79), adding that ‘It
would not be the first time a man in ecclesiastical dress called
on a woman while her husband was away’ (p. 80). Such
tongue-in-cheek comments have the effect of discrediting the
‘Doppers’,4 whose leaders may not be as godly or holy as Postcolonial literature is often concerned with cruelty against
subjugated people of a colonised land. The opening chapter
of Bosman’s novel, Willemsdorp (written in 1951 but first
published in 1977), shows the Afrikaner escaping the English,
through the Great Trek, to where ‘the might of the British http://www.literator.org.za Open Access empire could not reach them’ (Bo
include the cruelty of the British conc
Afrikaner women and children were
coercing the Boer guerrilla soldiers
each small town there is a Boer War
children of the concentration camps
heal all wounds’ (Bosman 1977:8). T
cruelty is that generations of futu
women would carry the pain and b
colonial policy. Postcolonial writing c
on the legacies of colonialism throug
after decolonisation. Postcolonial discourse centres on the
control and exploitation of the colo
land. In Chapter 5 of Willemsdorp
Afrikaner as a ‘refugee’, fleeing f
exploitation:
And in the end, the thousand miles t
enough. A thousand miles by ox-wa
was the only form of transport that th
they thought that a distance of a
themselves and the English – an ox-
would be enough. They could not fo
the petrol engine. 9.The Natives Land Act was passed in 1913, by the Union Government. It allocated
less than one-tenth of South African land to black people (who at the time were a
majority at 61% of the population). It limited land acquisition amongst black people
and resulted in land dispossession. The Act also made it an illegal practice for black
people to make use of white land and to recompense the landowner in any form
except for labour. 10.Willemsdorp was initially written for publication in the United States of America
and for an American readership. The use of this term is by the character Cyril Stein
and its use is thus being represented by Bosman. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War Independent, prosperous African farmers who
owned land and livestock were forced to surrender these
possessions and reduced to migrant mine labourers or wage
labourers on white-owned farms (Ross 1999:88). These
concerns are synonymous with postcolonial themes of
dispossession, displacement and exploitation of the colonised
people and their land. Although Bosman is critical of such
laws, he also sympathises with the Afrikaner, whose deep
love and appreciation for the land come from their forebears,
the first Dutch settlers in the 1600s. Afrikaners view
themselves as ‘belonging’ to Africa, regarding it as their
indigenous home, as Bosman explains in his critical personal
writings: Bosman’s concern with the injustice of land dispossession
resulting from Black-Boer land conflicts and the Natives Land
Act (No. 27 of 1913)9 was because of the long-term effect of
such laws. Independent, prosperous African farmers who
owned land and livestock were forced to surrender these
possessions and reduced to migrant mine labourers or wage
labourers on white-owned farms (Ross 1999:88). These
concerns are synonymous with postcolonial themes of
dispossession, displacement and exploitation of the colonised
people and their land. Although Bosman is critical of such
laws, he also sympathises with the Afrikaner, whose deep
love and appreciation for the land come from their forebears,
the first Dutch settlers in the 1600s. Afrikaners view
themselves as ‘belonging’ to Africa, regarding it as their
indigenous home, as Bosman explains in his critical personal
writings: 12.This Act permitted the government to establish separate residential areas based on
racial categories. Members from other races were barred from living in areas not
assigned to them. A hindrance to hybridity: The apartheid colonial
state Typical in postcolonial writing is the theme of hybridity. Mambrol (2016:n.p.) notes that, as ‘One of the most widely
employed and most disputed terms in postcolonial theory’, it
‘commonly refers to the creation of new transcultural forms
within the contact zone produced by colonization’. The present
study explores cultural and linguistic hybridity through the
theme of racism in Willemsdorp, Bosman’s own hybridity and
the stylistic aspects of his writing, which is characterised by the
inclusion of Afrikaans and African words in his English texts. Issues relating to the mixing of races and co-habitation are
central to the novel. The main storyline concerns inter-racial
relationships in the context of the Immorality Act (No. 21 of
1950)11 and the Group Areas Act (No. 41 of 1950),12 both passed
by the newly elected apartheid government the year before the
novel was written. These policies, implemented by the
Afrikaners as the previously colonised people and now the
colonisers, feature in the novel as examples of racial
discrimination that deliberately hinders cultural hybridity or
multiculturalism. Hybridity, discrimination and racism
similarly feature in the discourse of postcolonial writers such
as Bhabha (1994) and Fanon ([1952]1986), which is discussed in
the analysis of Willemsdorp. [T]he Afrikaner accepts himself as part of Africa. Out of his own
traditions and history and background, out of the stones and the
soil and the red guts of Africa, he is fashioning a literature that
has not reached a very high inspirational level […] but that has
struck an authentic note, somehow, and that you can feel has got
a power in it that must become an enduring part of the
Afrikaner’s national heritage. (Bosman 2003:168–169) However, Bosman acknowledged that Africa belongs to
Africans. This is successfully portrayed in the description of
the ‘African Woman’, Marjorie Jones, in Willemsdorp, as the
character Cyril Stein admires her: 11.This Act prohibited intercourse or sexual relationships between white people and
non-white people, which included black, coloured or Asian people. The colonising power: Bosman’s depiction of the
political and sociological impacts of the South
African War By the end of this story, Bosman presents the Afrikaners’
awareness of the irony when they realise that black people,
too, have a love for and an attachment to their land: Lena Cordier, who leaves Willemsdorp to teach in the city of
Johannesburg, relays a tragic story of the plight of ‘poor
white’ Afrikaner schoolchildren: [T]hey came from very poor families. And you could see that
their parents tried to keep them decent. And that was what
seemed so awful about it, because it was so hopeless – poor
whites trying to keep up a show of respectability. Your people
and my people are there in the slums of Johannesburg. You’ve
got no idea how awful it is. (Bosman 1977:146–147) 8.The ‘Poor White Problem’ was a social phenomenon officially named by the 1932
Carnegie Commission. Open Access http://www.literator.org.za Page 6 of 11 Original Research and claim Africa – a highly political statement to make in
light of the time in which Bosman wrote this novel. ... picking up handfuls of soil and pressing it together. We felt the
deep loam in it, and saw how springy it was and we let it trickle
through our fingers. And we could remember only that it was for
sowing. I understood then how, in an earlier war, the Mtosas had
felt, they who were also farmers. (Bosman 1991:164) ... picking up handfuls of soil and pressing it together. We felt the
deep loam in it, and saw how springy it was and we let it trickle
through our fingers. And we could remember only that it was for
sowing. I understood then how, in an earlier war, the Mtosas had
felt, they who were also farmers. (Bosman 1991:164) Bosman, like later postcolonial writers, was clearly concerned
with and critical of the coloniser’s power, control and conquest
of the colonised and of the socio-political impacts that follow. Bosman, like later postcolonial writers, was clearly concerned
with and critical of the coloniser’s power, control and conquest
of the colonised and of the socio-political impacts that follow. Bosman’s concern with the injustice of land dispossession
resulting from Black-Boer land conflicts and the Natives Land
Act (No. 27 of 1913)9 was because of the long-term effect of
such laws. Cultural hybridity: Strictly taboo [I]n the movement of her hips, there was, for all the world, to
take note of Africa ... The African woman’s backside, Cyril
Stein was thinking to himself. It was like the shape of the
African continent on the map. From the loins of the negro10
woman would spring all the future generations of the people
of the African continent. The white man would come and go. His brief sojourn and his passing would leave behind few
traces. In the loins of the black woman, the history and the
destiny of Africa were wrapped up. The white man would
come and go and be forgotten. Africa, wombed in the negro
woman’s pelvis, was secure. Africa would go on forever. (Bosman 1977:90–91) Willemsdorp was written during a politically volatile time in
South Africa. Its first posthumous publication in 1977
contained significant cuts, which according to Gray (in
Bosman 1998:216), contain ‘painful revelations of illegal
brutality on the part of the South African Police’. This
brutality is a direct result of enforcing the aforementioned
statutes. As such, the content was censored drastically by its
publishers. Gray continues: [O]mitting them effectively got rid of the swingeing exposure
Bosman intended of such routine practices. An element of
tyranny, of menace, simply disappeared from the book – which to say the least, is unfortunate. (Gray, in Bosman 1998:216) It is clear, from the passage above, that Bosman felt that
Africans have a rightful place in Africa – the verb ‘wombed’
emphasising that it is indeed their natural home. The repeated
phrase, ‘the white man would come and go’, conveys the
message that the colonists would never successfully conquer It is clear, from the passage above, that Bosman felt that
Africans have a rightful place in Africa – the verb ‘wombed’
emphasising that it is indeed their natural home. The repeated
phrase, ‘the white man would come and go’, conveys the
message that the colonists would never successfully conquer The year 1977, as Sunday Times journalist, Pendock (1999)
recalls, was: The year 1977, as Sunday Times journalist, Pendock (1999)
recalls, was: [T]he dark night of Apartheid. Soweto had exploded the previous
year and 1977 saw a clampdown on the press. World and Weekend
World were closed down and their editors placed in ‘preventative
detention’. (p. 15) [T]he dark night of Apartheid. Soweto had exploded the previous
year and 1977 saw a clampdown on the press. Page 7 of 11 Page 7 of 11 It is, therefore, in this context that the publishers of
Willemsdorp, Human & Rousseau, excluded the numerous
passages on police brutality and only published the full
version in 1998.13 It is, therefore, in this context that the publishers of
Willemsdorp, Human & Rousseau, excluded the numerous
passages on police brutality and only published the full
version in 1998.13 Once the affair between Marjorie and Charlie begins, his
conscience is ruled by the indoctrination of the government’s
law and by his own heritage. Charlie feels tainted and is
‘filled with self-loathing’ (Bosman 1977:80). The phrase, ‘He
felt low’, is repeated several times, but this does not stop him
from physically engaging further with Marjorie, as though he
instinctively knows, on a human rather than a racial level
that as two consenting adults, they had not faulted: The setting of the novel is a small town in the Northern
Transvaal. Snyman (2012:61) observes that ‘small-town
society was for Bosman the ideal vehicle for commentary on
social and moral issues in an era of political turmoil in South
Africa’. Willemsdorp clearly reveals Bosman’s critique of the
contentious, racist policies and laws passed by the new
government in 1950 and 1951. [H]e knew, of course. It wasn’t that there was anything wrong
per se with his relations with Marjorie. At least, it was rotten, and
all that, stinking, and all that. But, it wasn’t just that. He was, in
spite of all kinds of liberal and even egalitarian views that he
might hold, still, at heart, a Boer and a Calvinist. Charlie
Hendricks knew that about himself. He was the editor of a Union
Party newspaper. And intellectually he recoiled from the
Volksparty tenets. But in his blood, he was a Boer. And, he was
sleeping with a kaffir woman. The generations of Boer ancestry
were stronger than he was. He felt a lost soul. (Bosman 1977:133) Bosman’s parody targets government officials responsible
for enforcing the laws that separated cultures and races in
South Africa. From his awful grammar and unsophisticated
use of language to his nonsensical, nocturnal investigations
whereby he uses a torch to identify footprints in the dark,
which he feels could only belong to a non-white person,
Detective Sergeant Brits functions as Bosman’s parodic
weapon to convey his critique of the apartheid state and its
absurd racial laws. 13.Except for the scenes on police brutality and the use of an indigenous African word
(explained later), the two versions are almost identical. In the analysis, I make use
of the 1977 version in order to highlight the differences when compared with the
1998 version. Page 7 of 11 He informs Charlie Hendricks, the editor
of one of the town’s local newspapers, of his ‘instructions
from the new Government to clean up the place’ (Bosman
1977:24) by apprehending people who contravened the
Immorality Act. Charlie’s own hybrid identity, as revealed in the above
passage, leads to his psychological strife. This reflects what,
postcolonial author, Bhabha (1994) notes, occurs in a hybrid
identity: [T]wo contradictory and independent attitudes inhabit the same
place, one takes account of reality, the other is under the influence
of instincts which detach the ego from reality. This results in the
production of a multiple and contradictory belief. (p. 132) In view of Bosman’s critique of this Act, a significant scene in
Willemsdorp highlights the potential dangers of hybridity in
the colonial context. When Marjorie Jones, a coloured
prostitute, pays Charlie Hendricks a visit at his apartment, he
does not invite her to sit down, for, as we are told by the
narrator, it ‘would be out of the question’ (Bosman 1977:79)
for a white man to offer a coloured woman a seat in his home: In presenting Charlie’s predicament, Bosman demonstrates
how the racial ideologies of the time, many of which were
used to justify colonialism, shaped the ways in which white
people viewed themselves in relation to non-white people. Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: [B]ut otherwise, when he lay with her on the divan, Charlie
Hendricks could not detect that she was essentially different
from a white girl that might have come to lie on the divan in his
arms. And – although of this, he was unconscious – he had also
solved the problem as to how a white man had to act when a
coloured girl came into his room. He couldn’t offer her a chair,
but he could, the divan. (Bosman 1977:80) [I]ntellectual uncertainty and anxiety that stems from the fact
that disavowal is not merely a principle of negation or elision; it
is a strategy for articulating contradictory and coeval statements
of belief. (p. 132) [I]ntellectual uncertainty and anxiety that stems from the fact
that disavowal is not merely a principle of negation or elision; it
is a strategy for articulating contradictory and coeval statements
of belief. (p. 132) Charlie clearly experiences similar uncertainty and anxiety in
his emotional and psychological struggle as he crosses racial
and cultural boundaries. In this scene, the narrator’s opinions reflect those of the
author. Davis (2006) investigates the source of multiple
ironies in Bosman’s texts, focusing on the nature of the
relationship between the author and the narrator and
highlights Bosman’s lack of consistency in applying his
authorial voice. Davis explains that at times, the distance
between the author and narrator is a notable and definite
one, whilst at other times, there is no clear distinction as to
whether it is Bosman or the narrator’s voice (or both) that is
heard between the lines (as is the case in many of his short
stories). It is clear in this scene that the authorial voice
highlights the hypocrisy of the racist values held by white
men and reflects their ideology regarding the inequality of
races. In this scene, the narrator’s opinions reflect those of the
author. Davis (2006) investigates the source of multiple
ironies in Bosman’s texts, focusing on the nature of the
relationship between the author and the narrator and
highlights Bosman’s lack of consistency in applying his
authorial voice. Cultural hybridity: Strictly taboo World and Weekend World were closed down and their editors placed in ‘preventative
detention’. (p. 15) World were closed down and their editors placed in preventative
detention’. (p. 15) Open Access http://www.literator.org.za Original Research Original Research Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: ‘Dr Pee’, doing house calls to
check on Lena’s health after the abortion, becomes
‘embarrassing’ to her: It is in this Third Space, according to Bhabha, that a community
can exist as a cultural hybrid, creating new meanings and new
identities. It is in this Third Space, according to Bhabha, that a community
can exist as a cultural hybrid, creating new meanings and new
identities. The consequence of crossing these boundaries and
experiencing social and political interaction in the ‘Third
Space’ between Marjorie, Charlie and Dap van Zyl,
precipitates the prejudice that Marjorie encounters, which
leads to her untimely demise. The envisioned unity of a
‘Third Space’, which Bhabha believes hybridity could bring,
could not materialise in current South African conditions, as
Bosman poignantly points out. Marjorie Jones (believed to
have been illegitimately fathered by a white man named
Jones) is a product of miscegenation and personifies the
notion of cultural hybridity – a concept not welcomed in this
colonial context and thus symbolically renounced through
her murder (Bosman 1997:127). [A]fter all, he was an Indian. And she could not help but wonder
what the landlady of Repton House was beginning to think of
his visits. Lena was sure that the land lady would be prepared to
overlook Dr Pee’s presence in the building if she knew that his
coming around was connected only with a matter of procuring
an abortion. But she would never tolerate his paying social calls
in the place. (Bosman 1977:174–175) Bosman foregrounds the ideo-cultural indoctrination of
white superiority, as endorsed through colonialism. As a
result of the broader racial ideologies of the time, which
negated the idea of cultural hybridity, both Charlie and Lena
display anxiety about being sexually or romantically
associated with people across the colour bar. Bosman foregrounds the ideo-cultural indoctrination of
white superiority, as endorsed through colonialism. As a
result of the broader racial ideologies of the time, which
negated the idea of cultural hybridity, both Charlie and Lena
display anxiety about being sexually or romantically
associated with people across the colour bar. Whilst Bhabha’s idea of hybridity is generally a favourable
one, Fanon’s ([1952] 1986) theorisation of inter-cultural or
inter-racial desire is, by contrast, based on the view that the
consequence of attempts at hybridity is often the self-
destruction of the colonised. Fanon believed that inter-racial
sexuality under colonial conditions was fundamentally
pathological. 14.The concept of ‘epistemic violence’ was introduced to postcolonial studies in
Spivak’s essay ‘Can the Subaltern Speak?’ (1988), where it refers to the Westerners’
exclusion or marginalisation of the voice of indigenous people through projecting
Eurocentric knowledge about Third-World countries. 15.The important postcolonial concern of ‘othering’ – a concept coined by Spivak in
her essay, ‘The Rani of Sirmur’ (1985) – refers to engendering marginality of an
individual or a group that does not fit in with the norms of a dominant social group. Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: The black woman who desires a white man
suffers under the delusion that his body is a bridge to wealth
and access (Fanon [1952] 1986). Marjorie’s actions echo
Fanon’s theorisation of inter-racial desire as a form of self-
destruction. He also elaborated on the objectification of
women through his theory of colonisation. Bergner (1995:77)
explains that in Fanon’s Black Skin, White Masks, women are
considered as objects (just as the colonised are considered
objects) in terms of their sexual relationships with men. Nicholls (2012), in her Fanonian analysis of colonising the
female body, explains: In an attempt to prevent hybridity and multiculturalism,
government vigorously implemented the Group Areas Act,
which brought an end to racially mixed residential areas by
creating physical boundaries between white and non-white
people. The latter were forced to leave their homes and were
relocated to specified areas. This legislation by the new
colonising power could also be considered a form of what
Bunch (2015) calls ‘distributive epistemic violence’, a category
adapted from the postcolonial scholar Spivak’s (1988) term,
‘epistemic violence’.14 Bunch (2015) refers to the withholding,
from the marginalised ‘other’,15 of education, land and other
resources that allow for a productive life. In this way, the
‘otherness’ is entrenched and further separates the
marginalised from the dominating group. Epistemic violence,
in whatever form, subjugates and controls the ‘other’ and has
its roots in wide-ranging discriminatory practices that help
the dominant group to maintain power and control. [A]s I read Fanon, his deconstruction of colonisation and his
development of a theory of decolonisation are grounded in an
analysis of power relations that is as applicable to gender as it is
to conquest of foreign territories. (n.p.) [A]s I read Fanon, his deconstruction of colonisation and his
development of a theory of decolonisation are grounded in an
analysis of power relations that is as applicable to gender as it is
to conquest of foreign territories. (n.p.) Korf (2010) argues that part of the apartheid government’s
moral justification for the policy of separate development
included the upliftment and development of Africans, albeit
separately from whites. However, she explains, ‘during
Malan’s tenure [from 1948 to 1954], Apartheid seemed to
consist of anti-measures, and little came of the promises to
“uplift” Africans’ (Korf 2010:464–465). Bosman’s novel lends itself to a similar analysis when one
reflects on Cyril Stein’s admiration of Marjorie’s body in the
extract discussed earlier (Bosman 1977:90–91). Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: Davis explains that at times, the distance
between the author and narrator is a notable and definite
one, whilst at other times, there is no clear distinction as to
whether it is Bosman or the narrator’s voice (or both) that is
heard between the lines (as is the case in many of his short
stories). It is clear in this scene that the authorial voice
highlights the hypocrisy of the racist values held by white
men and reflects their ideology regarding the inequality of
races Hybridity was anathema from the perspective of Afrikaner
Calvinism and the government. Thus, in the novel, the
government (and the ideologies it was based on) is presented
as the major obstacle hindering cultural hybridity, especially
through its implementation of The Immorality Act of 1927,
amended later again in 1950. Even though laws had been
passed and serious penalties put into place for miscegenation,
Bosman reveals how certain characters in the novel are
compelled to disobey these laws, cross the racial boundaries
and are in fact not opposed to multiculturalism. As an attractive coloured woman, Marjorie represents the
proverbial forbidden fruit to which the white Afrikaner male
characters, such as Charlie and Dap van Zyl, are deeply
attracted. This attraction and interaction occur in what http://www.literator.org.za Page 8 of 11 Page 8 of 11 Bhabha (1994) later termed the ‘Third Space’, where new
identities can be constructed, combining the identities and
cultures of all individuals interacting in that space: show how hybridity is negated by political agendas is
represented through Marjorie’s pathological behaviour and
self-destruction – a colonised woman who was not only
sexually objectified and exploited but also conquered by the
colonist/white male, through her tragic death. [I]t is that Third Space, though unrepresentable in itself, which
constitutes the discursive conditions of enunciation that ensure
that the meaning and symbols of culture have no primordial
unity or fixity; that even the same signs can be appropriated,
translated, rehistoricised and read anew. (Bhabha 1994:37) Bosman’s irony is searing where miscegenation, which is
presented as an act of ‘immorality’ where Marjorie is
concerned, is made to seem lighter when compared to Lena
Cordier, a white woman, who has an abortion, which is
performed by an Indian doctor. http://www.literator.org.za Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: At first glance,
it seems to be a compelling image of an African woman
representing the power of creation and the sustainability of
Africans in Africa. However, a closer Fanonian analysis
suggests that this white man’s admiration, in fact,
deconstructs her body, reducing it to a sexual object with the
sole function of fulfilling male desire. Bosman’s attempt to Bosman’s novel lends itself to a similar analysis when one
reflects on Cyril Stein’s admiration of Marjorie’s body in the
extract discussed earlier (Bosman 1977:90–91). At first glance,
it seems to be a compelling image of an African woman
representing the power of creation and the sustainability of
Africans in Africa. However, a closer Fanonian analysis
suggests that this white man’s admiration, in fact,
deconstructs her body, reducing it to a sexual object with the
sole function of fulfilling male desire. Bosman’s attempt to Open Access http://www.literator.org.za Page 9 of 11 Original Research Page 9 of 11 Original Research could assume the guise of an insider-figure and comment on
Afrikaans writing in Afrikaans. (De Kock 2001:204) The description in Willemsdorp of the ‘Native location’ where
Marjorie lives reflects these postcolonial aspects of
entrenching ‘otherness’ by withholding resources, neglecting
to uplift Africans and disempowerment through separate
development, and Bosman (1977) here refers implicitly to the
results of the implementation of the Group Areas Act: In ‘straddling two cultures’, as Leff (2014:24) notes, Bosman’s
‘hyphenated identity’ allowed him to bring hybridity into his
texts, especially in relation to language. [T]he houses were a haphazard collection of wood-and-iron and
unbaked brick structures, with holes in the walls to take the
place of windows. A lot of them were mud huts with flat roofs of
corrugated iron held down with huge stones. (p. 153) Amongst stylistic devices characteristic of postcolonial
authors is that of language variance, which, according to
Ashcroft, Griffiths and Tiffin (2002:50), ‘abrogates the
privileged centrality of English’. This technique is evident in
Bosman’s stories, which bring Afrikaans words into his
English texts. The inclusion of an African indigenous
language, although minimal, is also evident in Bosman’s
original version of Willemdsorp: ‘Back home in his khaya
[home] in the location, Josias did not sleep very easily that
night’ (Bosman 1998:167, my emphasis). The metonymic
function of language variance is a strong feature of
postcolonial writing. Bhabha (1994) further explains that in cases of hybrid identity,
the person experiences: The practice of carrying words over
from the mother tongue into the adopted literary form is
regarded as an ‘insertion of the truth of culture into the text’
(Ashcroft et al. 2002:51). The word ‘khaya’ is unglossed in
Bosman’s text, thus inserting the culture that it signifies and
pointing to the importance for him of cultural hybridity. Charlie Hendricks realises that should he father a child with
Marjorie, the child would have to live in the township as a
‘half caste’ according to the law. The plight of such a child is
powerfully conveyed in his thoughts: He was not prepared to go through life with the feeling that there
was that illegitimate coloured child – born into the world without
hope and without opportunity. Born into a world in which all the
doors were shut. His child, his own child, begotten in sin and
disgustingness, that child is a human being. And that human
being a coloured bastard, bred in the nigger location, there was
no other place for a part-white child to grow up in. And with no
future – no matter what talents that child had, was there an
avenue that would afford a part-white, part-coloured child any
hope of self-expression? (Bosman 1977:141) Untranslated foreign words in a text hold the power and
presence of the culture that they signify and therefore have
the important function of inscribing difference (Ashcroft
et al. 2002). Ashcroft et al. (2002:65) further explain that: Bosman’s implicit political views on separate development as
implemented in the Group Areas Act, as well as on apartheid,
are highlighted in the concluding rhetorical question. These
views prefigure Loomba’s (1998:2) view of the coloniser’s role
in ‘unforming or re-forming the communities’. Bosman’s bleak
description of the locations also prefigures subsequent
postcolonial
discourse
relating
to
racism,
difference,
subjugation and suppression of colonised people. [T]he choice of leaving words untranslated in post-colonial texts
is a political act, because while translation is not inadmissible in
itself, glossing gives the translated word, thus the ‘receptor’
culture, the higher status. (p. 51) These techniques are present in Bosman’s writing. By mixing
untranslated/un-glossed Afrikaans (and a few African)
words into his English texts, Bosman takes an anti-colonial
stance, consciously promoting a ‘South African English’ and
challenging the authority of the metropolitan language: References Ashcroft, B., Griffiths, G. & Tiffan, H., 2002, The empire writes back, 2nd edn.,
Routledge, London. Ashcroft, B., Griffiths, G. & Tiffan, H., 2003, The post-colonial studies reader, 2nd edn.,
Routledge, London. Bergner, G., 1995, ‘Who is that masked woman? or, The role of gender in Fanon’s
“Black skin, white masks”’, PMLA 110(1), 75–88. https://doi.org/10.2307/463196 Bhabha, H.K., 1994, The location of culture, Routledge, London. Bosman, H.C., 1977, Willemsdorp, Human & Rousseau, Cape Town. Bosman, H.C., 1987, Ramoutsa road, Ad. Donker, Johannesburg. [A]ttempt to define himself in terms of his disengagement from
the codes, religious, social and political, to which South African
whites in the first half of the twentieth century subscribed. (p. 113) Bosman, H.C., 1991, Unto dust: Stories, 3rd completely reset edn., Human & Rousseau,
Cape Town. Bosman, H.C., 1998, Willemsdorp, Human & Rousseau, Cape Town. Bosman, H.C., 2003, My life and opinions, Human & Rousseau, Cape Town. In doing so, it is clear that both authors were able to construct
their narratives, within a colonial period, from a post-colonial
viewpoint and successfully depicted the ‘darker underbelly
to the accepted notion of colonialism: what we now of course
call postcolonialism’ (Kimber 2018:122). Bunch, A.J., 2015, ‘Epistemic violence in the process of othering: real-world applications
and moving forward’, Scholarly Undergraduate Research Journal at Clark 2, viewed
09 December 2019, from https://commons.clarku.edu/surj/vol1/iss1/2. Clarke, J., 2015, ‘The “Baroque weaving machine”: Contrasting counterpoint in James
Joyce and Anthony Burgess’, in M.C. Carpentier (ed.), Joycean legacies, pp. 127–149,
Palgrave Macmillan, London. Chapman, M., 2006, Art talk, politics talk: A consideration of categories, University of
Kwazulu Natal Press, Scottsville, VA. Cuthbertson, G., 2006, ‘Oom Schalk’s Boer War: Herman Charles Bosman, Afrikaner
outsider,
writes
a
wider
war’,
Kleio
38(2),
142–163. https://doi. org/10.1080/00232080585380031 Bosman’s incisive rejections of the historical attempts around
him to essentialise culture, ethnicity and race and to suppress
various forms of hybridity are key to his satire. They provide
sufficient evidence to substantiate the view that he was
indeed a ‘proto-postcolonial author’ who, both thematically
and stylistically, repudiated the dominant colonial discourses
of his time. Davis, R., 2006, ‘Unstable ironies: Narrative instabilities in Herman Charles Bosman’s
“Oom Schalk Lourens” stories’, MA dissertation, Dept. of English, Rhodes University. De Kiewiet, C.W., 1957, A history of South Africa: Social and economic, Oxford
University Press, Oxford. De Kock, L., 2001, ‘English version of introduction’, in H.C. Page 10 of 11 Page 10 of 11 ‘Come on now, you kêrels [ fellows]’, the leader of the strong-arm
men said to Faded Blazer and Soiled Shirt – speaking to them in
Afrikaans – ‘you know we’re with you. But we’re getting a quid
each from the Union Party bastards to keep order at their
meeting. Go out quietly and we’ll be with you in the bar just
now. They’re just going to sing their bloody God Save the King
and then it will be all over. Come along now, Old Bul [bull]’... And those members of the audience who did not understand the
Boer language were suitably impressed. It was comforting to
think that the Union Party had as stewards determined men who
would brook no nonsense from hooligans. (Bosman 1977:14–15) Competing interests The author has declared that no competing interest exists. Acknowledgements The author would like to acknowledge Prof. Ian Bekker and
Prof. Nicholas Meihuizen for their supervisory contribution
to the Master of Arts in English dissertation on which the
article is based. Data availability No data (datasets, figures, etc.) are applicable with respect to
this article. Conclusion The main aim of this study was to investigate the proposed
description of Bosman as a ‘proto-postcolonial author’. The
focus on themes in Bosman’s texts shows them to resonate
with postcolonial concerns: the harsh and detrimental effects
of colonisation; racism in the colonial context and colonialism
as a hindrance to hybridity and multiculturalism. Bosman’s
criticism of British colonialism is accompanied by his
criticism of the reflexive nationalism of the Afrikaners that
culminated in the segregationist laws of the apartheid state. Disclaimer The views and opinions expressed in this article are those of
the author and do not necessarily reflect the official policy or
position of any affiliated agency of the author. Bosman explored the impact of colonial rule on the lives of
both black and white South Africans during different periods
in the country’s colonial history. Like Mansfield, whom
Kimber (2018:121) believed wrote about colonial New Zealand,
from a ‘post-colonial mind-set’, so did Bosman about colonial
South Africa. Whilst Kimber (2018:121) described Mansfield as
choosing to ‘dislocate’ herself from the cultural landscape in
which she grew up in New Zealand, Lenta (2003), from a
similar standpoint, describes Bosman’s: Funding information This research received no specific grant from any funding
agency in the public, commercial or not-for-profit sectors. Author’s contributions This scene clearly depicts how the ‘actual language of the
people’ as indicated by New (1978) reveals political tensions
and divisions between the English and the Afrikaner. This scene clearly depicts how the ‘actual language of the
people’ as indicated by New (1978) reveals political tensions
and divisions between the English and the Afrikaner. I declare that I am the sole author of this research article. Ethical considerations Typical stylistic features of postcolonial writing are thus in
evidence in Bosman’s pre-postcolonial works. Their
application anticipates one of the major themes of postcolonial
writing, hybridity, and shows Bosman’s acute awareness of
the ways in which colonialism in its various forms in South
Africa was a hindrance to hybridity. This article followed all ethical standards for research without
direct contact with human or animal subjects. Linguistic hybridity: A political act Willemsdorp depicts a divided South Africa, not just on a
racial level, between white and non-white people, but also
between the English and the Afrikaners. Bosman conveys
this division through the language differences between these
two groups in the town, accentuated by the exclusive names
of the two main newspapers: The Willemsdorp News, which
promotes the interests of the Union Party and English-
speaking supporters, and the Noordelike Transvaal Nuus,
which is the ‘vehicle of the Boer Volksparty’ (Bosman
1977:10). [T]he language in which I am writing is not pure English – it is
Afrikaans-English. It took me five years of writing for the
wastepaper basket, plus the ability to break a few English
grammar rules effortlessly, before I could write that way
automatically. (In Gray 2005:44) New (1978) (in Ashcroft et al. 2003:305) claims that ‘Literature
which uses the actual language – the sounds and syntax – of
the people becomes an arena in which the people’s political
and psychological tensions can find expression’ and explains
that the literary form that holds and reflects the verbal
tensions of the people ‘becomes a means of celebrating, or
exposing, or at least recognising and communicating
particular social realities’. In Bosman’s writing, the tension or
‘discord’, aptly described by Leff (2016:117), between the
English and the Afrikaners, is conveyed convincingly in the
election campaign scene in Willemsdorp. The young, Afrikaner
men cause a disruption by heckling the Union party speaker
and are escorted out by Afrikaner stewards: Bosman did not support the separation of the two languages
as an ideal. An anglicised Afrikaner with his own hybrid
identity, he advocated a multilingual society, where English
and Afrikaans could co-exist harmoniously and which was
inclusive of the indigenous languages. Leff (2014:24) refers to
Bosman as the ‘hybrid man’ and notes De Kock’s (2001)
interpretation of Bosman’s hybridity: [H]e could be an Afrikaner and a South African. He could be an
English-speaking author and an Afrikaans writer. He could
comment on Afrikaans letters as an English-speaker, and he http://www.literator.org.za Open Access Original Research References Bosman (ed.), Verborge
skatte: Herman Charles Bosman in / oor Afrikaans, pp. 190–210, Compiled by
Leon de Kock, Human & Rousseau, Cape Town. http://www.literator.org.za Open Access Page 11 of 11 Original Research Dugard, J., 1978, Human rights and the South African legal order, Princeton University
Press, Princeton, NJ. Meihuizen, N., 1991, ‘Bosman and self-conscious fiction’, Literator 12(1), 35–42. Miller, A., 2006, A storyteller’s story, viewed 28 May 2018, from https://mg.co.za/
article/2006-07-05-a-storytellers-story. Fanon, F., 1963, The wretched of the earth, Grove Weidenveld, New York, NY. New, W.H., 1978, ‘New language, new world’, in B. Ashcroft, G. Griffiths & H. Tiffin
(eds.), The postcolonial studies reader, pp. 303–308, Routledge, London. Fanon, F., 1986, Black skin, white masks, Pluto Press, London. Gray, S., 1986, Herman Charles Bosman, McGraw-Hill, Johannesburg. Ngugi, W.T., 1986, Decolonising the mind. The politics of language in African literature,
East African Educational Publishers, Nairobi. Gray, S., 2005, Life sentence: A biography of Herman Charles Bosman, Human &
Rousseau, Cape Town. Nicholls, T., 2012, ‘Concerning violence against women: A Fanonian analysis of
colonizing the female body’, e-cadernos CES 16, 2012. https://doi.org/10.4000/
eces.1047 Hayden, S., 2002, ‘Dancing the Tiekiedraai: A socio-historic approach to Bosman’s
Bushveld narratives’, MA dissertation, Dept. of English Language and Literature,
University of Cape Town. Oliver, E., 2010, ‘Afrikaner Christianity and the concept of empire’, Verbum et Ecclesia
31(1), Art. #393. https://doi.org/10.4102/ve.v31i1.393 Katrak, K.H., 1989, ‘Decolonizing culture: Toward a theory for postcolonial women’s
texts’, Modern Fiction Studies 35(1), 157–179. https://doi.org/10.1353/mfs.0.0420 Opperman-Lewis, H., 2016, Apartheid: Britain’s bastard child, Reach, Wandsbeck. Kimber, G., 2018, ‘A cycle of dislocation: Katherine Mansfield, modernism, and proto-
postcolonialism’, in P. Gill & F. Kläger (eds.), Constructing coherence in the British
short story cycle, pp. 104–124, Routledge, New York, NY. Overbey, K.E., 2012, ‘Postcolonial’, Studies in Iconography 33, 145–156. Pendock, N., 1999, ‘Death brings Bosman’s pen back to life: review of Willemsdorp by
H.C. Bosman’, Sunday Times, 10 January, p. 15. Korf, L., 2010, ‘DF Malan: A political biography’, PhD thesis, Stellenbosch, University of
Stellenbosch. Rao, R., 2007, ‘Postcolonial cosmopolitanism: Between home and the world’, PhD
thesis, Dept. of Politics & International Relations, University of Oxford. Leff, C., 2014, ‘Bosman as verbindingsteken: Hybridities in the writing of Herman
Charles Bosman’, MA dissertation, Dept. of English, Rhodes University. Ross, R., 1999, A concise history of South Africa, Cambridge University Press, Cambridge. References oss, R., 1999, A concise history of South Africa, Cambridge University Press, Ca Leff, C., 2016, ‘Herman Charles Bosman: A man of profound contradictions’, English in
Africa 43(1), 109–129. https://doi.org/10.4314/eia.v43i1.6 Snyman, M.S., 2009, ‘The small-town novel in South African English literature
(1910–1948)’, DLitt thesis, Dept. of English, University of Pretoria. Snyman, S., 2012, ‘“Willemsdorp” by Herman Charles Bosman: The small-town locale
as fictional vehicle for commentary on social and moral issues in the South African
historical context’, Tydskrif vir Letterkunde 49(2), 60–71. https://doi.org/10.4102/
ve.v31i1.393 Lenta, M., 2003, ‘White South African and latter-day Bohemian: Two editions of
Herman Charles Bosman’, Current Writing: Text and Reception in Southern Africa
15(1), 109–122. https://doi.org/10.1080/1013929X.2003.9678146 Loomba, A., 1998, Colonialism/Postcolonialism, Routledge, London. Spivak, G.C., 1985, ‘The Rani of Sirmur: An essay in reading the archives’, History and
Theory 24(3), 247–272. https://doi.org/10.2307/2505169 Mackenzie, C., 1999, The oral-style South African short story in English: A.W. Dryson to
H.C. Bosman, Rodopi, Amsterdam. Spivak, G.C., 1988, ‘Can the subaltern speak?’, in P. Williams & L. Chrisman (eds.). Colonial discourse and post-colonial theory: A reader, pp. 66–111, Columbia
University Press, New York, NY. Mackenzie, C., 2003, A preface to Herman Charles Bosman’s ‘A Boer Rip Van Winkel’,
Rand Afrikaans University, Johannesburg. Mambrol, N., 2016, Literary theory and criticism notes: Postcolonialism, viewed 28
May 2018, from https://literariness.org/2016/04/06/postcolonialism/. Stam, R. & Spence, L., 1983, ‘Colonialism, racism and representation’, Screen 24(2),
2–20. https://doi.org/10.1093/screen/24.2.2 http://www.literator.org.za http://www.literator.org.za Open Access
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https://www.nature.com/articles/srep35729.pdf
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miR-200 family controls late steps of postnatal forebrain neurogenesis via Zeb2 inhibition
|
Scientific reports
| 2,016
|
cc-by
| 9,449
|
miR-200 family controls late steps
of postnatal forebrain neurogenesis
via Zeb2 inhibition Christophe Beclin1,†, Philipp Follert1,†, Elke Stappers2, Serena Barral1,3, Nathalie Coré1,
Antoine de Chevigny1, Virginie Magnone4, Kévin Lebrigand4, Ute Bissels3,
Danny Huylebroeck2,5, Andreas Bosio3, Pascal Barbry4, Eve Seuntjens2,6 & Harold Cremer1 received: 17 May 2016
accepted: 04 October 2016
Published: 21 October 2016 During neurogenesis, generation, migration and integration of the correct numbers of each neuron sub-
type depends on complex molecular interactions in space and time. MicroRNAs represent a key control
level allowing the flexibility and stability needed for this process. Insight into the role of this regulatory
pathway in the brain is still limited. We performed a sequential experimental approach using postnatal
olfactory bulb neurogenesis in mice, starting from global expression analyses to the investigation of
functional interactions between defined microRNAs and their targets. Deep sequencing of small RNAs
extracted from defined compartments of the postnatal neurogenic system demonstrated that the miR-
200 family is specifically induced during late neuronal differentiation stages. Using in vivo strategies
we interfered with the entire miR-200 family in loss- and gain-of-function settings, showing a role of
miR-200 in neuronal maturation. This function is mediated by targeting the transcription factor Zeb2. Interestingly, so far functional interaction between miR-200 and Zeb2 has been exclusively reported in
cancer or cultured stem cells. Our data demonstrate that this regulatory interaction is also active during
normal neurogenesis. Fine-tuning of gene expression is a fundamental requirement for the control of developmental processes. This
is particularly evident during nervous system development, where stem cell populations generate a multitude
of neuronal and glial cell types in a temporally and quantitatively perfectly orchestrated manner. After their
generation, precursors migrate to their respective target structures and form functional connections with their
environment. Neurogenesis continues into postnatal and adult stages in defined regions of the mammalian brain,
making the control and stabilization of regulatory processes a lifelong requirement1. It is evident that complex
molecular networks, superposed levels of control and tight interactions between regulatory mechanisms guard
induction and maintenance of neurogenesis. MicroRNAs (microRNAs) represent one key control level providing
the needed flexibility and stability2. l
Dicer mutant mouse lines have been widely used to show the general involvement of the microRNA pathway
in brain development and function3–7. Specific microRNAs have been implicated in the control of neurogenesis at
different levels. First, they act at the level of initiation of differentiation and the progression of progenitors towards
a differentiated state. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 17 May 2016
accepted: 04 October 2016
Published: 21 October 2016 Results
i
RN microRNA expression in the OB neurogenic system: the miR-200 family. We investigated the
expression pattern and dynamics of microRNA expression in the OB neurogenic system through the miRNome
analysis of defined compartments. We isolated: 1. dorsal and lateral periventricular tissue at P1 (mainly radial
glia and transit amplifying cells) and P6 (containing in addition ependymocytes), 2. RMS tissue at P15 (mainly
tangentially migrating neuronal precursors and tunnel glia), 3. OB tissue, depleted of the anterior RMS, at P15
and P28 (containing mainly OB neurons and glial cells). Deep sequencing identified 151 microRNAs with an
expression level of at least 1000 reads per million (triplicate average) in at least one tissue.f For these 151 miRNAs, we considered the ratios of expression between different samples, thereby comparing
spatial and temporal expression in the VZ-SVZ as well as the temporal evolution along the RMS towards the OB. The results, presented as a heat map, are shown in Fig. 1a. Heat map columns represent the different sample com-
binations selected for comparison. Vertical clustering in the heat map groups miRNA exhibiting similar spatial
and temporal expression during the neurogenic process.h g
g
Among these 151 microRNAs, some showed stable expression along the neurogenic sequence. This is the case
for the neuronal microRNAs miR-9 and miR-124 (red fringes), which maintained high expression in all samples
analyzed, in agreement with their general role in the control of the neurogenic sequence progression20. Other
microRNAs, like for example the pro-dopaminergic microRNAs miR-7a13 (blue fringe), showed highly dynamic
expression changes both, in space (dorsal vs lateral stem cell compartment) and in time (VZ-SVZ vs OB). p
g
p
(
p
)
(
)
A second interesting observation concerns the behavior of microRNAs families that share common seed
sequences. In general, all members of such families show highly similar expression patterns, demonstrated by
tight clustering in the heat map, indicating a common function in the system.hih g
g
p
g
y
This is exemplified by the miR-34/miR-449 family (Fig. 1b). The family is composed of 6 microRNAs, coded
by three independent genomic loci. All members play roles in the differentiation of multiciliated cells in several
structures21–23. We found among the 151 microRNAs expressed above threshold four members, which were all
strongly induced in the P6 VZ-SVZ samples compared to P1. This induction parallels the appearance of multicil-
iated ependymocytes along the ventricular wall during the first postnatal week24. www.nature.com/scientificreports/ generate large amounts of neuronal precursors that, after their amplification migrate tangentially within the ros-
tral migratory stream (RMS) into the OB. Once arrived in their target structure they migrate radially into the
granular and glomerular layers where they differentiate into interneurons that use GABA, dopamine or glutamate
as their neurotransmitters17,18. This neurogenic process presents major experimental advantages making it a unique tool for the study of
neurobiological problems. First, the process is permanent and not restricted to a small time window in utero. Second, stem cells producing defined neuron populations are regionalized and can be efficiently labeled and
manipulated by targeted brain electroporation19. Third, different compartments containing cells at distinct stages
of the neurogenic process (stem cells, amplifying progenitors, migrating precursors and mature neurons) are spa-
tially separated and can be isolated. Thus, the system is particularly suited to systematically approach the complex
regulatory processes that underlie the fine-tuning of neurogenesis by microRNAs. Here, we focus on the role of
microRNAs in late steps of neuronal differentiation.i pf
We generated a complete profile of microRNA expression, based on deep sequencing of small RNAs,
in the principal compartments of this neurogenic system. Using this unique dataset we identified a family
of microRNAs, the miR-200 family, that is specifically expressed at late neurogenic stages but absent from
immature differentiation intermediates. We used an in vivo approach to perform gain-and loss-of function
with the entire miR-200 family leading to promotion or inhibition of neuronal differentiation, respectively. Finally, we show that miR-200 microRNAs function in this context by targeting the zinc-finger transcription
factor Zeb2. miR-200 family controls late steps
of postnatal forebrain neurogenesis
via Zeb2 inhibition For example, miR-124 and the miR-9/miR-9* duplex inhibit the expression of molecular
components that oppose neuronal differentiation8–12. Second, they act at the level of neuronal phenotype. This is
exemplified by the regulation of dopaminergic fate determination in the forebrain by miR-7a targeting Pax613 or
the repartition between inter-neurons and motoneurons in the spinal cord controlled through the targeting of
Olig2 by miR-17–3p14. Third, microRNAs act at the level of synaptogenesis and synaptic function. For example,
miR-134 inhibits dendritogenesis and spine formation15,16. However, it is likely that additional microRNAs con-
trol specific steps of neurogenesis between fate determination at the NSC level and synaptogenesis. pi
p
g
y
p g
Here we investigate the expression and function of microRNAs during postnatal olfactory bulb (OB) neu-
ogenesis. In this system pre-determined neuronal stem cells in the ventricular/subventricular zone (VZ-SVZ) 1IBDM, Aix-Marseille Université, CNRS, UMR7288, 13288 Marseille, France. 2Laboratory of Molecular Biology, Dept
Development and Regeneration, KULeuven, 3000 Leuven, Belgium. 3Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany. 4CNRS and University Nice Sophia Antipolis, IPMC, Sophia Antipolis, France. 5Dept Cell Biology, Erasmus
MC, 3015 CN Rotterdam, The Netherlands. 6GIGA-Neurosciences, Université de Liège, 4000 Liège, Belgium. †These
authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.B. (email: christophe.beclin@univ-amu.fr) 1 Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 www.nature.com/scientificreports/ Results
i
RN p
y
y
g
gi
p
Another family of microRNAs that is tightly regulated during postnatal OB neurogenesis is the miR-200 fam-
ily, that has been implicated in neurogenesis in cultured cells25 and sensory neurons26. Indeed, the five members
of the miR-200-family were exclusively expressed in the OB and densely clustered in the heat map representation
(Fig. 1b,c). In mice, three members (miR-429, miR-200a, miR-200b) reside in one intergenic cluster on chro-
mosome 4. These showed particularly high expression levels (Fig. 1c). miR-200c and miR-141 are localized on
chromosome 6 and were expressed at lower levels (Fig. 1c).f p
g
While micro-dissection before sequencing allowed enriching the samples for the different cell populations
of the forebrain neurogenic system, these samples were still heterogeneous, containing, for example, contami-
nating neurons and glial cells. Therefore we aimed at refining miR-200 family expression in the system combin-
ing transgenic and sorting approaches. First, we investigated microRNA expression analyses in the OB neuron
sub-populations. In GAD67-GFP knock-in mice the GAD67 promoter drives GFP expression in the GABAergic
lineage (Fig. 2a)27. We micro-dissected tissue from the OB and performed GFP based FACS sorting after disso-
ciation. These analyses identified three distinguishable cell populations: I) A small population of cells express-
ing high amounts of GFP (GFP-high). These were positive for the neuron marker GluR2 and the precursor
marker Doublecortin (Dcx) demonstrating an immature OB neuron identity (Fig. 2b). II) Cells expressing low
amounts of GFP (GFP-low). These expressed GluR2 but not DCX and were therefore likely mature OB neurons. III) Cells that were GFP-negative (GFP-neg; Fig. 2a). These did not express significant levels of either GluR2 or
DCX (Fig. 2b), thus likely representing glia and GluR2 negative neurons28. qRT-PCR analyses to detect miR-
200b and miR-141 as representative members of each of the two miR-200 clusters showed strongest expression
in the mature GABAergic (GFP-low) population (Fig. 2c), in agreement with the deep-sequencing data (Fig. 1). This demonstrates that miR-200b and miR-141, and therefore likely the entire miR-200 family, are present in the
postnatal neurogenic lineages and that their expression level increases with maturation. The observation that Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 2 www.nature.com/scientificreports/ Figure 1. miRNome profiling by deep-sequencing. (a) Heatmap showing the results of deep sequencing
analyses. Only microRNAs representing more than 0.1% of total microRNAs in at least one tissue are shown. Results
i
RN Number of reads for a microRNA in a given tissue was obtained by averaging the different sample repetitions. Columns describe the ratios between tissues selected for comparison. Proximity of vertical position indicates
the similarity of expression profile of detected microRNAs and was determined using the MeV application60. (b) Close-up of specific regions of the heat map highlighting the miR-200-family a group of microRNAs
preferentially expressed in the OB and the miR-34 family that is induced during ciliogenesis. miR-34a does not
cluster in the heatmap due to strong expression in OB glia. (c) Histogram representing the absolute number of
reads per tissue obtained in the deep sequencing analyzis for each member of the miR-200 family. All miR-200
family members are exclusively expressed in the OB but not in the stem cell or migratory compartments. Figure 1. miRNome profiling by deep-sequencing. (a) Heatmap showing the results of deep sequencing Figure 1. miRNome profiling by deep-sequencing. (a) Heatmap showing the results of deep sequencing
analyses. Only microRNAs representing more than 0.1% of total microRNAs in at least one tissue are shown. Number of reads for a microRNA in a given tissue was obtained by averaging the different sample repetitions. Columns describe the ratios between tissues selected for comparison. Proximity of vertical position indicates
the similarity of expression profile of detected microRNAs and was determined using the MeV application60. (b) Close-up of specific regions of the heat map highlighting the miR-200-family a group of microRNAs
preferentially expressed in the OB and the miR-34 family that is induced during ciliogenesis. miR-34a does not
cluster in the heatmap due to strong expression in OB glia. (c) Histogram representing the absolute number of
reads per tissue obtained in the deep sequencing analyzis for each member of the miR-200 family. All miR-200
family members are exclusively expressed in the OB but not in the stem cell or migratory compartments. Figure 1. miRNome profiling by deep-sequencing. (a) Heatmap showing the results of deep sequencing
analyses. Only microRNAs representing more than 0.1% of total microRNAs in at least one tissue are shown. Number of reads for a microRNA in a given tissue was obtained by averaging the different sample repetitions. Columns describe the ratios between tissues selected for comparison. Proximity of vertical position indicates
the similarity of expression profile of detected microRNAs and was determined using the MeV application60. Results
i
RN (b) Close-up of specific regions of the heat map highlighting the miR-200-family a group of microRNAs
preferentially expressed in the OB and the miR-34 family that is induced during ciliogenesis. miR-34a does not
cluster in the heatmap due to strong expression in OB glia. (c) Histogram representing the absolute number of
reads per tissue obtained in the deep sequencing analyzis for each member of the miR-200 family. All miR-200
family members are exclusively expressed in the OB but not in the stem cell or migratory compartments. miR-200b and miR-141 are also expressed in the GFP-neg fraction indicates that both micro-RNAs are present
either in the glial fraction or in GAD67 negative neurons in the OB, like glutamatergic interneurons or projection
neurons such as mitral cells. Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 3 www.nature.com/scientificreports/ Figure 2. MicroRNA expression in OB subpopulations. (a) Sagittal section of a GAD67-GFP
forebrain. Diagrams are dot plots of the FACsorting experiment performed on wild-type (left)
knock-in mice (right). Three cell populations were sorted from GAD67-GFP knock-in mouse b
for subsequent qRT-PCR analyses. (b) qRT-PCR characterization of the three populations. Glu
on both, GABAergic neuronal progenitors and fully differentiated neurons. Doublecortin is exc
expressed in neuronal progenitors. GFP negative cells do not significantly express these neuron Figure 2. MicroRNA expression in OB subpopulations. (a) Sagittal section of a GAD67-GFP knock-in mouse
forebrain. Diagrams are dot plots of the FACsorting experiment performed on wild-type (left) and GAD67-GFP
knock-in mice (right). Three cell populations were sorted from GAD67-GFP knock-in mouse brain and used
for subsequent qRT-PCR analyses. (b) qRT-PCR characterization of the three populations. GluR2 is expressed
on both, GABAergic neuronal progenitors and fully differentiated neurons. Doublecortin is exclusively
expressed in neuronal progenitors. GFP negative cells do not significantly express these neuronal markers. Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 4 www.nature.com/scientificreports/ (c) qRT-PCR analysis of the expression of miR-200b and miR-141 in the three sorted populations showing a
preferential expression in the GFP-low fraction. (d) qRT-PCR characterization of the two purified cell fractions
issued from the MACS experiment designed to discriminate neuronal vs glial fraction from the OB based. Neuronal (NeuN, GluR2, DCX) and glial (GFAP, Olig1, Olig2) markers validate the expected neuronal and
glial identities. (e) qRT-PCR analysis demonstrates that miR-200b and miR-141 are enriched in the neuronal
fraction. Results
i
RN 3a).hi y
g
We validated the constructs using a luciferase assay system. The 3′UTR of the zinc finger/homeodomain
transcription factor Zeb2, a well-characterized miR-200 target30, was cloned downstream the firefly luciferase
gene in the pmiRGlo vector (Promega). Co-transfection of HeLa cells with miR-200-gof and the resulting plas-
mid strongly repressed luciferase activity. Simultaneous expression of the miR-200-sponge was able to partially
restore luciferase activity (Fig. 3b), altogether demonstrating that both vectors were functional. We then used in
vivo brain electroporation to introduce the miR-200-sponge or miR-200-gof constructs into the OB neurogenic
system. y
First, we analyzed the consequences of miR-200 inhibition on neuronal differentiation in the OB using the
miR-200-sponge. As miR-200 expression occurs during late stages of OB neurogenesis, we analyzed the electro-
porated cells at 15 dpe, a time point of advanced maturation. Knockdown of miR-200 significantly increased the
percentage of electroporated cells in the OB that were negative for NeuN, a marker for mature neurons (control:
1.99% ± 0,43%; miR-200-sponge: 7.32% ± 1.76%; Fig. 3c,d).fi p
g
g
Second, we investigated if expression of the miR-200 members at early stages of OB neurogenesis was suffi-
cient to induce premature neuronal differentiation. To this end we electroporated miR-200-gof into the lateral
ventricular wall and analyzed their progeny. A major step in the neurogenic sequence is the transformation of proliferating progenitors into post-mitotic
migrating neuroblasts. We thus investigated whether premature expression of miR-200 can induce premature exit
of cell-cycle. We measured BrdU incorporation 2 days after electroporation in control and miR-200 gof condi-
tions and found that the percentage of BrdU positive cells was significantly decreased in miR-200 gof conditions
(Fig. 3e). g
We then investigated the phenotype of electroporated cells in the RMS at 4 days and 7 days
post-electroporation. At these stages all GFP-positive cells displayed the typical morphology of migrating neu-
roblasts (Fig. 3f). In the control situation, calretinin, a late-appearing marker for defined subpopulations of OB
interneurons31–34 was absent from the RMS (4 dpe, 0.17% ± 0.17%; 7 dpe, 0.64% ± 0.19, Fig. 3f,g), in accord-
ance with its expression in mature neurons. However, 4 days after miR-200-gof electroporation 4.79% ± 1.15%,
(Fig. 3g) of the GFP positive cells in the RMS expressed calretinin and this percentage increased to 8.96%; ± 1.82%
at 7 dpe (Fig. 3f,g). Generic markers of differentiation like NeuN and Map2 were unchanged at this time point. Results
i
RN For b-e the qPCR values shown in the histograms result from 2 (b,d) or 3 (c,e) qPCR experiments
(4 wells per condition in each experiment) (f) Electroporation of an expression construct driving GFP with
regulatory sequences of the human miR-429/miR-200a/miR-200b cluster leads to GFP-labeled cells in the OB. Scale bar: 70 μm. Error bars: sem. To address the latter point, we used Magnetic Activated Cell Sorting (MACS, Miltenyi) to separate neuronal
from glial cells after dissociation of the OB of 1-month-old mice and characterized the resulting cell populations
by qRT-PCR. As expected, the glial-enriched population expressed high levels of GFAP, Olig1 and Olig2, whereas
the neuronal fraction showed strong expression of the neuronal markers NeuN, GluR2 and DCX (Fig. 2d). qRT-PCR analyses demonstrated that miR-200b and miR-141 expression were highest in the neuronal fraction. In
the glial fraction miR-200b was expressed at a very low level whereas miR-141 presence was significant (Fig. 2e). g
p
y
p
gi
( g
)
Finally, we introduced the human sequence upstream of the miR-200b/miR-200a/miR-429 cluster29 upstream
of a GFP-cassette. The resulting plasmid was introduced in neural stem cells in the wall of the forebrain ventricle,
together with a control vector (pCAGGS-Tomato), using postnatal brain electroporation19. Twenty-four days
later a subpopulation of mature granule neurons derived from these transfected stem cells produced both, GFP
and tomato proteins, demonstrating activity of the promoter fragment in postnatal generated neurons of the OB
(Fig. 2f). Taken together, the above results demonstrate that miR-200 microRNAs expression increases with mat-
uration in the postnatal neuronal lineage that generates OB interneurons. miR-200 family microRNAs regulate neuronal differentiation. Next, we investigated the function
of the miR-200 family in the control of neuronal differentiation. The miR-200 family contains two different seed
sequences (differing in only one nucleotide) and both sequences are present in the two genomic loci. Together
with their synchronized expression this suggested a redundant or cooperative function of the miR-200 family
members in the OB. Therefore we developed an approach to interfere in parallel with the entire family in both,
gain- and loss-of-function settings. g
g
First, we constructed an in vivo expression vector that generates a single transcript containing the two genomic
regions harboring the miR-200 family clusters under the control of the chicken β-actin promoter (miR-200-gof,
Fig. 3a). Second, we designed a miR-200-sponge that contained four repeats capable to bind each of the miR-200
family members (Fig. Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 Results
i
RN At 15 dpe in both, control and miR-200-gof conditions the percentage of GFP + cells expressing calretinin was
approximately 2% suggesting that the calretinin expressing neuroblasts at 4 and 7 dpe after miR-200-gof electro-
poration either downregulated calretinin or died at later stages. p
g
g
Altogether these results show that knockdown of miR-200 family microRNAs interferes with terminal neu-
ronal differentiation while their premature expression induces in a subset of postnatally generated precursors
defined aspects of neuronal maturation: cell-cycle exit and premature expression of a mature neuron marker. Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 5 www.nature.com/scientificreports/ Figure 3. In vivo functional analysis of miR-200 microRNAs. (a) Representation of the two vectors designed
to over-express (miR-200-gof) or down-regulate (miR-200-sponge) the expression of all miR-200 family
members in parallel (b) Luciferase assay performed on HeLa cells transfected with the Zeb2-UTR vector
together with control vectors (control condition), with the miR-200 expression vector alone (miR-200-gof)
or with the miR-200 expression vector and the miR-200 sponge plasmid (miR-200-gof + miR-200 sponge
condition). Data represent the mean ± s.e.m of values from 4 wells. miR-200 sponge partially rescues the Figure 3. In vivo functional analysis of miR-200 microRNAs. (a) Representation of the two vectors designed
to over-express (miR-200-gof) or down-regulate (miR-200-sponge) the expression of all miR-200 family
members in parallel (b) Luciferase assay performed on HeLa cells transfected with the Zeb2-UTR vector
together with control vectors (control condition), with the miR-200 expression vector alone (miR-200-gof)
or with the miR-200 expression vector and the miR-200 sponge plasmid (miR-200-gof + miR-200 sponge
condition). Data represent the mean ± s.e.m of values from 4 wells. miR-200 sponge partially rescues the Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 6 www.nature.com/scientificreports/ inhibitory activity of the miR-200 expression vector. (c) Fluorescent images showing OB neurons 15 days
after lateral co-electroporation of pCX-GFP and pCX-mcs2 control vector (left column) or pCX-GFP and
miR-200 sponge vector (right column). Arrows indicate cells expressing both GFP and NeuN, asterisk shows
a cell positive for GFP but negative for the late pan-neuronal marker NeuN. (d) Mean of GFP + cells that do
not express NeuN (n = number of animals analyzed). Difference between groups were analyzed using Man
and Whitney test (P = 0.009023). (e) Ratios of GFP + cells showing BrdU integration 2 days after lateral
electroporation of a GFP vector. Difference between groups were analyzed using Man and Whitney test. Results
i
RN (f) Fluorescent images showing neuroblasts in the RMS 7 days after lateral co-electroporation of pCX-GFP
and pCX-mcs2 control vector or pCX-GFP and miR-200-gof stained for calretinin. Only in the miR-200 over-
expression condition GFP + cells expressing calretinin are detected. (g) Ratios of GFP + cells co-expressing
calretinin at 4 and 7 dpe (n = number of animals analyzed). Differences between groups were analyzed using
Man and Whitney test. Scale bars: 30 μm in (c,e). Mir-200 family target Zeb2 in the OB neurogenic system. Next, we aimed at analyzing the regulatory
mechanism that underlies the differentiation-inducing function of miR-200 family microRNAs in the system. The
best-characterized targets of the miR-200 family, albeit in cancer backgrounds, are the zinc finger proteins Zeb1
and Zeb230. This interaction is a key regulatory mechanism for epithelial-mesenchymal transition, thereby con-
trolling cell migration, stem-cell properties, apoptosis and senescence30. In situ hybridization data demonstrated
that Zeb2 transcripts are particularly strongly expressed in the entire VZ/SVZ-RMS-OB system (Fig. 4a; from
Allen brain atlas: http://mouse.brain-map.org). Moreover, immunofluorescence using a Zeb2 specific antibody
demonstrated high protein levels in the system (Fig. 4b). We asked if premature expression of miR-200 family
microRNAs interfered with Zeb2 levels in neuronal precursors. The miR-200-gof and a GFP- expression vectors
were co-electroporated into the SVZ and Zeb2 expression was analyzed in the RMS at 4 dpe. GFP-expressing cells
showed significantly less Zeb2 immunoreactivity when miR-200-gof was present (Fig. 4b,c). Moreover, transgenic
co-expression of Zeb2 rescued the miR-200 mediated induction in calretinin expression (Fig. 4d). Altogether,
these data strongly indicated that the miR-200 induced increase in neuronal differentiation in the RMS was medi-
ated by inhibition of Zeb2. y
Finally, we used mouse genetics to further reinforce the link between Zeb2 expression and calretinin induc-
tion in postnatal OB neurogenesis. Gsh2-Cre mice target Cre recombinase to SVZ-progenitors that generate
neurons for the OB35. We crossed Gsh2-Cre mice to Zeb2-floxed animals36 and analyzed calretinin expression
in system. While the periventricular region of control mice was almost devoid of calretinin immunoreactiv-
ity at P5, Gsh2Cre/Zeb2fl mice showed significantly increased numbers of calretinin positive cells surrounding
the lateral ventricles and extending into the RMS (Fig. 4e,f). We isolated the Cre-targeted cells by FACS of the
micro-dissected SVZ at P2 from control and mutant forebrains, and measured the expression level of calretinin
by RT-qPCR. Discussion In cancer cells the interaction between miR-200 and Zeb proteins is a key regulatory event in the control of
epithelial-mesenchymal transition (EMT) and has been extensively implicated in the metastasis of different can-
cer types. A role in regulation of developmental processes has been repeatedly proposed, but so far not been
demonstrated30. Here we show for the first time that this regulatory pathway is active in vivo to control a develop-
mental process, the maturation of new neurons. MicroRNAs appear to be particularly abundant and strongly regulated in the developing and adult brain
as concluded from microRNA profiling in crude extracts of total brain tissue or specific structures like cortex
at successive embryonic and post-natal stages37–40. Moreover, several specific microRNAs were shown to regu-
late neurogenesis in vivo. For example miR-9 and miR-124 are general regulators of the neurogenic process12,41
whereas other microRNAs were shown to regulate specific steps during neurogenesis (for review: refs 2 and 42). Finally few microRNAs have been shown to regulate neuronal fate decisions, as exemplified by miR-7a and miR-
17-3p13,14,43. However, our understanding of the in vivo role of the microRNA pathway in neurogenesis is still
limited.h This limitation is mostly due to technical issues, since the particular molecular structure of microRNAs ren-
ders traditional approaches difficult. For example, the lack of polyA-tails prevents the use of classical linear ampli-
fication protocols and therefore genomic analyses with limited amounts of material. Moreover, the small size and
the strong sequence homology between microRNA molecules makes in situ hybridization experiments more
problematic. Therefore, a global representation of dynamic microRNA expression along neuron differentiation
from neural stem cell to mature neurons has, to the best of our knowledge, not been reported. g
p
Here we use the unique features of postnatal OB neurogenesis to investigate the expression of microRNAs dur-
ing neurogenesis at high resolution. Indeed, during postnatal neurogenesis in the OB, the main neurogenic stages
are spatially distinct and can be physically isolated: the VZ-SVZ region contains mitotic progenitors, post-mitotic
neuroblasts migrate in the RMS and young neurons terminally differentiate and integrate into the OB circuitry18. Our deep sequencing approach described the dynamics and regionalization of all known microRNAs dur-
ing the different phases of the forebrain neurogenic process. Among the microRNAs expressed in the system
some appeared stably expressed, whereas other are tightly regulated. Results
i
RN The genetic inactivation of Zeb2 resulted in a more than 14-fold up-regulation of calretinin mRNA
(p = 0.0041, n = 6, Fig. 4g), suggesting that it induced the premature maturation of OB interneurons. We con-
clude that regulation of Zeb2 by miR-200 family microRNAs regulates neuronal maturation during postnatal
neurogenesis. Discussion We focused our functional analysis on the Here we use the unique features of postnatal OB neurogenesis to investigate the expression of microRNAs dur-
ing neurogenesis at high resolution. Indeed, during postnatal neurogenesis in the OB, the main neurogenic stages
are spatially distinct and can be physically isolated: the VZ-SVZ region contains mitotic progenitors, post-mitotic
neuroblasts migrate in the RMS and young neurons terminally differentiate and integrate into the OB circuitry18. g
y
g
yf
g
y
Our deep sequencing approach described the dynamics and regionalization of all known microRNAs dur-
ing the different phases of the forebrain neurogenic process. Among the microRNAs expressed in the system
some appeared stably expressed, whereas other are tightly regulated. We focused our functional analysis on the Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 7 www.nature.com/scientificreports/ Figure 4. miR-200 induces calretinin expression through Zeb2 inhibition. (a) Zeb2 mRNA (red) is widely
expressed in the forebrain with particularly prominent presence in the SVZ and RMS. (b) Images showing
GFP cells in the RMS stained with Zeb2 antibody 4 days after in vivo electroporation in control or miR-200-
gof conditions. (c) Quantification of mean Zeb2 staining intensity 4 days after in vivo electroporation cells at
4 dpe in control or miR-200-gof conditions. This showed a significant reduction in Zeb2 protein expression
in neuronal precursors, regardless of their calretinin expression status. Differences between groups of cells
were analyzed pairwise with a t-test (control vs miR-200 calretinin positive P < 2.2e-16; control vs miR-200
calretinin negative P < 2.2e-16); n = number of cells used for analysis; an = number of animals from which
analyzed cells were issued. (d) Zeb2 expression normalizes the miR-200-gof mediated induction in calretinin
expression. Differences between groups were analyzed pairwise with the Man and Whitney test (control (n = 5
animals) vs miR-200 (n = 5 animals) P = 0.008816, miR-200 (n = 5 animals) vs miR-200 + Zeb2 (n = 7 animals)
P = 0.04236). (e) Calretinin immunostaining of coronal forebrain section through the SVZ of Gsh2-Cre;
Zeb2+/+ (wt) or Gsh2-Cre; ZebFl/Fl knockout (Zeb2 −/−) animals at P5 at the level indicated in the schema. (f) The number of calretinin immunoreactive cells in the aSVZ. is much higher in knockout (Zeb2 −/−) than in
control (wt) animals. Discussion (b) Images showing
GFP cells in the RMS stained with Zeb2 antibody 4 days after in vivo electroporation in control or miR-200-
gof conditions. (c) Quantification of mean Zeb2 staining intensity 4 days after in vivo electroporation cells at
4 dpe in control or miR-200-gof conditions. This showed a significant reduction in Zeb2 protein expression
in neuronal precursors, regardless of their calretinin expression status. Differences between groups of cells
were analyzed pairwise with a t-test (control vs miR-200 calretinin positive P < 2.2e-16; control vs miR-200
calretinin negative P < 2.2e-16); n = number of cells used for analysis; an = number of animals from which
analyzed cells were issued. (d) Zeb2 expression normalizes the miR-200-gof mediated induction in calretinin
expression. Differences between groups were analyzed pairwise with the Man and Whitney test (control (n = 5
animals) vs miR-200 (n = 5 animals) P = 0.008816, miR-200 (n = 5 animals) vs miR-200 + Zeb2 (n = 7 animals)
P = 0.04236). (e) Calretinin immunostaining of coronal forebrain section through the SVZ of Gsh2-Cre;
Zeb2+/+ (wt) or Gsh2-Cre; ZebFl/Fl knockout (Zeb2 −/−) animals at P5 at the level indicated in the schema. (f) The number of calretinin immunoreactive cells in the aSVZ. is much higher in knockout (Zeb2 −/−) than in
control (wt) animals. (g) qRT-PCR analysis in FACS sorted SVZ cells from P2 animals reveals a massive increase
in calretinin mRNA expression in knockout (Zeb2 −/−) compared to control (wt) animals. In (f,g) difference
between groups was analyzed using t- test. Scale bars: 1 mm in a, 20 μm in (b) 200 μm in (e). miR-200 family. All members of this family, despite being coded by two independent loci, are induced and func-
on at late stages of the OB neurogenic process. g
g
p
miR-200 family members are major regulators of tumorigenesis, notably through the capacity to inhibit the
transcription factors Zeb1 and Zeb2, two major factors controlling epithelial–mesenchymal-transition44–47. Our
immunohistological analyses and in situ data (the Allen Brain project) demonstrate strong Zeb2 expression in
the forebrain neurogenic system. Moreover, both, Zeb2 loss-of-function and miR-200 gain-of-function led to a
comparable phenotype, the premature expression of the late neuronal subtype marker calretinin. Two alternative explanations can be proposed for this observation. First, repression of Zeb2 by miR-200
microRNAs has a direct impact on differentiation of at least a subfraction of neuronal progenitors. Discussion (g) qRT-PCR analysis in FACS sorted SVZ cells from P2 animals reveals a massive increase
in calretinin mRNA expression in knockout (Zeb2 −/−) compared to control (wt) animals. In (f,g) difference Figure 4. miR-200 induces calretinin expression through Zeb2 inhibition. (a) Zeb2 mRNA (red) is widely
expressed in the forebrain with particularly prominent presence in the SVZ and RMS. (b) Images showing
GFP cells in the RMS stained with Zeb2 antibody 4 days after in vivo electroporation in control or miR-200-
gof conditions. (c) Quantification of mean Zeb2 staining intensity 4 days after in vivo electroporation cells at
4 dpe in control or miR-200-gof conditions. This showed a significant reduction in Zeb2 protein expression
in neuronal precursors, regardless of their calretinin expression status. Differences between groups of cells
were analyzed pairwise with a t-test (control vs miR-200 calretinin positive P < 2.2e-16; control vs miR-200
calretinin negative P < 2.2e-16); n = number of cells used for analysis; an = number of animals from which
analyzed cells were issued. (d) Zeb2 expression normalizes the miR-200-gof mediated induction in calretinin
expression. Differences between groups were analyzed pairwise with the Man and Whitney test (control (n = 5
animals) vs miR-200 (n = 5 animals) P = 0.008816, miR-200 (n = 5 animals) vs miR-200 + Zeb2 (n = 7 animals)
P = 0.04236). (e) Calretinin immunostaining of coronal forebrain section through the SVZ of Gsh2-Cre;
Zeb2+/+ (wt) or Gsh2-Cre; ZebFl/Fl knockout (Zeb2 −/−) animals at P5 at the level indicated in the schema. (f) The number of calretinin immunoreactive cells in the aSVZ. is much higher in knockout (Zeb2 −/−) than in
control (wt) animals. (g) qRT-PCR analysis in FACS sorted SVZ cells from P2 animals reveals a massive increase
in calretinin mRNA expression in knockout (Zeb2 −/−) compared to control (wt) animals. In (f,g) difference
between groups was analyzed using t- test. Scale bars: 1 mm in a, 20 μm in (b) 200 μm in (e). Figure 4. miR-200 induces calretinin expression through Zeb2 inhibition. (a) Zeb2 mRNA (red) is widel
d
h f
b
h
l l
h
Z
d
(b)
h Figure 4. miR-200 induces calretinin expression through Zeb2 inhibition. (a) Zeb2 mRNA (red) is widely
expressed in the forebrain with particularly prominent presence in the SVZ and RMS. Material and Methods Mouse lines. Mice carrying floxed Zeb2 alleles (Zeb2fl/fl)54 were crossed to the RCE reporter mice55. Resulting
progeny was subsequently crossed with Gsh2-Cre mice56 to generate Gsh2-Cre; RCE; Zeb2fl/fl mutant mice
or Gsh2-Cre; RCE; Zeb2fl/wt control mice. Animal experiments were carried out in accordance to European
Communities Council Directive and approved by French ethical committees (Comité d’Ethique pour l′expéri-
mentation animale n°14; permission number: 62-12112012). Plasmid constructs. The pCX-Cre and pCX-GFP vectors are derived from pCX-MCS257. To generate the vec-
tor expressing gfp under the control of the human miR-200b/miR-200a/miR-429 regulatory sequence we subcloned
gfp from pCX-GFP into the pGL3-1574/ + 120 vector obtained from addgene. The miR-200 expression vector
(miR-200-gof) was generated by PCR amplification of both miR-200 clusters from CD1 mouse genomic DNA and
sub-cloning of amplified fragments into pCX-MCS2. The sponge construct was designed according to58 with 4 rep-
etitions of 2 oligonucleotides (5′-GACACATCGTTACTCTCAGTGTTAGACACGGCATTACTCTCAGTATTA
and 5′-GACTTCATCATTACTCCCAGTATTAGACCCATCTTTACTCTCAGTGTTA) partially complementary
to any member of the miR-200 family were placed behind a destabilized GFP gene in pCX-d2-GFP plasmid. Zeb2 3′UTR was PCR-amplified from CD1 mouse brain cDNA and cloned into the pMir-Glo vector
(Promega) to generate the 3′UTR-Zeb2 pmiRGlo vector. y
y
p
g
p
p
Zeb2 3′UTR was PCR-amplified from CD1 mouse brain cDNA and cloned into the pMir-Glo vector
Promega) to generate the 3′UTR-Zeb2 pmiRGlo vector. RNA extraction and deep sequencing. Total RNA was extracted from CD1 mice using the miRNeasy kit
(Qiagen). RNA was extracted from dorsal or lateral VZ-SVZ at P1 and P6, from RMS at P15 and P28 and from
OB at P15 and P28. All samples were dissected in triplicate. Deep sequencing analysis were performed on the
15–50 bp RNA molecules using the Applied Biosystems SOLiD™ System. For each sample, results were normal-
ized for each microRNA as number of reads per million. Results were submitted to GEO (GSE60817). Cell sorting (FACS, MACS), qRT-PCR and luciferase assay. For isolation of OB interneurons from
GAD67-GFP knock-in mice59, whole bulbs of P30 animals were dissected and dissociated by Trypsin/DNAse
digestion. GFP cells were purified using MoFlow (Beckman-Coulter) flow cytometer. For Zeb2 mutant analyses,
lateral SVZ of P2 control brains were dissected and dissociated by Papain (Sigma)/DNAse digestion. GFP Cells
(provided by the RCE locus) were sorted using a FACSAriaI (BD Biosciences). (p
y
)
g
(
)
To separate neuronal from glial cells by MACS, OB from P30 CD1 mice were subjected to Trypsin/DNAse
dissociation. Discussion Indeed, in
the developing cortex conditional deletion of Zeb2 induced premature neuronal and glial differentiation48. Such
a role for Zeb2 in the differentiation process would account for the appearance of calretinin positive cells in the
RMS in the context of miR-200 overexpression. It would also explain the lack of differentiation, as measured by
decreased NeuN staining, when miR-200 is inhibited. Second, induction of calretinin expression in the RMS
might be a consequence of slowed neuronal migration. Indeed, it has been shown that interfering with migration Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 8 www.nature.com/scientificreports/ through knockdown of DCX leads to the appearance of calretinin positive cells in the RMS49. Moreover, direct
roles of Zeb2 in in migration of metastatic cancer cells30,50 and cortical interneurons36 have been shown. g
Another question concerns the observation that only a small fraction of neuronal precursors shows altered
differentiation after interference with miR-200 expression. The quantity of NeuN negative cells in the OB after
miR-200 knockdown increases by only 5%, while premature expression of the family induces calretinin in less
than 10% of all transfected cells. It should be noted that such minor alterations, typical for the fine-tuning func-
tion of microRNAs, would likely be missed in the analysis of other neurogenic processes, which do not permit
the same high-resolution analysis. The limited effects might be due to the fact that only a subfraction of the
transfected cells are responsive to either inhibition or increase of miR-200 microRNAs. Our finding that the
miR-200 promoter fragment that we used to drive GFP expression is only active in a small fraction of the trans-
fected neurons supports this potential lack of competence. Alternatively, it is possible that other microRNAs have
redundant functions in the system. In line with this idea, we found that the miR-183/96/182 cluster, a group of
microRNAs that has been implicated in the maintenance of retinal neuron integrity51,52 and shares common pre-
dicted targets53, appears as nearest neighbors with the miR-200 family in our heat map representation (Fig. 2a,b). Functional studies using tools targeting both groups of microRNAs in the forebrain compartments will be nec-
essary to address this issue. References References
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b ll
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RNA J E
M d
(
) ,
Rf
p
progenitors during early neurogenesis in the mouse embryonic neocortex. Development 135, 3911–3921 (2008). 7. Schaefer, A. et al. Cerebellar neurodegeneration in the absence of microRNAs. J Exp Med 204, 1553–1558 (2007). . Schaefer, A. et al. Cerebellar neurodegeneration in the absence of g
p
8. Zhao, C., Sun, G., Li, S. & Shi, Y. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cel
fate determination. Nat Struct Mol Biol 16, 365–371 (2009).h 9. Packer, A. N., Xing, Y., Harper, S. Q., Jones, L. & Davidson, B. L. The bifunctional microRNA miR-9/miR-9* regulates REST and
CoREST and is downregulated in Huntington’s disease. J Neurosci 28, 14341–14346 (2008). Material and Methods was performed using ImageJ software on a single z-plan focused on the nucleus (chosen using DAPI staining). A ROI was subsequently drawn inside the nucleus area and the mean intensity of Zeb2 staining signal was then
measured across the ROI. Material and Methods Both neuronal and glial enriched fractions were recovered from single cell suspension containing
approximately 1 × 106 cells using the “Neuron Isolation Kit” (Miltenyi). RNAs were extracted from sorted cells using the mRNeasy or miRNeasy kit (Qiagen). cDNAs were prepared
using superscript-III (Life-Technologies) and qPCR was performed using SYBR-GreenER qPCR SuperMix
(Life-Technologies), except for Zeb2KO sorted cells which were performed on a LightCycler 480 Instrument
(Roche) using SYBR Green PCR Master Mix (Roche). Beta-Actin was used as reference gene. Primers sequences
are given in SI. MicroRNA qPCR was performed using LNA-qRT-PCR system from Exiqon and using U6 as
reference gene.t g
Luciferase assay was performed on HeLa cells 48 h after Lipofectamine 2000 (Life-Technologies) mediated
transfection using the Dual-Luciferase Reporter Assay (Promega) and a Luminometer (Berthold Technologies). Immunohistochemistry and Image analysis. Brain sections and staining experiments were performed
as in19 except those performed on Gsh2-Cre; RCE; Zeb2fl/fl mice brains processed as in48. Primary antibodies
used are: Calretinin (rabbit, Swant, 1/1000), GFP (chicken, Aves, 1/500), mouse IgG1 anti-NeuN (Millipore,
1:100), rat Igg2a anti-BrdU (AbD Serotec (Oxford B), 1/1000). Images were taken using a fluorescence micro-
scope (Axiolmager Z1, ApoTome system, Zeiss) except for Gsh2-Cre; RCE; Zeb2fl/fl sections (Leica DMR micro-
scope) and for spine density measurement (laser confocal scanning microscope, LSM510, Zeiss - magnification:
63x). Data in graphics are presented as mean ± s.e.m of values obtained on n samples (*P < 0,05. **P < 0,01,
***P < 0,001). For BrdU incorporation analysis, animals at 2 dpe were injected once with a BrdU solution (50 μg/g
body weight, Sigma, Saint-Louis MO) 2 hours before perfusion. BrdU staining was performed after 15 min
incubation at 37° in 2N HCl-0.5%. In Fig. 4c, Zeb2 expression level per transfected cell was assessed as fol-
lows. Transfected cells in the RMS were identified based on GFP expression. Quantification of Zeb2 staining Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 9 www.nature.com/scientificreports/ was performed using ImageJ software on a single z-plan focused on the nucleus (chosen using DAPI staining). A ROI was subsequently drawn inside the nucleus area and the mean intensity of Zeb2 staining signal was then
measured across the ROI. was performed using ImageJ software on a single z-plan focused on the nucleus (chosen using DAPI staining). A ROI was subsequently drawn inside the nucleus area and the mean intensity of Zeb2 staining signal was then
measured across the ROI. References d l
l
f
d
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forebrain]. Med Sci (Paris) 29, 153–155 (2013). Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 10 © The Author(s) 2016 Additional Informationi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Beclin, C. et al. miR-200 family controls late steps of postnatal forebrain neurogenesis
via Zeb2 inhibition. Sci. Rep. 6, 35729; doi: 10.1038/srep35729 (2016). How to cite this article: Beclin, C. et al. miR-200 family controls late steps of postnatal forebrain neurogenes
via Zeb2 inhibition. Sci. Rep. 6, 35729; doi: 10.1038/srep35729 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:35729 | DOI: 10.1038/srep35729 11 www.nature.com/scientificreports www.nature.com/scientificreports Corrigendum: miR-200 family
controls late steps of postnatal
forebrain neurogenesis via Zeb2
inhibition
Christophe Beclin, Philipp Follert, Elke Stappers, Serena Barral, Nathalie Coré,
Antoine de Chevigny, Virginie Magnone, Kévin Lebrigand, Ute Bissels, Danny Huylebroeck,
Andreas Bosio, Pascal Barbry, Eve Seuntjens & Harold Cremer
Scientific Reports 6:35729; doi: 10.1038/srep35729; published online 21 October 2016; updated on 21 December
2016 Scientific Reports | 6:39368 | DOI: 10.1038/srep39368 Corrigendum: miR-200 family
controls late steps of postnatal
forebrain neurogenesis via Zeb2
inhibition Christophe Beclin, Philipp Follert, Elke Stappers, Serena Barral, Nathalie Coré,
Antoine de Chevigny, Virginie Magnone, Kévin Lebrigand, Ute Bissels, Danny Huylebroeck,
Andreas Bosio, Pascal Barbry, Eve Seuntjens & Harold Cremer The original version of this Article contained a typographical error in the spelling of the author Nathalie Coré,
which was incorrectly given as Coré Nathalie. This has now been corrected in the PDF and HTML versions of
the Article. This work is licensed under a Creative Commons Attribution 4.0 International License. The images
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users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016
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https://openalex.org/W4384830638
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https://sciendo.com/pdf/10.2478/hppj-2023-0009
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Behavioral and histopathological changes of <i>Clarias gariepinus</i> as a predatory fish against <i>Culex pipiens</i> larvae following exposure to sublethal concentration of quinclorac and bensulfuron-methyl based herbicide
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Hellenic Plant Protection Journal
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cc-by
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1 Plant Protection Department, Faculty of Agriculture,
Assiut University, Assiut 71526, Egypt.
2 Pathology Department, Faculty of Veterinary Medi-
cine, Assiut University, Assiut 71526, Egypt.
3 Fish Diseases and Management Department, Facul-
ty of Veterinary Medicine, Assiut University, Assiut
71526, Egypt.
* Corresponding author: ibrahim.mahmoud@agr.aun.
edu.eg Behavioral and histopathological changes of Clarias gariepinus
as a predatory fi sh against Culex pipiens larvae following
exposure to sublethal concentration of quinclorac and
bensulfuron-methyl based herbicide Behavioral and histopathological changes of Clarias gariepinus
as a predatory fi sh against Culex pipiens larvae following
exposure to sublethal concentration of quinclorac and
bensulfuron-methyl based herbicide I.A. Mohamed1,*, M. Fathy1, A.I.A. Farghal1, S.A.H. Temerak1, S.Kh. Abd El-Ghaff ar2 and
S.K.A. Idriss3 I.A. Mohamed1,*, M. Fathy1, A.I.A. Farghal1, S.A.H. Temerak1, S.Kh. Abd El-Ghaff ar2 and
S.K.A. Idriss3 Summary Clarias gariepinus is one of the widespread culturable freshwater fi sh species in Africa,
which is prevalent in various natural and human-made aquatic habitats including rice-fi sh system. This
fi sh species displays predation potential on the aquatic stages of mosquitoes. Bensulfuron-methyl and
quinclorac are herbicide active substances that have been extensively applied in rice culture in Egypt
and other countries worldwide. This study assessed the adverse eff ects of sublethal concentration of
a commercial herbicide formulation containing quinclorac and bensulfuron-methyl on the predation
potential of C. gariepinus female and male predatory fi sh on Culex pipiens mosquito larvae. Also, stom-
ach and intestine histopathology of the treated fi sh was investigated. The exposure of C. gariepinus to
sublethal concentration of quinclorac and bensulfuron-methyl based herbicide produced detrimen-
tal eff ects on prey consumption and histopathological changes in the stomach and intestine of the
fi sh. The mosquito consumption by the treated female and male fi sh decreased signifi cantly compared
to the untreated fi sh of both sexes. The histological changes in the intestines were hyperplasia of the
intestinal epithelium and goblet cells; edema of lamina propria and broad intestinal villi, and distor-
tion in intestinal villi in comparison to control. The stomach histopathology changes were necrosis
and sloughing of mucosal epithelium with severe damage of sub-mucosa. Thus, the tested herbicide
at sublethal concentration on C. gariepinus decreased the prey consumption on mosquito larvae and
caused histopathological alterations in the fi sh that may impair its digestive physiology. These fi nd-
ings suggest a threat of the tested herbicide to C. gariepinus survival and potential as a native success-
ful biocontrol agent against Cx. pipiens larvae. Additional keywords: Clarias gariepinus, Culex pipiens, herbicide, histopathology, predatory fi sh, prey Hellenic Plant Protection Journal 16: 67-78, 2023
DOI 10.2478/hppj-2023-0009 Introduction gariepinus against mosquito larvae was
signifi cantly aff ected by the mosquito gen-
era, larvae number exposed, hours of feed-
ing, and the size of predatory fi sh. Interest-
ingly, predation of larvivorous fi shes is also
negatively impacted by the presence of tox-
icant chemical residues, mainly pesticides
(insecticides and herbicides) in their aquat-
ic habitats (Kerby et al., 2012). Sublethal con-
centrations of diff erent chemical pesticides
induced larger adverse eff ects on the be-
havior of predatory fi sh than on their prey
(Kerby et al., 2012). For example, the insec-
ticide diazinon at sub-lethal concentrations
signifi cantly reduced the activity and attack
rates of Gambusia affi nis (Baird and Girard)
on Pseudacris regilla (Baird and Girard) tad-
pole prey which likely refl ects the negative
eff ects of the pesticide on the predatory be-
havior of G. affi nis (Kerby et al., 2012). Also,
several herbicides produced adverse eff ects
on diff erent freshwater fi sh such as chang-
es in mobility, feeding, orientation behavior,
enzymes, biochemical and hematological
modifi cations, histopathological disturbanc-
es, oxidative stress, genotoxicity, endocrine
toxicity, and neurotoxicity (Ullah et al., 2014;
Stanley and Preetha, 2016; Fathy et al., 2019;
Saleh et al., 2022). Clarias gariepinus is a native African
freshwater fi sh, which is prevalent in various
natural and human-made aquatic habitats
or even in sewage systems in urban regions
worldwide, especially in African and Asian
countries (Ponzoni and Nguyen, 2008). It is
an omnivore fi sh that regularly feeds on a
large variety of aquatic invertebrates, aquat-
ic insect larvae and pupae, small fi shes, al-
gae, and aquatic plants (Ghosh et al., 2008;
Ponzoni and Nguyen, 2008). It is a hardy
fi sh and tolerant to diffi cult environmental
conditions (e.g., polluted water) (Ponzoni
and Nguyen, 2008). The immature stages of
mosquitoes may occur in aquatic habitats of
C. gariepinus (Gashaw et al., 2008). All these
reasons make C. gariepinus a suitable candi-
date biocontrol agent against aquatic ani-
mal pests, mainly mosquitoes. In Ethiopia,
C. gariepinus has the potential to control the
larvae of Culex spp. and Anopheles arabien-
sis Patton (Chala et al., 2016) and the aquat-
ic snail Biomphalaria pfeiff eri Krauss (Gashaw
et al., 2008). Egypt is the largest producer of vari-
ous edible fi sh mainly O. niloticus, C. gariepi-
nus, and Mugil cephalus L. in Africa (Soliman
and Yacout, 2016). Introduction widely used as exotic predators against
mosquitoes in diff erent aquatic habitats all
over the world (Pyke et al., 2008). However,
negative impacts of Gambusia spp. on sev-
eral aquatic benefi cial invertebrates, am-
phibians, and other native fi sh species have
been recorded in many countries (Pyke et
al., 2008). This has led researchers to look for
diff erent indigenous predatory fi sh species
in local aquatic environments in Africa, Asia,
and South America (Ghosh and Dash, 2007;
Chandra et al., 2008). Therefore, about 315
diff erent fi sh species, from many diff erent
countries, have been recorded as being ef-
fective and potentially suitable indigenous
predatory fi sh for mosquito control (Ghosh
and Dash, 2007). For example, many edible
fi sh such as Clarias gariepinus (Burchell), Ore- Several fi sh species have been used success-
fully as eff ective, low-cost, and eco-friendly
biocontrol approaches against aquatic stag-
es of mosquitoes (Chandra et al., 2008; Bhat-
tacharjee et al., 2009). Since 1900, Gambusia
spp. and Poecilia spp. are native larvivorous
fi sh in American regions and have been © Benaki Phytopathological Institute Mohamed et al. 68 ochromis mossambica (Peters), Oreochromis
spilurus (Gunther), Oreochromis niloticus L.,
Tilapia zillii (Gervais), and Ctenopharyngdon
idella (Valenciennes) are farmed in natural
habitats or human-made aquatic culture for
human consumption and they also provide
eff ective control of mosquito larvae and pu-
pae of Anopheles, Aedes, and Culex species
(Chandra et al., 2008; Chala et al., 2016; Abe-
be et al., 2018; Das et al., 2018; Mohamed et
al., 2021). humans and is the most widely distributed
mosquito in temperate regions worldwide,
including Egypt (Mohamed et al., 2021).i ochromis mossambica (Peters), Oreochromis
spilurus (Gunther), Oreochromis niloticus L.,
Tilapia zillii (Gervais), and Ctenopharyngdon
idella (Valenciennes) are farmed in natural
habitats or human-made aquatic culture for
human consumption and they also provide
eff ective control of mosquito larvae and pu-
pae of Anopheles, Aedes, and Culex species
(Chandra et al., 2008; Chala et al., 2016; Abe-
be et al., 2018; Das et al., 2018; Mohamed et
al., 2021). Predation of larvivorous fi shes may be
aff ected by diff erent biotic and abiotic fac-
tors, which are related to the fi sh (preda-
tor), mosquito (prey), and its aquatic hab-
itats (Yildirim and Karacuha, 2007; Chala et
al., 2016; Mohamed et al., 2021). Chala et al. (2016) indicated that the predation effi cacy
of C. Culex pipiens larvae Field populations of Cx. pipiens larvae
were collected from the sewage treatment
plant in the Arab El-Madabegh region, As-
siut city, Egypt. The samples were trans-
fered to the laboratory and kept at 25 ± 1°C. The collected Cx. pipiens larvae were fed on
a diet containing fi ne ground dry bread and
yeast and then late third to early fourth in-
star larvae were used in this study. Introduction In Egypt, rice-fi sh farming
produced about 51.31% of O. niloticus and
29.93% of C. gariepinus from the total pro-
duction of rice-fi sh system in 2012 (Soliman
and Yacout, 2016). However, according to
the best of our knowledge, limited studies
have been published assessing the predato-
ry effi ciency of C. gariepinus against aquat-
ic stages of mosquitoes, and no studies on
the mosquito Culex pipiens L. Culex pipiens is
a main effi cient vector of lymphatic fi laria-
sis worms and several serious arboviruses to Bensulfuron-methyl (sulfonylurea) and
quinclorac (quinoline) are among the most
commonly and widely applied selective
herbicides in rice, various agricultural crops,
and turfgrass lands worldwide. The residues
of both herbicides are persistent in diff erent
aquatic systems that may result in adverse
eff ects on aquatic biota, mainly fi sh (Okamo-
to et al., 1998). Bensulfuron-methyl has been
detected in water of rice fi elds at a range
of <0.01–139.97 μg/L (Parveen et al., 2005), © Benaki Phytopathological Institute Sublethal eff ect of herbicide on the mosquito predator Clarias gariepinus 69 Faculty of Agriculture, Assiut University. The
female and male fi sh were stocked separate-
ly in 70 L boxes and acclimated for 4 weeks
under laboratory conditions at 25 ± 1°C and
12:12 h (L:D) photoperiod. while quinclorac was present at 1.34–6.97
μg/L (Resgalla et al., 2007). Recently, both
herbicides are mixed in a post-emergence
herbicide (trade name Repare®), which has
also been extensively applied in rice culture
in Egypt and other countries worldwide (APC
2021). Exposure of O. niloticus to a sublethal
dose of bensulfuron-methyl and quinclorac
individually altered the behavior of fi sh, in-
duced oxidative stress, and damaged the liv-
er and spinal cord (Fathy et al., 2019; Saleh et
al., 2022). The 96-h exposure of silver catfi sh,
Rhamdia quelen to LC50 of quinclorac (395
mg/L) elicited adverse behavioral changes,
oxidative stress, biochemical alternations,
and induced histological lesions in the liver,
kidney, and gill on the fi sh (Miron et al., 2005;
Persch et al., 2017; Persch et al., 2018). Histo-
pathological examination variables are fre-
quently used as potential biomarkers in the
pesticide toxicological studies to provide
information about organ damage and the
physiological, functional, and health status
of fi sh after exposure to these toxic agents
(Vander Oost et al., 2003; Fathy et al., 2019;
Saleh et al., 2022). C. Determination of sublethal concentra-
tion of herbicide on C. gariepinus The tested commercial mixture her-
bicide formulation was Repare® 18% TB
(16.5% quinclorac and 1.5% bensulfuron-
methyl; Starchem Chemical Manufacturing,
Egypt). A preliminary experiment was con-
ducted to defi ne the sub-lethal concentra-
tion of the herbicide on C. gariepinus fi sh. In this experiment C. gariepinus fi sh (72 fi sh,
28.5–30.0 cm and 167–180 g) were divided
to six experimental groups (12 fi sh/group),
the non-exposed fi sh (control) group and
fi ve experimental groups exposed to dif-
ferent concentrations of the tested quinclo-
rac and bensulfuron-methyl herbicide. Each
group of C. gariepinus fi sh was kept in three
separate 70 L boxes (4 fi sh/box). In the non-
exposed control group, the fi sh remained
in water, while in the herbicide treatment
groups, the fi sh were exposed to 390, 780,
1560, 3120 and 6240 mg of Repare® 18% TB/L
water for 96 hours. Fish were maintained in
the laboratory at the same conditions as de-
scribed above. Mortality was recorded ev-
ery 24 hours over 96 h and dead fi sh were
removed from boxes immediately. Finney’s
probit analysis (Finney, 1971) was used to es-
timate lethal concentration (LC50 and LC90)
values of C. gariepinus after 96 h exposure
using SPSS software version 16.0 (SPSS Inc.,
Chicago, IL, USA, 2). This study was designed in order to eval-
uate the eff ects of sublethal concentration of
a quinclorac and bensulfuron-methyl based
commercial herbicide on the predation po-
tential of C. gariepinus against Cx. pipiens lar-
vae under laboratory conditions and to as-
sess the histopathological changes in the
stomach and intestine of the fi sh. Introduction gariepinus is continu-
ously exposed to a great range of pesticide
residues (e.g. herbicides), in its aquatic habi-
tats, especially present in or surrounding ag-
ricultural regions like rice-fi sh culture (Cha-
la et al., 2016). © Benaki Phytopathological Institute Histopathological studies i After the acclimatization, eighteen fe-
males (30.0–30.76 cm and 180.00 ± 2.77 g)
and eighteen males (29.50–31.00 cm and
162.5 ± 2.51 g) of C. gariepinus were selected
for the experiments. The aforementioned
female and male fi sh were divided into two
groups (9 fi sh/sex/group) as follows: the
fi rst group was the non-exposed fi sh (con-
trol) and the second group was exposed to
the determined sub-lethal concentration of
quinclorac and bensulfuron-methyl based
mixture herbicide in water (390 mg of Re-
pare® 18% TB/L) for 15 days and placed in 70
L boxes. The water in boxes was changed ev-
ery 48 h and the herbicide was again add-
ed to maintain the concentration constant
as described by Doherty et al. (2016) and
Hamed and Osman (2017). Fish were fed
once a day with commercial dry food pel-
lets (25% crude protein, Al-Salam Compa-
ny, Egypt). On the day 14 of exposure, all C. gariepinus fi sh in all groups were left over
without food for 24 h to starve. After the ex-
posure period and before the predation ex-
periment, the treated fi sh of each sex were
transferred carefully to separate boxes con-
taining clean water to wash the fi sh off her-
bicide before starting the predation exper-
iments. Each fi sh was placed in 8 L water
with 600 mosquito larvae for 12h, from 7:30
a.m. to 7:30 p.m. (day time predation exper-
iment) and then the same fi sh was trans-
ferred to new box with fresh 8 L water and
new batch of 600 larvae from 7:30 p.m. to
7:30 a.m. (night time predation experiment). This procedure was repeated every 12 hours
for a total period of 5 days (120 hours). g
Three C. gariepinus fi sh from each of the
pre-treated and untreated (control) groups
were randomly selected after 15 days of ex-
posure. Tissue samples of the stomach and
intestine were anatomically dissected from
each fi sh in both groups, rapidly removed,
washed with neutral saline, and fi xed in neu-
tral buff ered formalin 10%. Post-fi xed tissue
specimens of each group were dehydrated in
a graded alcohol series, cleared with methyl
benzoate, and then embedded in a paraffi n
wax (Bancroft and Stevens, 1982). Thin sec-
tions were cut at 5 μm thickness and stained
with the hematoxylin–eosin stain (HE) tech-
nique (Bancroft et al., 1996). Collection and maintenance of the fi sh
Clarias gariepinus i Females and males of C. gariepinus fi sh
were collected from a private local fi sh farm
in Assiut Governorate, Egypt, and trans-
ferred to the Environmental Toxicology lab-
oratory in the Plant Protection Department, 70 Mohamed et al. between treated and untreated group at a=
0.05 signiffi cance level. Predatory potential of C. gariepinus fi sh
on Culex pipiens larvae after exposure to
sublethal concentration of herbicide Histopathological studies i All sections in
each group were histopathologically exam-
ined and photographed using an Olympus
CH30 microscope. Sublethal concentration of tested herbi-
cide on C. gariepinusi g
p
All fi sh exposed to 3120 and 6240 mg Re-
pare® 18%/L water died within 24 h, but for
those exposed to 390 and 780 mg of the her-
bicide there was no mortality after 24 h and
even after 96 h of treatment, respectively. The concentration of 1560 mg/L produced
41.67% mortality after 24 h and 96 h of treat-
ment. The LC50 of the herbicide against C. gariepinus at 96 h was 1607 mg/L (Table 1). The selected sublethal concentration of Re-
pare® herbicide in our study was 390 mg/L,
which is almost the 1/4 LC50 value at 96 h
and approximately 1/27 from the recom-
mended application rate (10416.67 mg Re-
pare® 18%/L) of the herbicide for rice weeds
in Egypt (APC, 2021). The total number of Cx. pipiens larvae
consumed by both pre-treated and un-
treated female and male fi sh groups was
recorded every 12 h over 120 hours. The
means of Cx. pipiens larvae consumption by
pre-treated and untreated female and male
C. gariepinus fi sh groups were estimated and
expressed as mean ± SE. The t-test for inde-
pendent samples was performed to com-
pare the mean number of consumed larvae Predatory potential of C. gariepinus fi sh
on Culex pipiens larvae after exposure to
sublethal concentration of herbicide © Benaki Phytopathological Institute Predatory potential of C. gariepinus fi sh
on Culex pipiens larvae after exposure to
sublethal concentration of herbicide The data regarding the predatory ef-
fi cacy of the treated female and male of C. Sublethal eff ect of herbicide on the mosquito predator Clarias gariepinus 71 gariepinus fi sh after exposure to subleth-
al concentration of quinclorac and bensul-
furon-methyl based herbicide (390 mg Re-
pare® 18%/L) and the control against third
to fourth -instar larvae of Cx. pipiens under
laboratory conditions are showed in Table
2. The sublethal concentration of the test-
ed herbicide aff ected negatively the feed-
ing potential of the female and male of C. gariepinus. The daily consumption (sum of
day and night time records) of pre-treated
and untreated females of C. gariepinus on Cx. pipiens larvae was 669.17 ± 3.72 and 1018.17
± 10.27 larvae, respectively, i.e., signifi cantly
reduced by 34.28%. Also, the daily consump-
tion of pre-treated and untreated males of C. gariepinus on Cx. pipiens larvae was 471.25 ±
10.78 and 595.92 ± 24.92 larvae, respective-
ly, i.e., signifi cantly reduced by 20.92%. The
Table 1. Lethal concentrations (LC50 and LC90)
rac and bensulfuron-methyl based herbicide
exposure. Value
Concentration (mg/L)
Lo
LC50
1607.0
154
LC90
1941.0
177
Table 2. Predation of untreated and treated fe
posure with sublethal concentration of quinclo
(390 mg Repare® 18%/L water) for 15 days aga
Fish sex
Treatment
Mean Fish
size (±SE) (cm)
Mean Fish
weight (±SE) (
Female
Untreated
30.00 ± 1.38
180.00 ± 2.77
Treated
30.76 ± 0.33
180.00 ±2.77
P-value
Male
Untreated
29.50 ± 1.50
162.50 ± 2.51
Treated
31.00 ± 1.00
162.50 ± 2.51
P-value
Adult
Untreated
29.75 ± 1.44
171.25 ± 2.64
Treated
30.88 ± 0.67
171.25 ± 2.64 gariepinus fi sh after exposure to subleth-
al concentration of quinclorac and bensul-
furon-methyl based herbicide (390 mg Re-
pare® 18%/L) and the control against third
to fourth -instar larvae of Cx. pipiens under
laboratory conditions are showed in Table
2. The sublethal concentration of the test-
ed herbicide aff ected negatively the feed-
ing potential of the female and male of C. gariepinus. The daily consumption (sum of
day and night time records) of pre-treated
and untreated females of C. gariepinus on Cx. pipiens larvae was 669.17 ± 3.72 and 1018.17
± 10.27 larvae, respectively, i.e., signifi cantly
reduced by 34.28%. Also, the daily consump-
tion of pre-treated and untreated males of C. gariepinus on Cx. Predatory potential of C. gariepinus fi sh
on Culex pipiens larvae after exposure to
sublethal concentration of herbicide pipiens larvae was 471.25 ±
10.78 and 595.92 ± 24.92 larvae, respective-
ly, i.e., signifi cantly reduced by 20.92%. The gariepinus fi sh after exposure to subleth-
al concentration of quinclorac and bensul-
furon-methyl based herbicide (390 mg Re-
pare® 18%/L) and the control against third
to fourth -instar larvae of Cx. pipiens under
laboratory conditions are showed in Table
2. The sublethal concentration of the test-
ed herbicide aff ected negatively the feed-
ing potential of the female and male of C. gariepinus. The daily consumption (sum of
day and night time records) of pre-treated
and untreated females of C. gariepinus on Cx. pipiens larvae was 669.17 ± 3.72 and 1018.17
± 10.27 larvae, respectively, i.e., signifi cantly
reduced by 34.28%. Also, the daily consump-
tion of pre-treated and untreated males of C. gariepinus on Cx. pipiens larvae was 471.25 ±
10.78 and 595.92 ± 24.92 larvae, respective-
ly, i.e., signifi cantly reduced by 20.92%. The daily larval consumption by the pre-treat-
ed and untreated C. gariepinus was 570.21 ±
2.68 and 794.65 ± 5.93, respectively, regard-
less the gender (Table 2), hence, the subleth-
al concentration of the herbicide reduced
the consumption potential of C. gariepinus
on Cx. pipiens larvae by 28.23%. Larval consumption by the pre-treated
and untreated female of C. gariepinus was
higher than in the pre-treated male by 1.71
and the untreated male fi sh by 1.42 times. The larval consumption by the pre-treat-
ed and untreated male fi sh was also higher
during the nighttime by 1.91 and 1.71 times
than during the daytime, respectively. Histopathological studies Histopathological studies In the present study, no visible changes
or no pathologic changes were observed in Table 1. Lethal concentrations (LC50 and LC90) along with 95% confi dence limits of quinclo-
rac and bensulfuron-methyl based herbicide (Repare® 18%) on Clarias gariepinus after 96 h
exposure. Value
Concentration (mg/L)
95% confi dence limits
Slope±S.E. Lower
Upper
LC50
1607.0
1542.72
1794.84
0.004 ± 0.001
LC90
1941.0
1770.05
2957.69
0.004 ± 0.001
Table 2. Predation of untreated and treated females and males of Clarias gariepinus after ex-
posure with sublethal concentration of quinclorac and bensulfuron-methyl based herbicide
(390 mg Repare® 18%/L water) for 15 days against third-fourth instar larvae of Culex pipiens. Fish sex
Treatment
Mean Fish
size (±SE) (cm)
Mean Fish
weight (±SE) (g)
Mean (±SE) number of consumed Cx. pipiens
larvae per fi sh
Day time†
Night time†
Daily‡
Female
Untreated
30.00 ± 1.38
180.00 ± 2.77
461.50 ± 3.04
556.67 ± 10.97
1018.17 ± 10.27
Treated
30.76 ± 0.33
180.00 ±2.77
309.67 ± 7.20
359.50 ± 5.79
669.17 ± 3.72
P-value
0.001*
0.0001*
0.0001*
Male
Untreated
29.50 ± 1.50
162.50 ± 2.51
196.38 ± 5.89
374.75 ± 1.25
571.13 ± 4.64
Treated
31.00 ± 1.00
162.50 ± 2.51
174.13 ± 6.64
297.13 ± 4.14
471.25 ± 10.78
P-value
0.013*
0.0001*
0.001*
Adult
Untreated
29.75 ± 1.44
171.25 ± 2.64
328.94 ± 0.39
465.71 ± 5.70
794.65 ± 5.93
Treated
30.88 ± 0.67
171.25 ± 2.64
241.90 ± 2.51
328.31 ± 4.63
570.21 ± 2.68
P-value
0.001*
0.0001*
0.0001*
Signiffi cant diff erences between means using t-test (p < 0.05) are indicated with an asterisk (*). † Day time (from 7.30
am to 7.30 pm) and night time (from 7.30 pm to 7.30 am). ‡ Daily is the sum of day and night time records. Table 1. Lethal concentrations (LC50 and LC90) along with 95% confi dence limits of quinclo-
rac and bensulfuron-methyl based herbicide (Repare® 18%) on Clarias gariepinus after 96 h
exposure. Table 1. Lethal concentrations (LC50 and LC90) along with 95% confi dence limits of quinclo-
rac and bensulfuron-methyl based herbicide (Repare® 18%) on Clarias gariepinus after 96 h
exposure. Value
Concentration (mg/L)
95% confi dence limits
Slope±S.E. Lower
Upper
LC50
1607.0
1542.72
1794.84
0.004 ± 0.001
LC90
1941.0
1770.05
2957.69
0.004 ± 0.001 Table 2. Histopathological studies Predation of untreated and treated females and males of Clarias gariepinus after ex-
posure with sublethal concentration of quinclorac and bensulfuron-methyl based herbicide
(390 mg Repare® 18%/L water) for 15 days against third-fourth instar larvae of Culex pipiens. Mean (±SE) number of consumed Cx pipiens Table 2. Predation of untreated and treated females and males of Clarias gariepinus after
posure with sublethal concentration of quinclorac and bensulfuron-methyl based herbic
390 mg Repare® 18%/L water) for 15 days against third-fourth instar larvae of Culex pipie Table 2. Predation of untreated and treated females and males of Clarias gariepinus after ex-
posure with sublethal concentration of quinclorac and bensulfuron-methyl based herbicide
(390 mg Repare® 18%/L water) for 15 days against third-fourth instar larvae of Culex pipiens. Fish sex
Treatment
Mean Fish
size (±SE) (cm)
Mean Fish
weight (±SE) (g)
Mean (±SE) number of consumed Cx. pipiens
larvae per fi sh
Day time†
Night time†
Daily‡
Female
Untreated
30.00 ± 1.38
180.00 ± 2.77
461.50 ± 3.04
556.67 ± 10.97
1018.17 ± 10.27
Treated
30.76 ± 0.33
180.00 ±2.77
309.67 ± 7.20
359.50 ± 5.79
669.17 ± 3.72
P-value
0.001*
0.0001*
0.0001*
Male
Untreated
29.50 ± 1.50
162.50 ± 2.51
196.38 ± 5.89
374.75 ± 1.25
571.13 ± 4.64
Treated
31.00 ± 1.00
162.50 ± 2.51
174.13 ± 6.64
297.13 ± 4.14
471.25 ± 10.78
P-value
0.013*
0.0001*
0.001*
Adult
Untreated
29.75 ± 1.44
171.25 ± 2.64
328.94 ± 0.39
465.71 ± 5.70
794.65 ± 5.93
Treated
30.88 ± 0.67
171.25 ± 2.64
241.90 ± 2.51
328.31 ± 4.63
570.21 ± 2.68
P-value
0.001*
0.0001*
0.0001*
Signiffi cant diff erences between means using t-test (p < 0.05) are indicated with an asterisk (*). † Day time (from 7.30
am to 7.30 pm) and night time (from 7.30 pm to 7.30 am). ‡ Daily is the sum of day and night time records. Mohamed et al. 72 the stomach (normal histological structure
in Figure 1A, 1B) and the intestine (Figure
2A) of C. gariepinus in the control group. Ex-
posure for 15 days to 390 mg Repare® 18%/L
altered the stomach histology resulting in
a focal area of damage in stomach muco-
sa (Figure 1C, 1D), accompanied by necrosis
and sloughing of mucosal epithelium that the stomach (normal histological structure
in Figure 1A, 1B) and the intestine (Figure
2A) of C. gariepinus in the control group. © Benaki Phytopathological Institute Histopathological studies Ex-
posure for 15 days to 390 mg Repare® 18%/L
altered the stomach histology resulting in
a focal area of damage in stomach muco-
sa (Figure 1C, 1D), accompanied by necrosis
and sloughing of mucosal epithelium that causes severe damage to sub-mucosa (Fig-
ure 1C, 1D). After 15 days, the fi sh intestinal
in the treated group showed: hyperplasia of
intestinal epithelium and goblet cells (Fig-
ure 2B), edema of lamina propria and broad
intestinal villi in some cases (Figure 2C), and
distortion in intestinal villi (Figure 2D) in
comparison to control. Figure 1. Stomach sections of Clarias gariepinus (H & E stain, 400×): A and B) Control stomach showing normal mucosa
with a single layer of columnar epithelial cells (CEC) and gastric glands (GG), sub mucosa (SM), and muscle layer (ML). C) and
D) Exposed to 390 mg Repare® 18%/L for 15 days showing necrosis (circle), sloughing of mucosal epithelium (arrow). Scale
bars: A, C, D (100 μm) and B (20 μm). Figure 1. Stomach sections of Clarias gariepinus (H & E stain, 400×): A and B) Control stomach showing normal mucosa
with a single layer of columnar epithelial cells (CEC) and gastric glands (GG), sub mucosa (SM), and muscle layer (ML). C) and
D) Exposed to 390 mg Repare® 18%/L for 15 days showing necrosis (circle), sloughing of mucosal epithelium (arrow). Scale
bars: A, C, D (100 μm) and B (20 μm). © Benaki Phytopathological Institute Sublethal eff ect of herbicide on the mosquito predator Clarias gariepinus 73 f
q
p
g
p
Figure 2. Intestine sections of Clarias gariepinus (H & E stain): A) Control intestine tissue showing normal lamina propria
(LP), and normal columnar epithelial cells (CEC) with distinct nucleus. B, C, D) Exposed to 390 mg Repare® 18%/L for 15 days;
B) Showing hyperplasia of intestinal epithelium (yellow star) with hyperplasia of goblet cells (GC) (arrow); C) Showing Ede-
ma of lamina propria (star) and broad intestinal villi (double arrow); D) Showing damaged CEC (arrow) and distortion of villi
and lamina propria (oval). Magnifi cation: A (100×) and B, C, D (400×). Scale bars: A, D (100 μm) and B, C (20 μm). Figure 2. Intestine sections of Clarias gariepinus (H & E stain): A) Control intestine tissue showing normal lamina propria
(LP), and normal columnar epithelial cells (CEC) with distinct nucleus. Histopathological studies B, C, D) Exposed to 390 mg Repare® 18%/L for 15 days;
B) Showing hyperplasia of intestinal epithelium (yellow star) with hyperplasia of goblet cells (GC) (arrow); C) Showing Ede-
ma of lamina propria (star) and broad intestinal villi (double arrow); D) Showing damaged CEC (arrow) and distortion of villi
and lamina propria (oval). Magnifi cation: A (100×) and B, C, D (400×). Scale bars: A, D (100 μm) and B, C (20 μm). Discussion Tthe herbicide at 1/2 of the 96-h
LC50 (72.6 mg/L) caused behavioral and mor-
phological changes and induced severe path-
ological alterations in the gill, heart, muscle,
perigastric organ, midgut, and stomach of
the fi sh (Yu et al., 2017). The 96-h LC50 of ben-
sulfuron-methyl on Cyprinus carpio was 1620
mg/L (Rahmani et al. 2020) and the 96-h LC50
of quinclorac on Rhamdia quelen fi ngerlings
was 395 mg/L (Miron et al. 2005).i Female and male of C. gariepinus ex-
posed to sublethal dose of the tested quin-
clorac and bensulfuron-methyl based her-
bicide showed decreased predation rates
on Cx. pipiens larvae compared to non-ex-
posed fi sh. This might be associated with the
direct deleterious eff ect of the herbicide on
the behavioral, physiological, pathological,
and metabolic functions in the treated fi sh,
e.g. to reduce the activity and change swim-
ming speed behavior (rest and slow swim)
that may decrease the attack capability rate
on mosquito larvae. The presence of chemi-
cal contaminants such as heavy metals and
pesticides in aquatic habitats may aff ect the
survival and predatory ability of predatory
fi sh by decreasing the predation rates, alter-
ing their swimming ability or reducing their
vigor (Kerby et al., 2012; Monde et al., 2016;
Yofukuji et al., 2021). Changes in ecological
functions of mobility and predation may be
due to the inhibitory eff ect of the toxic com-
pounds on fi sh’s acetylcholinesterase (Ba-
naee, 2012). The hybrid catfi sh, which was
exposed to sub-lethal doses of endosulfan
insecticide (0.03-1.0 μg/L) exhibited behav-
ioral changes and decreased its predation
capacity on Bulinus globosus (Morelet) prey
(Monde et al., 2016). Kerby et al. (2012) found
that diazinon insecticide at low concentra-
tions signifi cantly decreased the predation
rate of mosquitofi sh G. affi nis against tad- This is the fi rst report for the predatory
activity of C. gariepinus fi sh collected in Egypt
against Cx. pipiens larvae under laboratory
conditions, indicating its potential use as an
eco-friendly and eff ectively indigenous bio-
control agent against the aquatic develop-
mental stages of Cx. pipiens. The larvivorous
effi ciency of C. gariepinus has been reported
in Ethiopia against larvae of both An. arabien-
sis and Culex sp. in laboratory and semi-fi eld
experiments; the larvae consumption by the
fi sh was signifi cantly increased during the
nighttime than during the daytime (Chala et
al., 2016). Discussion posure of C. gariepinus fi sh species to sub-
lethal concentration (almost 1/4 of the LC50
concentration for 96 h exposure) of a com- This is the fi rst report indicating that the ex- © Benaki Phytopathological Institute © Benaki Phytopathological Institute Mohamed et al. 74 mercial herbicide containing quinclorac and
bensulfuron-methyl has negative eff ects on
the predation potential of the fi sh, female
and male. Kerby et al. (2012) indicated that
exposure of G. affi nis to sublethal concentra-
tions (0.5 and 1.0 mg/L) of the pesticide diaz-
inon for 48 h decreased its consumption by
90-100% on P. regilla tadpole prey, whereas
the presence of the pesticide resulted in a
signifi cant reduction in activity and attack
rates of G. affi nis against the target prey. mercial herbicide containing quinclorac and
bensulfuron-methyl has negative eff ects on
the predation potential of the fi sh, female
and male. Kerby et al. (2012) indicated that
exposure of G. affi nis to sublethal concentra-
tions (0.5 and 1.0 mg/L) of the pesticide diaz-
inon for 48 h decreased its consumption by
90-100% on P. regilla tadpole prey, whereas
the presence of the pesticide resulted in a
signifi cant reduction in activity and attack
rates of G. affi nis against the target prey. 2008; Chala et al., 2016; Mohamed et al., 2021),
or the presence of diff erent chemical and
physical stressors such as pesticides, light,
and salinity in aquatic habitats (Kerby et al.,
2012; Yofukuji et al., 2021). In our study, the untreated female of C. gariepinus consumed more Cx. pipiens larvae
than the untreated male fi sh. Moreover, the
daily predation rates of the pre-treated and
untreated groups of female C. gariepinus
against Cx. pipiens larvae were higher than
those of the male fi sh in both groups, indi-
cating that the predation rates of C. gariepi-
nus were strongly associated with the sex of
fi sh. Similarly, mosquito larval consumption
is strongly correlated with the sex of preda-
tory fi sh Poecilia reticulata (Seng et al., 2008;
Saleeza et al., 2014). In the case of bensulfuron-methyl and
acetochlor mixture rice herbicide, the LC50
values against Procambarus clarkii fi sh were
191.25 and 145.24 mg/L at 24 and 96 h, re-
spectively. © Benaki Phytopathological Institute Discussion In India, C. gariepinus exhibited a
highest capacity to feed on An. stephensi (Li-
ston) larvae compared with C. idella, Cyprinus
carpio L., and O. niloticus (Ghosh et al., 2005). Earlier studies demonstrated that the preda-
tion rates of C. gariepinus and other indige-
nous and exotic larvivorous fi sh species were
greatly associated with diff erent biological
traits such as the biotype, size, developmen-
tal stages and sex of the larvivorous fi sh, and
the genera, developmental stages and pop-
ulation density of mosquito prey (Seng et al., Sublethal eff ect of herbicide on the mosquito predator Clarias gariepinus 75 poles by reducing the fi sh activity and vigor. poles by reducing the fi sh activity and vigor. the midgut of the predator that impaired its
digestibility (Silva et al., 2021). Samanta et al. (2016b) described similar lesions formation
in villi of Anabas testudineus (Bloch) after ex-
posure to glyphosate herbicide. poles by reducing the fi sh activity and vigor. Pesticides and other chemical toxicants
in water can enter the digestive tracts of fi sh
during feeding and can also cause histo-
pathological injuries in the digestive organs
of fi sh that may negatively infl uence the fi sh
feeding activity (Banaee 2012; Yu et al., 2017;
Saleh et al., 2022). Histological analyses in
the present study showed histological alter-
nations in the stomach and the intestine tis-
sue of C. gariepinus fi sh after exposure to the
quinclorac and bensulfuron-methyl based
herbicide at a sublethal concentration of 390
mg Repare® 18%/L water for 15 days. Similar-
ly, Samanta et al. (2016a) reported that expo-
sure of O. niloticus to Almix® herbicide (met-
sulfuron-methyl+chlorimuron-ethyl) for 30
days led to degenerative changes like dis-
torted mucosal folds, damage in columnar
epithelial cells and submucosa, and merged
mucosal folds in stomach tissues. Patholog-
ical changes in the stomach, midgut, and
intestine were also found in Procambarus
clarkia (Girard) after exposure to the mixture
bensulfuron-methyl+acetochlor (Yu et al.,
2017). Exposure of Cirrhinus mrigala (Hamil-
ton) for 96 h to 1.5-3.0 μg/L fenvalerate in-
secticide altered intestine histology result-
ing in necrosis of the epithelial cells linning
intestinal villi, sloughing of the mucosal epi-
thelium and lymphocytic cell reaction in the
lamina propria (Velmurugan et al., 2007). p
g yp
In conclusion, this study showed that
the fi sh C. gariepinus collected from Egypt
can consume Cx. Discussion pipiens larvae, and fi sh fe-
males exhibit higher consumption capaci-
ty than the males. Our results suggest that
the native to Egypt fi sh, C. gariepinus could
be considered as a pioneer and a promis-
ing biocontrol agent against Cx. pipiens lar-
vae. However, exposure of C. gariepinus to
sublethal concentration of a herbicide con-
taining quinclorac and bensulfuron-meth-
yl (390 mg Repare® 18%/L water) for 15 days
reduced the predatory potential of the fi sh
female and male against mosquito larvae of
Cx. pipiens. The herbicide also caused histo-
pathological changes in the stomach and in-
testine of this predatory fi sh that may com-
promise predation and aff ect digestion and
absorption of nutrients. These fi ndings sug-
gest the necessity of appropriate application
of the tested herbicide according to the au-
thorized uses indicated in the label of the
product in agricultural lands and fi sh farms
in order to prevent or reduce harmful eff ects
on the fi sh. The eff ects of pesticides on pred-
atory fi sh, their prey, and their interactions
require further laboratory and fi eld studies. Fish stomach and intestine are primari-
ly responsible for the digestion of ingested
food materials and are vital organs, which
are aff ected by diff erent xenobiotic com-
pounds, mainly pesticides (Braunbeck and
Appelbaum, 1999). The histological chang-
es in stomach and intestine of C. gariepi-
nus exposed to quinclorac and bensulfuron-
methyl based herbicide could be related to
the deformity structures and functions of
these organs due to the herbicide toxicity
and cause a negative eff ect on the prey con-
sumption by the fi sh. The prey consumption
of a bug predator, Podisus nigrispinus (Dallas)
was decreased signifi cantly by prey expo-
sure to permethrin, thiamethoxam, and Ba-
cillus thuringiensis insecticides causing mor-
phological and histological alternation in The authors present sincere thanks to Dr
Youssef M. M. Omar (Plant Protection Depart-
ment, Faculty of Agriculture, Assiut University,
Egypt), for reviewing and improving the earlier
version of the manuscript. Compliance with ethical standards
All laboratory experiments were approved by
the Committee of the Faculty of Veterinary of
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bodia. Journal of Vector Ecology, 33: 139–144. Received: 27 September 2022; Accepted: 18 June 2023 Συμπεριφορικές αλλαγές και ιστοπαθολογικές αλλοιώσεις
στο ψάρι Clarias gariepinus ως αρπακτικό προνυμφών του
κουνουπιού Culex pipiens μετά από έκθεση σε υποθανατηφόρο
συγκέντρωση ζιζανιοκτόνου με δραστικές ουσίες quinclorac
και bensulfuron-methyl .A. Mohamed, M. Fathy, A.I.A. Farghal, S.A.H. Temerak, S.Kh. Abd El-Ghaff ar και
S.K.A. Idriss Περίληψη Το Clarias gariepinus είναι ένα καλλιεργούμενο είδος ψαριού του γλυκού νερού, ευρέως
διαδεδομένο στην Αφρική, το οποίο κυριαρχεί σε διάφορα φυσικά και ανθρωπογενή υδάτινα οικο- 78 Mohamed et al. συστήματα, συμπεριλαμβανομένου του μικτού συστήματος ιχυοκαλλιέργειας - καλλιέργειας ρυζιού. Αυτό το είδος ψαριού εμφανίζει αρπακτική ικανότητα στα υδρόβια στάδια ανάπτυξης των κουνου-
πιών. Οι bensulfuron-methyl και quinclorac είναι ζιζανιοκτόνες δραστικές ουσίες που έχουν εφαρμο-
στεί εκτενώς σε καλλιέργειες ρυζιού στην Αφρική και άλλες χώρες παγκοσμίως. Η μελέτη αυτή αξιο-
λόγησε τις αρνητικές επιδράσεις υποθανατηφόρου συγκέντρωσης ενός εμπορικά διαθέσιμου ζιζανιο-
κτόνου που περιέχει τις δραστικές ουσίες quinclorac and bensulfuron-methyl, στην αρπακτική ικανό-
τητα των θηλυκών και αρσενικών ατόμων του ψαριού Clarias gariepinus σε προνύμφες του κουνουπι-
ού Culex pipiens. Επίσης, μελετήθηκε η ιστοπαθολογία του στομάχου και του εντέρου των ψαριών στα
οποία έγινε η εφαρμογή του ζιζανιοκτόνου. Η έκθεση του C. gariepinus στην υποθανατηφόρο συγκέ-
ντρωση του ζιζανιοκτόνου με δραστικές ουσίες quinclorac και bensulfuron-methyl προκάλεσε αρνητι-
κές επιδράσεις στην κατανάλωση λείας και ιστοπαθολογικές αλλοιώσεις στον στόμαχο και στο έντερο
του ψαριού. Hellenic Plant Protection Journal 16: 67-78, 2023 © Benaki Phytopathological Institute © Benaki Phytopathological Institute Sublethal eff ect of herbicide on the mosquito predator Clarias gariepinus Η κατανάλωση προνυμφών κουνουπιών από τα θηλυκά και αρσενικά ψάρια στα οποία είχε
γίνει εφαρμογή του ζιζανιοκτόνου μειώθηκε σημαντικά σε σχέση με εκείνη των ψαριών και των δύο
φύλων που δεν εκτέθηκαν στο φάρμακο. Οι ιστοπαθολογικές αλλοιώσεις στο έντερο ήταν υπερπλα-
σία του επιθηλίου του εντέρου και των καλυκοειδών κυττάρων, οίδημα της υποβλεννογόνιου στοιβά-
δας και διεύρυνση των εντερικών λαχνών, και παραμόρφωση των εντερικών λαχνών, σε σύγκριση με
το μάρτυρα. Οι ιστοπαθολογικές αλλοιώσεις του στομάχου ήταν νέκρωση και αποκόλληση του επιθη-
λίου του βλεννογόνου με σοβαρή βλάβη του υποβλεννογόνου. Ως εκ τούτου, το δοκιμαζόμενο ζιζανι-
οκτόνο σε υποθανατηφόρο συγκέντρωση στο ψάρι C. gariepinus μείωσε την κατανάλωση προνυμφών
κουνουπιών και προκάλεσε ιστοπαθολογικές αλλοιώσεις στο ψάρι, οι οποίες ενδεχομένως επηρέασαν
τη φυσιολογίας της πέψης. Τα ευρήματα της μελέτης δείχνουν ότι το δοκιμαζόμενο ζιζανιοκτόνο μπο-
ρεί να αποτελέσει απειλή για την επιβίωση του ψαριού C. gariepinus και τη δυνατότητα χρήσης του ως
επιτυχούς ιθαγενή παράγοντα βιολογικής καταπολέμησης προνυμφών του κουνουπιού Cx. pipiens.
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https://openalex.org/W2884404516
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http://agritrop.cirad.fr/591719/1/journal.pone.0199547.pdf
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Predictive gravity models of livestock mobility in Mauritania: The effects of supply, demand and cultural factors
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PloS one
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cc-by
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RESEARCH ARTICLE Predictive gravity models of livestock mobility
in Mauritania: The effects of supply, demand
and cultural factors Gae¨lle Nicolas1*, Andrea Apolloni2, Caroline Coste3, G. R. William Wint4,
Renaud Lancelot2, Marius Gilbert1,3 1 Spatial Epidemiology Lab (SpELL), Universite´ Libre de Bruxelles, Brussels, Belgium, 2 International Center
for Agronomic Research and Development, CIRAD, Montpellier, France, 3 Fonds National de la Recherche
Scientifique, Brussels, Belgium, 4 Environmental Research Group Oxford (ERGO)—Department of Zoology,
University of Oxford, Oxford, United Kingdom * gaelle.nicolas6@gmail.com * gaelle.nicolas6@gmail.com * gaelle.nicolas6@gmail.com Editor: Rachata Muneepeerakul, University of
Florida, UNITED STATES Editor: Rachata Muneepeerakul, University of
Florida, UNITED STATES
Received: December 20, 2017
Accepted: June 8, 2018
Published: July 18, 2018
Copyright: © 2018 Nicolas et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Received: December 20, 2017
Accepted: June 8, 2018
Published: July 18, 2018 Copyright: © 2018 Nicolas et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: Data are part of a
previously published study referred as Apolloni
et al. (2018) and available on: https://doi.org/10. 5061/dryad.v4m56. The full citation of the
previously published work is: Apolloni A, Nicolas G,
Coste C, EL Mamy AB, Yahya B, EL Arbi AS, Baba
Gueya M, Baba D, Gilbert M, Lancelot R (2018). Towards the description of livestock mobility in
Sahelian Africa: Some results from a survey in
Mauritania. PLOS ONE 13(1): e0191565. Abstract Animal movements are typically driven by areas of supply and demand for animal products
and by the seasonality of production and demand. As animals can potentially spread infec-
tious diseases, disease prevention can benefit from a better understanding of the factors
influencing movements patterns in space and time. In Mauritania, an important cultural
event, called the Tabaski (Aïd el Kebir) strongly affects timing and structure of movements,
and due to the arid and semi-arid climatic conditions, the season can also influence move-
ment patterns. In order to better characterize the animal movements patterns, a survey was
carried out in 2014, and those data were analysed here using social network analysis (SNA)
metrics and used to train predictive gravity models. More specifically, we aimed to contrast
the movements structure by ruminant species, season (Tabaski vs. Non-Tabaski) and
mode of transport (truck vs. foot). The networks differed according to the species, and to the
season, with a changed proportion of truck vs. foot movements. The gravity models were
able to predict the probability of a movement link between two locations with moderate to
good accuracy (AUC ranging from 0.76 to 0.97), according to species, seasons, and mode
of transport, but we failed to predict the traded volume of those trade links. The significant
predictor variables of a movement link were the human and sheep population at the source
and origin, and the distance separating the locations. Though some improvements would be
needed to predict traded volumes and better account for the barriers to mobility, the results
provide useful predictions to inform epidemiological models in space and time, and, upon
external validation, could be useful to predict movements at a larger regional scale. OPEN ACCESS Citation: Nicolas G, Apolloni A, Coste C, Wint
GRW, Lancelot R, Gilbert M (2018) Predictive
gravity models of livestock mobility in Mauritania:
The effects of supply, demand and cultural factors. PLoS ONE 13(7): e0199547. https://doi.org/
10.1371/journal.pone.0199547 On this occasion, young
rams are slaughtered in most families on the 10th day of the month "dhou al-hija", a religious
holiday during the last month of the Muslim (lunar) calendar. The date of this festival changes
each year according to the Gregorian (solar) calendar and strongly structures the volume of
traded sheep during the year. Annual and seasonal differences are thus observed in the sheep
trade flows. The country, and the whole Sahel, was hit by severe droughts in the 1960’s, 70’s
and 80’s, and more limited droughts later on till 2017 [15]. The drought of 1970 was the main
climatic event for the area. Since then, the area has been mostly in deficit of rainfall [16]. The
series of droughts had a profound impact on the livestock (affecting 2/3 of the production)
and human population. Cattle population dropped, whilst the population of small ruminants
increased, the latter being more robust than the former to harsh climatic conditions. Apolloni
et al. [17] described the livestock trading mobility for the year 2014 in Mauritania, highlighting
that the main trading peak related to the Tabaski took place between August and December. During this period, the price of male lambs sharply increased, and the high demand strongly
affected the trading network structure. In this paper, we aimed to understand how the Tabaski festivity changed the trade network
i M
it
i
d t th
t f th
A
ll
i t l [17]
id d
h
i Funding: This study was funded by EU grant FP7-
613996 VMERGE and is catalogued by the
VMERGE Steering Committee as VMERGE000
(http://www.vmerge.eu). The contents of this
publication are the sole responsibility of the
authors and don’t necessarily reflect the views of
the European Commission. Competing interests: The authors have declared
that no competing interests exist. In this paper, we aimed to understand how the Tabaski festivity changed the trade network
in Mauritania compared to the rest of the year. Apolloni et al. [17] provided a comprehensive
study of the Mauritania survey data, characterizing the seasonal trade network and ruminant
flows within Mauritania, and between Mauritania and the neighboring countries. Here, we
first provided a complementary description of inner flows using social network analysis indi-
cators, by contrasting the Tabaski and non-Tabaski periods, the different ruminant species
and modes of transport. Livestock mobility in Mauritania distribution, growth and connectivity of human populations have also been changing rapidly
as result of demographic transitions, conflicts or migrations. Similarly, the distribution and
connectivity of traded animal populations are strongly influenced by agricultural intensifica-
tion and changes in trade patterns. The combined effect of these societal and environmental
changes taking place simultaneously is difficult to assess, but some, particularly mobility of
livestock and traditional trading practices, have been associated with the emergence and the
spread of infectious diseases [1–4], and can have strong socio-economic impacts [5,6]. In addi-
tion to these long-term trends, culture and tradition strongly shape societies at national,
regional and global scale. For example, human population movement during specific periods
such as Chinese Spring Festival [7], annual holidays [8], or religious feast around Christmas,
Ramadan, Thanksgiving or Hindu Holy feast are known to cause substantially affect global
mobility [9] with significant economic and epidemiological implications [10–12]. Large move-
ments of animal populations are also linked to changes in the spatial pattern of food demand,
which is anticipated by the market. In low-income countries, such as Sahelian African coun-
tries, rapid changes in demand for animal products linked to cultural and traditional events
therefore leads to a large number of animals–notably sheep, being slaughtered to meet the
seasonal food demand. As a consequence, in the months and weeks preceding such events,
trading of live animals is particularly intense. Due to the dry ecoclimate of the Sahelian area,
agriculture and breeding strongly depend on the amount of rainfall and the availability of
pasture. As a consequence, successive droughts also dramatically affect the Livestock flow. Another example is Madagascar, where kapsile is a traditional practice consisting in an animal
barters linked to the labour need in a period when breeders are cash-poor and which strongly
affects the patterns of trade flows [3,13]. In West Africa, similar practices, called loans, have
been described as a large number of short term animal exchanges for reproduction, food sup-
ply through milk production, animal traction, etc [14]. In Mauritania, the Muslim feast of
Tabaski or Aïd el-Kebir, is a major cultural and traditional event that strongly influence trad-
ing patterns and could have major impact on the spread of diseases. Introduction Many factors that may influence the dynamics and transmission of infectious diseases have
been rapidly changing over the last decades. Alongside climate and land use changes, often
considered in emerging infectious diseases literature as main drivers, other factors such as the PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Data collection A survey among veterinary officers was conducted in June 2015 by the National Office for Live-
stock Research and Development (ONARDEL) to collect their knowledge of ruminant trade
flows as reported in Apolloni et al. [17]. The survey aimed to monitor the movement patterns
during the year 2014. Its results were recorded as a series of trade flow events, with the following
information: i) the origin, ii) the destination, iii) the type of trade movement (farm, transit, mar-
ket), iv) the frequency (annual, monthly, weekly), v) the species (cattle–Bos indicus, sheep, goat
or camel—Camelus dromedarius), vi) the number of heads, vii) the date of the starting of the
event, viii) the transportation mode (by truck or on foot) and ix) the latitude/longitude coordi-
nates of the origin and destination. Three types of movements were recorded: i) between pasto-
ral areas for grazing and/or reproduction, ii) from farm to market, iii) or from market to
market. Transhumance movements aiming to gradually move herds for suitable pasture areas
were not included. The database was cross-checked against sanitary certificates, the scientific
documents describing transhumance patterns, and the knowledge of veterinarian staff [17]. Both national and international trade-flows were recorded and the transboundary movements
were double-checked through surveys on transit sites between Senegal and Mauritania. Study area Mauritania is situated in the hyper-arid (Sahara) and arid (Sahel) ecozones [18], with low
annual rainfall (0–400 mm) concentrated in a short rainy season (June-September). In the
northern part of the country, the driest one, only short-cycle plants grow. Livestock, mainly
camels and small ruminants, are reared moving around available water points and grazing
areas. The southern area, more humid and greener is mostly exploited by transhumant herds. Most of cattle population, being less mobile and demanding more water and nutrients, is con-
centrated in the southern area, mainly in the region around the river Senegal. Because of the
harsh conditions, mobility is a key aspect of animal rearing in Mauritania. Animals are moved
almost continuously among grazing areas to optimize the consumption of good quality nutri-
ents. In the absence of slaughterhouses, stocking facilities and road infrastructures, animals are
traded alive and butchered at consumption markets. Past droughts indirectly contributed to the growth of cities, in particular Nouakchott, due
to the migration of previous farmers and herders from the countryside to urban areas in search
of jobs. Because of this, Nouakchott, the capital city has seen its population dramatically
exploding during and following the drought years. As of today, almost one quarter of the total
population lives in the capital city (National Bureau of statistics http://www.ons.mr/) Maurita-
nia is still recovering from the latest food crisis in 2011, affecting almost 1 million of its habi-
tants. In 2018, the drought indicators were at the same levels as those of 2012, indicating that
the food emergency is not completely over. The inadequate levels of rainfall and the continu-
ous threat of the droughts force herders to sell their livestock, in particular small ruminants,
due to shortages of suitable feeding areas. Second, we developed predictive models to estimate the probability of
a trade connection between two spatial units (areas around markets and/or farms, etc.) based PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 2 / 21 Livestock mobility in Mauritania on their potential production and demand characteristics and different measures of the cost
distance between them. on their potential production and demand characteristics and different measures of the cost
distance between them. Additional data In 2013, the Ministry for Rural Development and Environment reported a population of 16.8
million sheep and goats, 1.8 million cattle, and 1.4 million camels for Mauritania to FAOSTAT 3 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Livestock mobility in Mauritania Fig 1. Population of human and ruminants in Mauritania. The size of the circle are proportional to the number of head located within the
Voronoï polygon related to the spatial location of the trading network. https://doi.org/10.1371/journal.pone.0199547.g001 Fig 1. Population of human and ruminants in Mauritania. The size of the circle are proportional to the number of head located within the
Voronoï polygon related to the spatial location of the trading network. https://doi.org/10.1371/journal.pone.0199547.g001 https://doi.org/10.1371/journal.pone.0199547.g001 [19]. Accordingly, and because no finer data was available at national level, we used the most
recent version of the Gridded Livestock of the World database (GLW), where the subnational
livestock statistics for Mauritania dates back to 2007, and were adjusted to match the FAO-
STAT 2010 national totals [20,21]. The WorldPop database was used for the human popula-
tion [22]. Both databases were aggregated at a spatial resolution of 0.083333 decimal degrees
(i.e. approximately 10 km at the equator) (Fig 1). To estimate the cost paths potentially affect-
ing mobility between different localities, we considered two main data sources: the friction
layer of Nelson accessibility map, which quantifies the time needed to travel through each
pixel [23], and the elevation from the GTOPO30 database (https://lta.cr.usgs.gov/GTOPO30). PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Analysis In this study, we only considered inner movements of cattle, camels, sheep and goats within
Mauritania. Social network analysis (SNA) [24] have proved to be of significant interest in animals
movements analyses in the past decade [25–28]. Here, it was first used to describe the trade
networks according to the species, the transport modality (using truck vs. walking), and the
season (Tabaski vs. non-Tabaski). These mentioned periods were defined as strongly influenc-
ing the livestock flow within the country by Apolloni et al. [17] who investigated the dataset
regarding the occurrence of the Muslim festival for the year 2014. A set of network parameters
were estimated for the different networks using the simplified definition provided in Wasser-
man and Faust, (1994) [24]: PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 4 / 21 Livestock mobility in Mauritania • Diameter: a network-level parameter representing the greatest number of links in the short-
est path between two nodes. • Average path length: a network-level parameter measuring the average number of steps
along the shortest paths of all possible nodes pairs, i.e. the average number of nodes an actor
has to trade through to connect to any other node. • The clustering coefficient: a node-level parameter of the density of local ties. It measures the
probability that neighboring nodes of a node are connected. • The density: a network-level parameter measuring the proportion of observed links among
the possible links between nodes, and indicates how strongly a network is connected. • Average degree: a network-level parameter quantifying the average number of links con-
nected with a node in a network. Besides these global measures, other centrality measures
highlighting the prominent role of nodes in the network were considered: node’s in- and
out-degree (the number of connection towards and from each node), node’s in and out-
weight (the volume of animal towards and from each node); node’s betweenness (the num-
ber of shortest path passing through the node) and node’s eigenvector centrality (scoring the
importance of nodes). Network’s vulnerability to target removal of nodes based on centrality measures and esti-
mates of the size of the largest connected component were tested. The removal of specific
nodes, and their links, cause the network to fragment in a set of smaller subnetworks. PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Analysis Eq (2) can be rewritten as a model (3) with an intercept (a flow that would still be pres-
ent when populations are equal to zero), and a set of predictor variables xk characterizing the
origin or destination, with their associated coefficients βk. MIGij ¼ pa
i pb
j
dg
ij
ð1Þ ð1Þ logðMIGijÞ ¼ api þ bpj gdij
ð2Þ
GijÞ ¼ b0 þ b1pi þ b2pj þ b3dij þ PK
k 4bkxk
ð3Þ logðMIGijÞ ¼ api þ bpj gdij
ð2Þ ð2Þ logðMIGijÞ ¼ b0 þ b1pi þ b2pj þ b3dij þ PK
k¼4bkxk
ð3Þ ð3Þ In this study, we first aimed to estimate the probability of a trade connection between two
nodes. Therefore, log(MIGij) was replaced with the logit of this probability and logistic regres-
sion was used to estimate the coefficients. As the response, all pairs of connected nodes were
coded with 1, and all other pairs of nodes were coded with 0. The analysis was split according
to the main structuring factors of the networks, i.e. species, transport modality and season. For
each sub-model, we tested seven combinations of predictors, considering them both at the ori-
gin and destination with inclusion of a distance estimator ((i) great-circle distance, (ii) cost-
path distance based on accessibility friction surface or (iii) cost-path distance based on eleva-
tion friction surface). The different combinations of predictors that were tested in the models
are shown in Table 1. The human and animal populations at the origin and destination were
extracted from the Worldpop and GLW raster layers within the Thiessens’ polygons around
each node (Fig 1). Thiessen’s polygons represent areas consisting of all points closer to the
node than to any other node. These were used because the nodes did not correspond to any
particular administrative division that could have been used (i.e. many nodes per admin unit). Although Thiessen’s polygon can produce somewhat misleading long shapes in desertic areas,
this is not necessarily a problem as the livestock and human population demographics would
be low in these areas anyway. In each sub-model, we first included the extracted animal popu-
lation of each species at both the source and destination (cattle, sheep, goats, and camels). We
used stepwise regression based on Akaike information criterion (AIC) to select a more parsi-
monious model, with the lowest AIC. Analysis The size
of the largest component can be thought as the maximum extent a disease can spread after the
implementation of the control measure (vaccination of animal in the areas surrounding the
nodes, market closure, etc.) [29]. Volkova et al. (2010) [30] introduced the notion of epidemic
threshold (q) in veterinarian epidemiology. This parameter estimates the (minimum) proba-
bility for a disease to be transmitted from one node to another to trigger an epidemic. The low-
est the epidemic threshold the higher is the risk of an epidemics. This quantity depends on the
heterogeneity of the network and the weight’s distribution. In the case of a weighted network
the epidemic threshold can be estimated as: q ¼
hwouti
hwin wouti Where h i indicates the average value and win, wout indicate node’s in- and out-weight,
respectively. Following the same procedure as in Lancelot et al. (2017) [31], the invasion
threshold for each month were estimated. Highlight on the role of occasional links were given
(connections appearing just once per year). We plotted path intersections between different species, transports modalities and seasons,
to highlight possible common or specific links for different combinations. In addition, metrics
quantifying these intersections were estimated, such as the pairwise percentage of common
and specific paths between two networks, respectively. Gravity models were used to estimate the probability of a link between two distinct nodes
according to their features. These models were developed in the field of socio-economics and
human migration studies [32,33]. They provide estimates for the flows of goods or people
between two nodes, as a function of node-level variables (e.g. population size, socio-economic
factors, etc.), and of the distance or movement cost between these nodes. Its most general for-
mulation (Eq 1) shows the analogy with Newton’s gravity law. MIGij is the flow between the
origin i and destination j, pi and pj are the population at the origin and destination, dij is the
distance between i and j, and α, β and γ are model parameters. The equation is linearized into PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 5 / 21 Livestock mobility in Mauritania Eq (2) by a log-transformation, and its coefficients can be estimated with generalized linear
models. Analysis In a second step, this analysis was repeated using the number of animals traded between
these two locations as the response variable. To better differentiate the factors influencing the
trade probability from those influencing its volume, the latter analysis was restricted to pairs of Table 1. General equation of the tested models. MODEL
GENERAL EQUATION
E1
Hpi + Hpj + dijg
E2
Lpi + Lpj + dijg
E3
Hpi + Hpj + Lpi + Lpj + dijg
E4
Hpi + Hpj + Lpi + Lpj + dija
E5
Hpi + Hpj + Lpi + Lpj + dijel
E6
Δ(Hpi, Hpj) + Δ(Lpi, Lpj) + dijg
E7
E3 + mode + mode: pi + mode: pj + mode: dijg
pi: population at the origin (L: livestock, H: human); pj: population at destination (L: livestock, H: human); dij: distance
(g: great circle distance, a: costhpath distance based on accessibility friction surface, el: costhpath distance based on
elevation friction surface), mode: transport modality (by truck or on foot). htt
//d i
/10 1371/j
l
0199547 t001 Table 1. General equation of the tested models. MODEL
GENERAL EQUATION
E1
Hpi + Hpj + dijg
E2
Lpi + Lpj + dijg
E3
Hpi + Hpj + Lpi + Lpj + dijg
E4
Hpi + Hpj + Lpi + Lpj + dija
E5
Hpi + Hpj + Lpi + Lpj + dijel
E6
Δ(Hpi, Hpj) + Δ(Lpi, Lpj) + dijg
E7
E3 + mode + mode: pi + mode: pj + mode: dijg
pi: population at the origin (L: livestock, H: human); pj: population at destination (L: livestock, H: human); dij: distance
(g: great circle distance, a: costhpath distance based on accessibility friction surface, el: costhpath distance based on
elevation friction surface), mode: transport modality (by truck or on foot). https://doi.org/10.1371/journal.pone.0199547.t001 Table 1. General equation of the tested models. https://doi.org/10.1371/journal.pone.0199547.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Analysis MODEL
GENERAL EQUATION
E1
Hpi + Hpj + dijg
E2
Lpi + Lpj + dijg
E3
Hpi + Hpj + Lpi + Lpj + dijg
E4
Hpi + Hpj + Lpi + Lpj + dija
E5
Hpi + Hpj + Lpi + Lpj + dijel
E6
Δ(Hpi, Hpj) + Δ(Lpi, Lpj) + dijg
E7
E3 + mode + mode: pi + mode: pj + mode: dijg pi: population at the origin (L: livestock, H: human); pj: population at destination (L: livestock, H: human); dij: distance
(g: great circle distance, a: costhpath distance based on accessibility friction surface, el: costhpath distance based on
elevation friction surface), mode: transport modality (by truck or on foot). pi: population at the origin (L: livestock, H: human); pj: population at destination (L: livestock, H: human); dij: distance
(g: great circle distance, a: costhpath distance based on accessibility friction surface, el: costhpath distance based on
elevation friction surface), mode: transport modality (by truck or on foot). PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 6 / 21 Livestock mobility in Mauritania locations with an existing trade link. All analyses were coded and carried out using R [34]. The
“sna” package was used to describe and analyse the trade network [35]. Table 2. Number of national and international traded animals in Mauritania. Values represent the number of animals moved for each given period. Movements are
defined as national if both the origin and destination are located within the Mauritanian border. Small ruminants (SR) include sheep and goats. Some of the records did
not differentiate the species individually and are counted as “Cattle and SR” or “Mixed”. Cattle
Cattle and SR
Camels
SR
Mixed
Total
Truck
67 240
43 760
1 105 870
1 216 870
Aug-Dec
34 398
19 119
843 047
896 565
international
2 400
2 120
410 500
415 020
national
31 998
16 999
432 547
481 545
Jan-July
32 842
24 641
262 822
320 305
international
9 600
3 280
980
13 860
national
23 242
21 361
261 842
306 445
Foot
1 801 846
1 660
598 034
3 334 755
96 563
5 832 859
Aug-Dec
342 444
277
112 740
716 645
19 740
1 191 847
international
283 403
28 011
518 420
829 835
national
59 041
277
84 729
198 225
19 740
362 012
Jan-July
1 459 402
1 383
485 294
2 618 110
76 823
4 641 012
international
119 1123
271 755
2 286 295
3 749 172
national
268 279
1 383
213 539
331 815
76 823
891 839
Total
1 869 086
1 660
641 794
4 440 625
96 563
7 049 729
https://doi.org/10.1371/journal.pone.0199547.t002 Table 2. Number of national and international traded animals in Mauritania. Values represent the number of animals moved for each given period. Movements are
defined as national if both the origin and destination are located within the Mauritanian border. Small ruminants (SR) include sheep and goats. Some of the records did
not differentiate the species individually and are counted as “Cattle and SR” or “Mixed”. locations with an existing trade link. All analyses were coded and carried out using R [34]. The
“sna” package was used to describe and analyse the trade network [35]. PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Results The dataset consisted in 2,219 trade movements involving 7.1 million head. The subset of
national movements, the focus of this analysis, included 1,178 movement events involving 2.1
million head (Table 2). International movements involved around 5 million head, sold or
bought to or from Senegal, Mali, Ivory Coast, Guinea Bissau and Morocco. As the destination
of these international movements within these countries was unknown, they could not be
included in the network analysis or gravity models. Within Mauritania, the trading network
was composed of 65 nodes and 84 unique paths. Transport by foot was the most represented
mode, corresponding to 83% of the animal flow (Tables 2 and 3). The foot and truck transport
modalities presented contrasted patterns. The largest share of foot movements being short to
medium distance (0–200 km), whereas the opposite was observed for movements by truck,
where the largest share was represented by movements > 500 km (Fig 2). Similarly, those con-
trasting patterns somewhat matched the seasonal pattern. The Tabaski period represented 41%
of the inner animal movements, with 57% of them by truck. In contrast, only 25% of the head
were moved by truck outside the Tabaski period (Table 2). The Mauritanian network was weakly connected. The density value indicated that 2% of
possible node pairs were actually connected, and the network level centrality parameters and
clustering coefficient were low (Table 3). However, each of the exchange networks (full net-
work, or species-specific sub-networks) contained a single component in which the average
length of the shortest path between node pairs was lower than 2 links, the maximum value
(diameter) being 5 links. The goat and camel trading networks were smaller with a diameter of
2 and 3 links. On average, in the full species network, a given node was directly connected with
approximately 2 other nodes on average (average degree). Both the in-degree and in-weight
distributions are right-skewed. Only 5 locations from the livestock trading network attracted
more than 50% of the connections, and are also the destination for more than 60% of the PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 7 / 21 Livestock mobility in Mauritania Fig 3. Network’s centrality measures of the Mauritanian’s livestock trade. Node size show the importance of the measured centrality values. in and out-weight
measures were scaled on the total volume of traded livestock; eigenvector centrality (centrality measure) were scored from 0 to 1, betweenness was considered for the
fraction of paths passing through the node. h
//d i
/10 13 1/j
l
0199 4
003 Fig 3. Network’s centrality measures of the Mauritanian’s livestock trade. Node size show the importance of the measured centrality values. in and out-weight Fig 3. Network’s centrality measures of the Mauritanian’s livestock trade. Node size show the importance of the measured centrality values. in and out-weight
measures were scaled on the total volume of traded livestock; eigenvector centrality (centrality measure) were scored from 0 to 1, betweenness was considered for the
fraction of paths passing through the node. https://doi.org/10.1371/journal.pone.0199547.g003 traded volume. Nouakchott, the main urban consumption market of the country, acts as hub
for livestock mobility with 18 links which concentrate around 1/3 of the traded animals’ total
volume (Fig 3, Table 4). With the highest centrality score, the capital city is the most important
node of the network. Almost 2/3 of the nodes has at most one outgoing link, whilst Aleg and traded volume. Nouakchott, the main urban consumption market of the country, acts as hub
for livestock mobility with 18 links which concentrate around 1/3 of the traded animals’ total
volume (Fig 3, Table 4). With the highest centrality score, the capital city is the most important
node of the network. Almost 2/3 of the nodes has at most one outgoing link, whilst Aleg and Table 4. List of nodes with highest values for centrality measures. Each column corresponds to a specific network (all species or by single species) Each line relates to a
specific centrality measure. Only the name of the node corresponding with the largest value for each measure is reported. In the case of multiple nodes with same value of
the measure, all the names are reported. Table 4. List of nodes with highest values for centrality measures. Each column corresponds to a specific network (all species or by single species) Each line relates to a
specific centrality measure. Only the name of the node corresponding with the largest value for each measure is reported. Livestock mobility in Mauritania ber of links of the national movement networks in Mauritania. Links are provided according to the transport modality and
-Tabaski: Jan–July). Table 3. Network parameters and number of links of the national movement networks in Mauritania. Links are provided according to the transport modality and
Tabaski periods (Tabaski: Aug–Dec; Non-Tabaski: Jan–July). ALL SPECIES
CATTLE
SR
SHEEP
GOAT
CAMEL
Network-level parameter
Number of nodes
65
65
65
65
65
65
Number of links
84
49
56
54
32
34
Diameter
5
4
5
5
3
2
Clustering coefficient
0.19
0.15
0.18
0.18
0.15
0.03
Average path length
1.84
1.56
1.73
1.73
1.34
1.13
Density
0.020
0.012
0.013
0.013
0.0077
0.0082
Average degree
2.65
1.51
1.72
1.66
0.985
1.05
Number of link
Transport modality
Truck
33
13
28
28
13
14
Foot
55
37
30
28
20
22
Intersection
2
2
2
2
1
2
Trading period
Non-Tabaski
73
45
46
44
29
31
Tabaski
57
31
39
39
19
21
Intersection
44
27
14
29
16
18 Fig 2. Great-circle distance of the national movements by truck (left) and on foot (right) recorded in the survey. Distance are given in
kilometers for each species (thick line: small ruminants, dashed line: cattle, thick dash line: camel). https://doi org/10 1371/journal pone 0199547 g002 Fig 2. Great-circle distance of the national movements by truck (left) and on foot (right) recorded in the survey. Distance are given in
kilometers for each species (thick line: small ruminants, dashed line: cattle, thick dash line: camel). https://doi.org/10.1371/journal.pone.0199547.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 8 / 21 In the case of multiple nodes with same value of
the measure, all the names are reported. ALL SPECIES
CATTLE
SR
CAMEL
INDEGREE
Nouakchott
Nouakchott
Nouakchott
Nouakchott
INWEIGHT
Nouakchott
Selibabi
Nouakchott
Zouerate
OUTDEGREE
Aleg
Aleg Kiffa
Aleg
Kiffa
Kiffa
Mmat Laakarich II
M. Lahjar
OUTWEIGHT
Tintane
Kaedi
Tintane
Nbeika
BETWEENNESS
Boutilimit
Adel-Bagrou
Boutilimit
Boutilimit
Mbout
Nbeika
EIGENVECTOR CENTRALITY
Nouakchott
Kaedi
Nouakchott
Nbeika
https://doi org/10 1371/journal pone 0199547 t004 9 / 21 Livestock mobility in Mauritania Table 5. Correlation coefficients among centrality measures. Pearson correlations coefficients among centrality measure. Only significant (p-value <0.05) coefficients
are reported. INDEGREE
OUTDEGREE
INWEIGHT
OUTWEIGHT
INDEGREE
0.88
OUTDEGREE
0.63
INWEIGHT
0.88
OUTWEIGHT
0.63
https://doi.org/10.1371/journal.pone.0199547.t005 Kiffa, in the southern region, are connected to other 7 localities, and Tintane has the largest
out-weight. Finally, the betweenness distribution is right-skewed and Boutilimit appears to be
the node with highest betweenness. Few of the centrality measures are significantly correlated
(p-value<0.05) and reported in Table 5. Results of the percolation analysis on networks cohesion are shown in Fig 4. Nodes are
removed based on the centrality measures (indegree, outdegree, incoming and outgoing Fig 4. Effect of targeted removal on the connected component size. Values on the x axis indicate the percentage of
nodes removed (cumulative), together with their links. Values on y axis indicate the percentage of nodes in the largest
connected sub-network, after the removal. Color indicates the removal procedure based on centrality measure score
(starting from the highest score nodes) or randomly. Shaded areas correspond to 95% CI for the random procedure. https://doi.org/10.1371/journal.pone.0199547.g004 Fig 4. Effect of targeted removal on the connected component size. Values on the x axis indicate the percentage of
nodes removed (cumulative), together with their links. Values on y axis indicate the percentage of nodes in the largest
connected sub-network, after the removal. Color indicates the removal procedure based on centrality measure score
(starting from the highest score nodes) or randomly. Shaded areas correspond to 95% CI for the random procedure. https://doi.org/10.1371/journal.pone.0199547.g004 Fig 4. Effect of targeted removal on the connected component size. Values on the x axis indicate the percentage of
nodes removed (cumulative), together with their links. Values on y axis indicate the percentage of nodes in the largest
connected sub-network, after the removal. Color indicates the removal procedure based on centrality measure score
(starting from the highest score nodes) or randomly. Shaded areas correspond to 95% CI for the random procedure. https://doi.org/10.1371/journal.pone.0199547.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 10 / 21 Livestock mobility in Mauritania Fig 5. Species-level movement networks. Each diagram represents the movement links that are specific to the species (red: by truck, dark
grey: by foot, both truck and foot: blue). The last plot shows the entire network of all species. https://doi.org/10.1371/journal.pone.0199547.g005 Fig 5. Species-level movement networks. All species: Association of small ruminants, Cattle and Camel trading networks. Each diagram represents the movement links that are specific to the species (red: by truck, dark
grey: by foot, both truck and foot: blue). The last plot shows the entire network of all species. Fig 5. Species-level movement networks. Each diagram represents the movement links that are specific to the species (red: by truck, dark
grey: by foot, both truck and foot: blue). The last plot shows the entire network of all species. https://doi.org/10.1371/journal.pone.0199547.g005 https://doi.org/10.1371/journal.pone.0199547.g005 volume, betweenness, eigenvector centrality) and, for comparison purposes, randomly. volume, betweenness, eigenvector centrality) and, for comparison purposes, randomly. Removing nodes in order of their incoming connections, incoming volume and centrality
result as the most effective strategies of fragmentation. In the first case, removing less than
20% of the nodes (13 nodes) results in decomposing network in a set of subnetworks whom
the largest one contains less than 10% of the nodes (7 nodes). Removing nodes in order of their incoming connections, incoming volume and centrality
result as the most effective strategies of fragmentation. In the first case, removing less than
20% of the nodes (13 nodes) results in decomposing network in a set of subnetworks whom
the largest one contains less than 10% of the nodes (7 nodes). In total, 94% of the goats trading network paths were shared with the sheep (Fig 5, Table 6). These two networks were merged into a single small ruminant trading network in the gravity
models. A high degree of overlap between pairs of species networks was highlighted (Table 6). However, overlapping only involved 20% of the full set of trading links (Fig 5, Table 6). the different species. The values correspond to the percentage (italic) and the number (bracket) of common links and
of species. Table 6. Comparison of the trading networks of the different species. The values correspond to the percentage (italic) and the number (bracket) of common links and
difference between the trading network of the pair of species. network SPECIES 1
network SPECIES 2
INTERSECTION
% (n)
DIFFERENCE SP1/SP2
% (n)
DIFFERENCESP2/SP1
% (n)
CATTLE/SR
49
56
65 (32)
35 (17)
43 (24)
CAMEL/SR
34
56
65 (22)
35 (12)
61 (34)
CAMEL/CATTLE
34
49
47 (16)
53 (18)
67 (33)
GOAT/SHEEP
32
54
94 (30)
6 (2)
44 (24)
ALL SPECIES
84
-
20 (17) Table 6. Comparison of the trading networks of the different species. ation of small ruminants, Cattle and Camel trading networks. https://doi.org/10.1371/journal.pone.0199547.t006 All species: Association of small ruminants, Cattle and Camel trading networks. https://doi.org/10.1371/journal.pone.0199547.t006 The values correspond to the percentage (italic) and
difference between the trading network of the pair of species. PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 11 / 21 Livestock mobility in Mauritania Among the remaining links, many were species-specific: 30% of the small ruminant (17/56),
33% of the cattle (16/49) and 32% of the camel (11/34) links were not included in any of the
other networks. Very few links were shared between the truck and foot networks and were
identical for all species (2/84, Table 6). The role of nodes could change depending on the species considered. Previous results
about most central nodes hold when we consider the “small ruminants only” network. Whilst
Nouakchott remains always the most connected node, most of the volume of cattle and camels
are directed towards Selibabi and Zouerate, respectively. The markets of Kaedi, for cattle, and
Nbeika, for camels, become more central in the network (see S1 File and Table 4). In term of
cohesiveness, specie-specific network are more vulnerable to the target removal of nodes. As
in the all species case target removal based on indegree and inweight are the most efficient pro-
cedures. In fact, in both cases, removing a limited quantity of nodes, 8 for camel’s network 10
for cattle’s one and 12 for small ruminants one, results in decomposing the respective networks
in a set of subnetworks whom the largest one contains at most 8 nodes. This is particularly rel-
evant for small ruminants and the cattle networks, whose largest subnetworks consist of 7 and
10 nodes, respectively, after the removal of only 3 most connected nodes. There was a high number of shared links between the Tabaski and non-Tabaski periods
(44/84, Table 6). Both Tabaski and non-Tabaski networks included truck and foot movements,
but their relative proportion was different. During the Tabaski period, there were fewer foot
movements and higher truck movements (Fig 6). Very few trade connections involved both
truck and foot movements in the two periods (Table 6, Figs 5 and 6). Fig 6. Movement networks according to the species and seasons. Note that for each season the links present in both defined
transportation mode are not plotted and are the same for both defined season (red: by truck, dark grey: by foot, nf: number of movement
links by foot, Jan-July: Non-Tabaski period; Aug-Dec: Tabaski period). Fig 7. Variations of network quantities along the year. Bottom: monthly epidemic threshold variations along the
year; Centre Volume of animals traded during the month; Top number of links active. For each month, we have
considered 2 networks: all network including occasional links appearing on that month; backbone, excluding
occasional link. The different colors correspond to the quantity evaluated for the specific network. https://doi.org/10.1371/journal.pone.0199547.g007 Fig 7. Variations of network quantities along the year. Bottom: monthly epidemic threshold variations along the
year; Centre Volume of animals traded during the month; Top number of links active. For each month, we have
considered 2 networks: all network including occasional links appearing on that month; backbone, excluding
occasional link. The different colors correspond to the quantity evaluated for the specific network. https://doi.org/10.1371/journal.pone.0199547.g007 As the network changes along the year so its proneness to diffuse diseases. Fig 7, present the
variation of the epidemic threshold (denoted q), estimated every month, along the year in
comparison with the number of links active and the volume of livestock traded. Since the net-
work can change along the year, particularly new active links around Tabaski, we considered
the backbone network from Apolloni et al. [17] (containing links present more than 2 months)
and the total network containing all the link active that month to elicit the role that Tabaski
plays on the risk of transmission. The epidemic threshold is at the lowest values between the
March and June, when the volume and the number of exchanges (links) is at maximum, and
around Tabaski, when a second peak of movements (mainly small ruminants) whose volume
is almost equal to the first peak, is observed. Occasional links, appearing only for the Tabaski
reason, decreases the invasion threshold and consequently the risk of disease spreading is
higher in this period. Table 7 presents the different sets of gravity models that were applied to the full network
(binary outcome: 1 if two nodes were connected and 0 otherwise), and to the networks broken
down by species, period and transport modality. The best results were obtained with models including human and animal populations at the
origin and destination, with a great-circle distance (model E3, Tables 1 and 7). Replacing this
distance with cost-distance functions of accessibility (E4) or elevation (E5) did not improve
the results. Fig 3 should be considered to see the redundancy between
transportation mode (blue edge). Fig 6. Movement networks according to the species and seasons. Note that for each season the links present in both defined
transportation mode are not plotted and are the same for both defined season (red: by truck, dark grey: by foot, nf: number of movement
links by foot, Jan-July: Non-Tabaski period; Aug-Dec: Tabaski period). Fig 3 should be considered to see the redundancy between
transportation mode (blue edge). https://doi.org/10.1371/journal.pone.0199547.g006 12 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Livestock mobility in Mauritania Fig 7. Variations of network quantities along the year. Bottom: monthly epidemic threshold variations along the
year; Centre Volume of animals traded during the month; Top number of links active. For each month, we have
considered 2 networks: all network including occasional links appearing on that month; backbone, excluding
occasional link. The different colors correspond to the quantity evaluated for the specific network. https://doi.org/10.1371/journal.pone.0199547.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 AIC
model
n link
n unique link
E1
E2
E3
E4
E5
E6
E7
ALL SPECIES
All
1178
140
1501.8
1604.1
1464.1
1488.9
1485.1
1461.9
1342.7
NTab
518
53
1339.8
1418.6
1303.3
1323.7
1323.6
1303.3
Tab
660
87
995.6
1133.7
979.8
983.1
982.9
976.1
Truck
735
121
427.2
601.1
405.0
408.9
407.3
416.9
Truck—NTab
443
91
315.7
470.7
302.4
309.1
305.4
307.8
Truck—Tab
257
40
175.2
280.5
172.0
174.9
172.2
175.2
Foot
129
21
899.3
885.9
877.5
951.1
934.7
885.6
Foot—NTab
478
81
843.8
832.2
821.8
886.8
874.0
832.1
Foot—Tab
182
41
460.4
454.7
455.2
498.3
484.9
455.7
SR
All
620
58
589.5
639.7
572.0
578.9
576.7
570.3
NTab
374
46
482.7
530.3
470.1
474.1
476.5
470.7
Tab
246
40
412.9
473.1
404.0
404.2
403.7
403.7
Truck
324
31
210.3
290.5
204.5
205.7
205.7
205.9
Foot
296
27
315.3
308.2
304.7
331.3
327.0
307.9
CT
All
341
47
494.1
526.9
480.6
474.9
487.2
489.4
NTab
214
43
465.0
492.4
451.3
444.5
455.8
459.3
Tab
127
31
342.3
384.2
329.3
322.5
328.0
340.2
Truck
109
13
107.2
146.7
105.6
105.9
106.0
106.0
Foot
232
34
305.7
295.8
296.0
304.1
307.9
307.7
CM
All
217
35
411.5
425.1
399.2
397.4
399.5
395.8
NTab
147
32
390
393.9
377.0
375.9
377.6
373.4
Tab
70
20
241.7
265.8
236.1
235.7
236.1
233.4
Truck
113
13
116.6
156.3
109.9
110.8
108.9
112.3
Foot
104
22
266.6
258.7
261.3
264.9
265.9
258.6
https://doi.org/10.1371/journal.pone.0199547.t007 Table 7. AIC values of the logistic regression models. The models are broken down by species (SR: small ruminants, CT: cattle, CM: camels), period (Tab: Tabaski,
NTab: Non-Tabaski) and transport modality. E1-E6 correspond to different models described in Table 1. Table 7. AIC values of the logistic regression models. The models are broken down by species (SR: small ruminants, CT: cattle, CM: camels), period (Tab: Tabaski,
NTab: Non-Tabaski) and transport modality. E1-E6 correspond to different models described in Table 1. Table 7. AIC values of the logistic regression models. The models are broken down by species (SR: small ruminan
NTab: Non-Tabaski) and transport modality. E1-E6 correspond to different models described in Table 1. Table 7. AIC values of the logistic regression models. The models are broken down by species (SR: small ruminants, CT: cattle, CM: camels), period (Tab: Tabaski,
NTab: Non-Tabaski) and transport modality. E1-E6 correspond to different models described in Table 1. AIC
model
n link
n unique link
E1
E2
E3
E4
E5
E6
E7
ALL SPECIES
All
1178
140
1501.8
1604.1
1464.1
1488.9
1485.1
1461.9
1342.7
NTab
518
53
1339.8
1418.6
1303.3
1323.7
1323.6
1303.3
Tab
660
87
995.6
1133.7
979.8
983.1
982.9
976.1
Truck
735
121
427.2
601.1
405.0
408.9
407.3
416.9
Truck—NTab
443
91
315.7
470.7
302.4
309.1
305.4
307.8
Truck—Tab
257
40
175.2
280.5
172.0
174.9
172.2
175.2
Foot
129
21
899.3
885.9
877.5
951.1
934.7
885.6
Foot—NTab
478
81
843.8
832.2
821.8
886.8
874.0
832.1
Foot—Tab
182
41
460.4
454.7
455.2
498.3
484.9
455.7
SR
All
620
58
589.5
639.7
572.0
578.9
576.7
570.3
NTab
374
46
482.7
530.3
470.1
474.1
476.5
470.7
Tab
246
40
412.9
473.1
404.0
404.2
403.7
403.7
Truck
324
31
210.3
290.5
204.5
205.7
205.7
205.9
Foot
296
27
315.3
308.2
304.7
331.3
327.0
307.9
CT
All
341
47
494.1
526.9
480.6
474.9
487.2
489.4
NTab
214
43
465.0
492.4
451.3
444.5
455.8
459.3
Tab
127
31
342.3
384.2
329.3
322.5
328.0
340.2
Truck
109
13
107.2
146.7
105.6
105.9
106.0
106.0
Foot
232
34
305.7
295.8
296.0
304.1
307.9
307.7
CM
All
217
35
411.5
425.1
399.2
397.4
399.5
395.8
NTab
147
32
390
393.9
377.0
375.9
377.6
373.4
Tab
70
20
241.7
265.8
236.1
235.7
236.1
233.4
Truck
113
13
116.6
156.3
109.9
110.8
108.9
112.3
Foot
104
22
266.6
258.7
261.3
264.9
265.9
258.6
https://doi.org/10.1371/journal.pone.0199547.t007 rather than their absolute values (E6) did not lead to improved models for the different break-
downs. In all models, and for all species networks, when the animal population was kept as a
predictor, the number of sheep was the most important predictor. rather than their absolute values (E6) did not lead to improved models for the different break-
downs. In all models, and for all species networks, when the animal population was kept as a
predictor, the number of sheep was the most important predictor. Table 8 presents the details of the final models broken down by species, season and trans-
port mode with the human and sheep population at the origin and destination and great-circle
distance as predictors. The predictive power of the models were moderate to very good,
according to the species, season and mode of transport, with AUC values ranging from 0.76 to
0.97. Similarly, although we noted some improvements for some combinations of species
and transport modalities, the use of the population difference between origin and destination PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 13 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Livestock mobility in Mauritania rather than their absolute values (E6) did not lead to improved models for the different break-
downs. In all models, and for all species networks, when the animal population was kept as a
predictor, the number of sheep was the most important predictor. Table 8 presents the details of the final models broken down by species, season and trans-
port mode with the human and sheep population at the origin and destination and great-circle
distance as predictors. The predictive power of the models were moderate to very good,
according to the species, season and mode of transport, with AUC values ranging from 0.76 to
0.97. Considering all species, seasons and transport modes, positive associations were found
between the probability of a trade connection and: i) low human population at the origin, ii)
high human population at the destination, iii) high sheep population at the origin, iv) low
sheep population at the destination, and v) a low great-circle distance (Table 8). The results
were similar for the small ruminants’ network model, whereas for the cattle model, the human
population at the origin and the sheep population at the destination were not significant. For
the camel network, the human population at the origin and the great-circle distance were not
significant. The sign of the significant effects was coherent across the sub-models, i.e. a higher
probability of trade event was always associated with low human population at the origin, or
high human population at the destination, a high population of sheep at the origin, a low pop-
ulation of sheep at the destination and a low great-circle distance, or a combination of these
effects. In addition, meaningful differences were noticed in some sub-models. For example, in
small ruminant models split by transport mode, human population at the destination, and
sheep population at the origin were not significant in the foot-movement sub-model. In
Table 7. AIC values of the logistic regression models. The models are broken down by species (SR: small ruminants, CT: cattle, CM: camels), period (Tab: Tabaski,
NTab: Non-Tabaski) and transport modality. E1-E6 correspond to different models described in Table 1. Multivariate linear model and significance of the parameters given for the selected model (E3). g
Hpop: Human population at the origin (i) and at destination (j); Spop: Sheep population at the origin (i) and at destination (j). https://doi org/10 1371/journal pone 0199547 t008 g
Hpop: Human population at the origin (i) and at destination (j); Spop: Sheep population at the origin (i) and at destination (j). Hpop: Human population at the origin (i) and at destination (j); Spop: Sheep population at the origin (i) and at destination (j). https://doi.org/10.1371/journal.pone.0199547.t008 contrast, both predictors at the origin were not significant in the truck-movement sub-model. Great-circle distance and both human and sheep population were not significant for the latter,
though. The observed and estimated trade links are illustrated in Fig 8 for these small rumi-
nant models for the Tabaski and non-Tabaski periods, and movements by trucks or on foot. During the non-Tabaski period, the fitted values correctly captured the co-existence of short-
and long-distance movements, whereas long-distance movements were prominent during the
Tabaski period. Similarly, the prediction of the truck or foot movements corresponded to their
respective long or short distances. None of the models correctly fitted the south/north
movements. None of the gravity models with the flow of animals as the response, and the same set
of predictors as in the presence/absence model detailed above, showed any significant
association. Considering all species, seasons and transport modes, positive associations were found
between the probability of a trade connection and: i) low human population at the origin, ii)
high human population at the destination, iii) high sheep population at the origin, iv) low
sheep population at the destination, and v) a low great-circle distance (Table 8). The results
were similar for the small ruminants’ network model, whereas for the cattle model, the human
population at the origin and the sheep population at the destination were not significant. For
the camel network, the human population at the origin and the great-circle distance were not
significant. The sign of the significant effects was coherent across the sub-models, i.e. a higher
probability of trade event was always associated with low human population at the origin, or
high human population at the destination, a high population of sheep at the origin, a low pop-
ulation of sheep at the destination and a low great-circle distance, or a combination of these
effects. In addition, meaningful differences were noticed in some sub-models. For example, in
small ruminant models split by transport mode, human population at the destination, and
sheep population at the origin were not significant in the foot-movement sub-model. In PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 14 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Livestock mobility in Mauritania Table 8. Multivariate linear model and significance of the parameters given for the selected model (E3). Intercept
Hpopi
Hpopj
Spopi
Spopj
Great-circle distance
AUC
All mode
All species
-4.239
-7.622 10−6
4.667 10−6
3.905 10−6
-5.104 10−6
-2.278 10−3
0.836
Small ruminants
-3.895
-1.0 10−5
4.92 10−6
2.45 10−6
-4.00 10−6
-2.129 10−3
0.840
Cattle
-4.619
-8.928 10−6
5.596 10−6
2.118 10−5
7.090 10−6
-4.416 10−3
0.884
Camel
-5.402
-1.966 10−6
4.189 10−6
4.699 10−6
-6.374 10−6
-5.632 10−4
0.790
All non-Tabaski
All species
-4.210
-1.069 10−5
4.555 10−6
4.196 10−6
-5.356 10−3
-2.415 10−3
0.840
Small ruminants
-3.809
-1.751 10−5
5.08 10−6
2.634 10−6
-3.575 10−6
-2.628 10−3
0.863
Cattle
-4.599
-1.239 10−5
5.417 10−6
2.244 10−5
4.982 10−6
-4.095 10−3
0.885
Camel
-5.563
-1.559 10−6
3.852 10−6
5.167 10−6
-6.139 10−6
-5.802 10−4
0.781
All Tabaski
All species
-5.355
-3.679 10−6
5.424 10−6
3.563 10−6
-4.107 10−6
-1.595 10−3
0.869
Small ruminants
-4.680
-5.409 10−6
5.408 10−6
1.985 10−6
-4.637 10−6
-1.378 10−3
0.863
Cattle
-6.179
-3.851 10−6
6.665 10−6
2.269 10−5
1.598 10−5
-3.287 10−3
0.901
Camel
-6.391
-4.458 10−7
5.102 10−6
4.862 10−6
-7.167 10−6
-2.454 10−4
0.837
Truck All period
All species
-5.841
-7.730 10−8
6.876 10−6
3.242 10−6
-2.072 10−5
1.126 10−3
0.934
Small ruminants
-5.432
-9.748 10−7
6.600 10−6
1.634 10−6
-8.942 10−6
8.968 10−4
0.91
Cattle
-2.574
-1.350 10−7
3.652 10−5
1.903 10−5
9.883 10−6
-1.049 10−2
0.945
Camel
-6.015
8.635 10−7
7.230 10−6
4.261 10−6
-3.349 10−5
1.590 10−3
0.966
Foot All period
All species
-2.665
-1.396 10−5
-1.435 10−6
4.702 10−6
-3.418 10−6
-5.783 10−3
0.830
Small ruminants
-2.225
-1.963 10−5
3.289 10−6
3.289 10−6
-2.996 10−6
-6.409 10−3
0.853
Cattle
-6.470
9.398 10−7
6.589 10−6
1.636 10−5
-1.105 10−4
8.720 10−4
0.908
Camel
-4.200
-3.373 10−6
-4.043 10−6
-4.940 10−6
-4.366 10−6
-1.982 10−3
0.759 Table 8. Discussion Though international movements were not addressed in this study, it is noteworthy that in
2014, the largest sheep exportation peak was observed during the “soudure”, i.e. the period sepa-
rating the end of familial cereal reserve saved after the previous harvest (millet, sorghum. . .)
from the next harvest. This was also the hot, dry season, when forage and surface water resources
were finished, and ruminant livestock starved. Therefore, the most obvious option for the Mau-
ritanian livestock farmers was to sell most of the offspring, only keeping the core of reproductive PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 15 / 21 Livestock mobility in Mauritania Fig 8. Observed and predicted movements links from the gravity model applied to the small ruminant networks. Models were applied to movements occurring within the non-Tabaski period (A), the Tabaski period (B), without
distinction of period (C-D): movements by truck (C) and by foot (D). https://doi.org/10.1371/journal.pone.0199547.g008 Fig 8. Observed and predicted movements links from the gravity model applied to the small ruminant networks. Models were applied to movements occurring within the non-Tabaski period (A), the Tabaski period (B), without
distinction of period (C-D): movements by truck (C) and by foot (D). https://doi.org/10.1371/journal.pone.0199547.g008 https://doi.org/10.1371/journal.pone.0199547.g008 https://doi.org/10.1371/journal.pone.0199547.g008 ewes and she-goats. In addition, many of the latter spent this season in the closest areas with
more abundant pastoral resources, i.e. in Senegal and Mali. Short- and mid-distance move-
ments–most of them by foot, thus allowed pastoralists to exploit more suitable environmental
conditions and reduce the economic cost of feeding the animals. In future years, when the
Tabaski feast occurs during the soudure period, the relative importance of truck vs. foot ewes and she-goats. In addition, many of the latter spent this season in the closest areas with
more abundant pastoral resources, i.e. in Senegal and Mali. Short- and mid-distance move-
ments–most of them by foot, thus allowed pastoralists to exploit more suitable environmental
conditions and reduce the economic cost of feeding the animals. In future years, when the
Tabaski feast occurs during the soudure period, the relative importance of truck vs. foot PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 16 / 21 Livestock mobility in Mauritania movements may change and add to each other. Moreover, as the Tabaski is a mobile festivity
which is celebrated annually among Muslims worldwide 70 days after Ramadan, a time slip of
10 days upstream occurs each year. Discussion An overlap with specific diseases vectors may thus occurs
depending on the occurrence of the feast (year-dependence of the epidemic risk). Around Sene-
gal river, Aedes mosquitoes show a peak in the periods of July-August and September during the
rainy season. Thanks to the happening of the Tabaski, held in the last years between September
and October, the risk of infection was elevated [36]. The risk remains high every year due to the
large volume of moved animals and the traders’ preference for truck transportation and its fast-
ness which allow viremic animal introduction on remote locations. The low values for the national network diameter, the presence of hubs and the low values
of the epidemic threshold, indicate that the network could be prone to transmission of dis-
eases. This means that also a lowly transmitted disease, once introduced in the national net-
work, could reach all nodes (the network’s single component) in a short amount of time (small
diameter). On the other hand, independently of the species considered, the mobility network
is prone to fragmentation due to targeted intervention based on nodes activity (in-degree and
in-weight). After the intervention, the network is decomposed in a set of smaller subnetworks,
and virus can circulate only among nodes of the same sub-network. Vaccinating animals in
largest markets (nodes with largest number of incoming animals) or closing these markets,
could result in a very effective way of controlling the epidemic spread. Regarding internal movements, the gravity models correctly predicted the probability of a
trade connection and their interpretation was straightforward. Locations with few sheep and
high human populations, i.e. urban consumption centers, acted as movement sinks. Con-
versely, areas with high sheep and low human populations, i.e. rural livestock farming areas,
acted as movement sources. For a similar level of production and demand, short distance
movements were more likely that long distance ones. In addition, Nouakchott, the capital city
located on the coast, strongly influenced the network structures. Almost a quarter of the total
population of Mauritania lives in Nouakchott (according to the National Statistical Office
http://www.ons.mr/) and because of this, it is the largest terminal market with an incoming
volume of almost 30% of the national one. In preparation for Tabaski, almost 50% of the total
traded small ruminants are sold in Nouakchott. The largest majority of livestock provisioning
Nouakchott markets comes from the South-Western area of Mauritania. PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Discussion The
cost-distance was estimated using the accessibility friction map which is based on the road net-
work and land-use data, two expected drivers of the livestock trade networks. However, the
friction layer might not be adapted to the specific constrains of animal movements in arid and
hyper-arid environments. For example, the presence of water points or stopover feeding sites
along the roads might be more relevant factors than those included in the global friction sur-
face. Therefore, further work is needed to build friction surfaces better suited to the specific
constrains of animal movement in this environment. The movement survey database also included volumes of the livestock trade flows between
locations. However, these quantitative data were not correctly predicted by any of the models. This failure might be related to two non-exclusive factors. First, when a link was established,
the volume of traded animals did not vary much, and not proportionally to the deficit in
demand or to the distance. Second, there might be noise in the data related to inaccurate
replies during the interviews with field veterinary officers. However, considering the outer
trade data (international movements), estimates from this survey closely matched importation
estimates from the Senegalese Veterinary Services for the Tabaski period. Because these data
were of crucial importance to stabilize sheep price (and thus prevent social troubles), all efforts
were made for an accurate monitoring of sheep importation. Therefore, this good match pro-
vided a partial validation for the quality of data produced by this survey, as well as the good
predictive power obtained with the presence/absence models. An important question underlying these analyses is the role that animal mobility might
play in the spread of animal and zoonotic diseases in the region. For example, animal move-
ments may contribute to the spread of Rift Valley fever (RVF) in the Sahelian region of Mauri-
tania, and from Mauritania to Senegal [37,38]. The network structure predicted by this model
may provide input for an epidemiological model of RVF or other important diseases affecting
the region, such as Peste des petits ruminants (PPR) [6]. Gravity models are an important method in economic analyses, used mainly to predict
bilateral flow of population and goods between two distant locations [39]. They were recently
adapted to describe the spread of biological agents [33,40–42]. To our knowledge, this study is
a first attempt to predict livestock mobility patterns. Discussion A continuous flux of
animals is ensured by stockists who collect animals at collection market and transfer them by
truck to the capital city. Here, stockists buy imported goods, arrived at the international port,
to sell at collection markets. It might look odd that the sheep population at the origin and destination of movements
was the best predictor for all species-level networks. A possible explanation might be that the
spatial distributions of all ruminant species were positively correlated, thus making the sheep
population a confounding factor for the other ruminant populations. This assumption was
corroborated by the dominance of small ruminant movements: 1.3 million vs. 0.39 million for
cattle and 0.37 million for camels. Therefore, the small ruminant network probably influenced
all other ruminant trade activities. The seasonal models had good predictive power for the models of both truck or foot move-
ments. These models captured the dominance of long-distance movements (truck movement
network), of short-distance movements (foot movement network) and different combinations
of short and long-distance movements (Tabaski vs non-Tabaski periods). However, the models
did not capture some of the trade links. For example, they failed to predict the link to the
northern city of Zouerate, which was involved in the small ruminant and camel trading net-
works. Located in the desert and with a low accessibility, this city has a large iron mining
industry, and virtually no local production. The working population there may be much higher PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 17 / 21 Livestock mobility in Mauritania than the population estimates in the Worldpop population database, which maps people
according to their residence, not their working place. Such high populations of workers may
generate high demand for small ruminant and camel meat, and camel milk. Armed conflicts
may also influence the pattern of demand with drivers not accounted for in the models. For
example, an important flow of human population was reported in south-eastern Mauritania
coming from Mali after the terrorist attack of January 2013. These refugees established camps
close to the border with Mali: their population may have influenced the pattern of demand
and production in a way that was not captured by the model. The use of cost-distance instead of great-circle distance did not improve the models. Discussion Besides obvious applications in the field
of pastoral economics, it opens new perspectives for predicting the transmission of pathogens
such as PPR or RVF viruses in animal meta-populations, or extending existing models of post-
vaccination immunity persistence at the population level [43]. Also, similar models might be
used at the regional level–e.g, between Maghreb or Sahel countries, or between Sahel and
Maghreb regions, etc.–to validate and compliment (un)available information on transbound-
ary animal movements. Acknowledgments This study was funded by EU grant FP7-613996 VMERGE and is catalogued by the VMERGE
Steering Committee as VMERGE000 (http://www.vmerge.eu). The contents of this publication
are the sole responsibility of the authors and don’t necessarily reflect the views of the European
Commission. Supporting information S1 File. Small ruminant, cattle and camel trading networks and related centrality measures
for Mauritania in 2014. Node size show the importance of the measured centrality values. in 18 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
July 18, 2018 Livestock mobility in Mauritania and out-weight measures were scaled on the total volume of traded livestock; eigenvector cen-
trality (centrality measure) were scored from 0 to 1, betweenness was considered for the frac-
tion of paths passing through the node. (DOCX) and out-weight measures were scaled on the total volume of traded livestock; eigenvector cen-
trality (centrality measure) were scored from 0 to 1, betweenness was considered for the frac-
tion of paths passing through the node. (DOCX) Author Contributions Conceptualization: Gae¨lle Nicolas, Marius Gilbert. Data curation: Gae¨lle Nicolas. Formal analysis: Gae¨lle Nicolas. Formal analysis: Gae¨lle Nicolas. Methodology: Gae¨lle Nicolas, Marius Gilbert. Methodology: Gae¨lle Nicolas, Marius Gilbert. Supervision: Marius Gilbert. Supervision: Marius Gilbert. Validation: Gae¨lle Nicolas. Validation: Gae¨lle Nicolas. Visualization: Gae¨lle Nicolas. Visualization: Gae¨lle Nicolas. Writing – original draft: Gae¨lle Nicolas. Writing – original draft: Gae¨lle Nicolas. Writing – review & editing: Gae¨lle Nicolas, Andrea Apolloni, Caroline Coste, G. R. William
Wint, Renaud Lancelot, Marius Gilbert. PLOS ONE | https://doi.org/10.1371/journal.pone.0199547
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ISSN 2617-409X Actual problems of modern medicine. Issue 11, 2023 ISSN 2617-409X DOI: https://doi.org/10.26565/2617-409X-2023-11-03
УДУ 616.155.3-02:616.248-053.2/.5 DOI: https://doi.org/10.26565/2617-409X-2023-11-03
УДУ 616.155.3-02:616.248-053.2/.5 Т. І.Лядова A, C, D , О. В. Волобуєва A, C, D, В. Г. Чернуський A, C, D,
М. М. Попов A, C, D, Г. В. Летяго B, E, К. В. Павлікова B, E
chernusky@karazin.ua Т. І.Лядова A, C, D , О. В. Волобуєва A, C, D, В. Г. Чернуський A, C, D,
М. М. Попов A, C, D, Г. В. Летяго B, E, К. В. Павлікова B, E
chernusky@karazin ua A – концепція та дизайн дослідження; B – збір даних; C – аналіз та інтерпретація даних; D – написання статті; E – редагування статті;
F – остаточне затвердження статті АНОТАЦІЯ. Відомо, що під впливом специфічних мітогенів сенсибілізовані Т-лімфоцити продукують в
культуральне середовище №199 цитокіни, які розподіляються на дві фракції: F1 та F2. Перша (F1) з них
визначає патогенез БА у дітей і цитокіни цієї фракції виробляються при імунопатологічних реакціях, а
друга (F2) відповідає за імунологічні параметри активації Т-системи імунітету та їх регуляції. Мета. Дослідити активність Т-лімфоцитів активованих бронхолегеневими антигенами та її значення у
дітей хворих на БА. АНОТАЦІЯ. Відомо, що під впливом специфічних мітогенів сенсибілізовані Т-лімфоцити продукують в
культуральне середовище №199 цитокіни, які розподіляються на дві фракції: F1 та F2. Перша (F1) з них
визначає патогенез БА у дітей і цитокіни цієї фракції виробляються при імунопатологічних реакціях, а
друга (F2) відповідає за імунологічні параметри активації Т-системи імунітету та їх регуляції. у
фр
р
р
у
р
друга (F2) відповідає за імунологічні параметри активації Т-системи імунітету та їх регуляції. Мета. Дослідити активність Т-лімфоцитів активованих бронхолегеневими антигенами та її значення у
дітей хворих на БА. ру
(
)
у
р
р
у
у
р
у
Мета. Дослідити активність Т-лімфоцитів активованих бронхолегеневими антигенами та її значення у
дітей хворих на БА. Матеріали та методи. Для визначення цитокінів сенсибілізованих Т-лімфоцитів, якісний склад цитокінів
Т-лімфоцитів в культуральній рідині №199 використовували дискфорез в поліакриламідному гелі у 105
дітей, хворих на БА та 20 здорових дітей у віці від 5 до 14 років зі специфічними антигенами бронхів і
легеневої тканини, а також з неспецифічним мітогеном ФГА фірми «Reanal». Результати. При БА у дітей більш кількісна наявність полос у дискфореграмах виявляється в залежності
від тяжкості перебігу захворювання та характеризується високою продукцією цитокінів F1-фракції
сенсибілізованими Т-лімфоцитами. Структурний аналіз цитокінів Т-лімфоцитів в дискфореграмах при
БА в умовах мітогенної активності сенсибілізованих Т-лімфоцитів комутованих з ліпополісахаридними
антигенами бронхів та легеневої тканини, показав розбіжності в залежності від тяжкості перебігу
захворювання у вигляді наявності двох полос, які визначені товщиною, при загальній нумерації від старту
мають порядковий номер 8 та 9. р
р
Висновки. Встановлено, що тяжкість перебігу БА корелює з вираженістю спектру продукції цитокінів
сенсибілізованих Т-лімфоцитів в сироватку крові у дітей, хворих на БА, та дозволяє з високою точністю
діагностувати тяжкість перебігу захворювання. Ключові слова: бронхіальна астма, діти, цитокіни сенсибілізованих Т-лімфоцитів, дискфорез,
поліакриламідний гель, гіперчутливість польного типу, бронхолегеневі антигени Ключові слова: бронхіальна астма, діти, цитокіни сенсибілізованих Т-лімфоцитів, дискфорез,
поліакриламідний гель, гіперчутливість польного типу, бронхолегеневі антигени Для цитування: Лядова ТІ, Волобуєва ОВ, Чернуський ВГ, Попов ММ, Летяго ГB, Павлікова КВ. © Лядова Т. І., Волобуєва О. В., Чернуський В. Г., Попов М. М., Летяго Г. В., Павлікова К. В., АРММ, 2023 21 21 A – концепція та дизайн дослідження; B – збір даних; C – аналіз та інтерпретація даних; D – написання статті; E – редагування статті;
F – остаточне затвердження статті ВИЗНАЧЕННЯ АКТИВНОСТІ Т-
ЛІМФОЦИТІВ АКТИВОВАНИХ БРОНХОЛЕГЕНЕВИМИ АНТИГЕНАМИ ТА ЇХ ІМУНОДІАГНОСТИЧНЕ ЗНАЧЕННЯ У ДІТЕЙ, ХВОРИХ
НА БРОНХІАЛЬНУ АСТМУ. Актуальні проблеми сучасної медицини. 2023;11:20-28. DOI: https://doi.org/10.26565/2617-409X-2023-11-03 Для цитування: Лядова ТІ, Волобуєва ОВ, Чернуський ВГ, Попов ММ, Летяго ГB, Павлікова КВ. ВИЗНАЧЕННЯ АКТИВНОСТІ Т-
ЛІМФОЦИТІВ АКТИВОВАНИХ БРОНХОЛЕГЕНЕВИМИ АНТИГЕНАМИ ТА ЇХ ІМУНОДІАГНОСТИЧНЕ ЗНАЧЕННЯ У ДІТЕЙ, ХВОРИХ
НА БРОНХІАЛЬНУ АСТМУ. Актуальні проблеми сучасної медицини. 2023;11:20-28. DOI: https://doi.org/10.26565/2617-409X-2023-11-03 Інформація про авторів
Тетяна Іванівна Лядова, д. м. н.,
професор, декан медичного факультету,
Харківський національний університет
імені В. Н. Каразіна, майдан Свободи, 6,
Харків,
Україна,
61022,
e-mail:
В’ячеслав Григорович Чернуський,
д. м. н., професор, професор кафедри
інфекційних
хвороб
та
клінічної
імунології,
медичний
факультет,
Харківський національний університет
імені В. Н. Каразіна, майдан Свободи, 6,
Ганна Володимирівна Летяго, к. м. н,
доцент,
доцент
кафедри
педіатрії,
медичний
факультет,
Харківський
національний університет імені В. Н. Каразіна, майдан Свободи, 6, Харків,
Україна,
61022,
e-mail: ВИЗНАЧЕННЯ АКТИВНОСТІ Т-ЛІМФОЦИТІВ АКТИВОВАНИХ
БРОНХОЛЕГЕНЕВИМИ АНТИГЕНАМИ ТА ЇХ ІМУНОДІАГНОСТИЧНЕ ЗНАЧЕННЯ
У ДІТЕЙ, ХВОРИХ НА БРОНХІАЛЬНУ АСТМУ A – концепція та дизайн дослідження; B – збір даних; C – аналіз та інтерпретація даних; D – написання статті; E – редагування статті;
F – остаточне затвердження статті ISSN 2617-409X Актуальні проблеми сучасної медицини. Випуск 11, 2023 фітогемаглютинином
(ФГА)
та
специфічними
ліпополісахаридними
антигенами бронхів та легеневої тканини або
(для ряду цитокінів) інтактних Т-лімфоцитів
з мітогеном. До неї відносяться: фактор
інгібуючий міграцію макрофагів; фактор
інгібуючий
розповсюдження
макрофагів;
фактор
агрегуючий
проліферацію;
хемотоксичний
фактор;
кластогенний
фактор; фактор, активуючий фагоцитоз;
цитотоксичний
фактор
та
ін. Фракція
F2 – друга група цитокінів, які присутні в
середині Т-лімфоцитів: фактор переносу;
проникністю
лімфовузлів;
бластогенний;
захищення макрофагів від некротизуючої дії
бактерій
та
ін. Ефекторні
цитокіни
Т-лімфоцитів дітей, хворих на БА фракції
F1
можуть
вивільнятися
під
впливом
специфічних
антигенів
бронхолегеневої
системи. Дана
група
цитокінів
сенсибілізованих
Т-лімфоцитів
може
набувати
антигенних
властивостей
та
проявляти цитотоксичну дію на клітинно-
тканинних
структурах
бронхолегеневої
системи, переводячи патологічний процес на
аутоімунну основу 1, 6. Таким чином,
цитокіни
фракції
F1
сенсибілізованих
Т-лімфоцитів має пряме відношення до
патогенезу захворювання, що раніше не
досліджувалося. Цей факт обумовив мету
нашої роботи. Вступ Вступ За останні роки у всьому світі, в тому числі
і в Україні, відмічається тенденція до
зростання захворюваності на бронхіальну
астму (БА) у дітей різних вікових груп та її
тяжкого перебігу. В зв’язку з цим проблема
діагностики
та
диференціації
ступенів
тяжкості перебігу БА набуває важливого
значення 1, 2, 5, 6. Нові погляди на
механізми розвитку БА дозволили розробити
концепцію її патогенезу, згідно якої основу
астми складає хронічне алергічне запалення
бронхолегеневої системи з домінуванням
алергічних реакцій швидкого (АРШТ) та
повільного типу (АРПТ) з включенням
аутоімунного компонента. Ця концепція
передбачає розробку нових підходів до
диференційної імунодіагностики ступенів
тяжкості перебігу та контролю ефективності
проведеної терапії 1, 5. Під впливом поліетіологічних факторів
Т-лімфоцити в сироватці крові дітей, хворих
на БА, продукують прозапальні цитокіни, які
визначають
специфіку
патологічного
процесу в бронхолегеневій системі. Згідно з
прийнятими
положеннями
цитокіни
Т-лімфоцитів, які характеризують АРПТ – це
розчинні молекули білкової природи, які
вивільняються
Т-лімфоцитами
або
екстрагуються з них і здатні здійснювати ряд
ефектів АРПТ. Проводячи оцінку ролі
клітинного
імунітету
як
захисного
і
пошкоджуваного механізму при БА у дітей,
ми
дослідили
цитокінову
функцію
Т-лімфоцитів в залежності від тяжкості
перебігу БА у дітей 3, 6, 7. Було показано,
що під впливом специфічних мітогенів
сенсибілізовані Т-лімфоцити продукують в
культуральне середовище №199 цитокіни, які
розподіляються відносно альбуміну на дві
фракції: F1 та F2. Перша (F1) з них визначає
патогенез БА у дітей і цитокіни цієї фракції
виробляються
при
імунопатологічних
реакціях,
а
друга
(F2)
відповідає
за
імунологічні параметри активації Т-системи
імунітету та їх регуляції. Фракція F1 – група
цитокінів,
що
найбільш
інтенсивно
вивчається. Вони
виявляються
після
інкубації in vitro T- лімфоцитів дітей, хворих
на
БА,
з
неспецифічним
мітогеном Мета – дослідити активність Т-лімфоцитів
активованих бронхолегеневими антигенами
та її імунодіагностичне значення у дітей,
хворих на БА. Матеріали та методи Інформація про авторів Харків,
Україна,
61022,
e-mail:
t.lyadova@karazin.ua,
ORCID
ID
https://orcid.org/0000-0002-5892-2599
Ольга Вікторівна Волобуєва, к. м. н.,
доцент, завідувач кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-
mail: o.volobyeva@karazin.ua, ORSID ID
https://orcid org/0000 0002 5569 1748
імені В. Н. Каразіна, майдан Свободи, 6,
Харків,
Україна,
61022,
e-mail:
chernusky@karazin.ua,
ORSID
ID
https://orcid.org/0000-0001-5657-9486. Микола Миколайович Попов, д. м. н.,
професор, професор кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-mail
: mykola m popov@karazin ua ORSID ID
Україна,
61022,
e-mail:
letyago@karazin.ua,
ORSID
ID
https://orcid.org/0000-0002-6327-1321. Ксенія
Вячеславівна
Павлікова,
к. м. н., асистент кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-
mail: k.pavlikova@karazin.ua, ORSID ID
https://orcid org/0000 0002 1228 4915 D
ID
599
к. м. н.,
р
Харків,
Україна,
61022,
e-mail:
chernusky@karazin.ua,
ORSID
ID
https://orcid.org/0000-0001-5657-9486. р
letyago@karazin.ua,
ORSID
ID
https://orcid.org/0000-0002-6327-1321. Ксенія
Вячеславівна
Павлікова, Харків,
Україна,
61022,
e-mail:
chernusky@karazin.ua,
ORSID
ID
https://orcid.org/0000-0001-5657-9486. t.lyadova@karazin.ua,
ORCID
ID
https://orcid.org/0000-0002-5892-2599
Ольга Вікторівна Волобуєва, к. м. н.,
Харків,
Україна,
61022,
e-mail:
chernusky@karazin.ua,
ORSID
ID
https://orcid.org/0000-0001-5657-9486. letyago@karazin.ua,
ORSID
ID
https://orcid.org/0000-0002-6327-1321. Ксенія
Вячеславівна
Павлікова, Ольга Вікторівна Волобуєва, к. м. н.,
доцент, завідувач кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-
mail: o.volobyeva@karazin.ua, ORSID ID
https://orcid.org/0000-0002-5569-1748. Ольга Вікторівна Волобуєва, к. м. н.,
доцент, завідувач кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-
mail: o.volobyeva@karazin.ua, ORSID ID
https://orcid.org/0000-0002-5569-1748. Ксенія
Вячеславівна
Павлікова,
к. м. н., асистент кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-
mail: k.pavlikova@karazin.ua, ORSID ID
https://orcid.org/0000-0002-1228-4915. Микола Миколайович Попов, д. м. н.,
професор, професор кафедри інфекційних
хвороб та клінічної імунології, медичний
факультет, Харківський національний
університет імені В. Н. Каразіна, майдан
Свободи, 6, Харків, Україна, 61022, e-mail
: mykola.m.popov@karazin.ua, ORSID ID
https://orcid.org/0000-0002-5759-9654. 22 ISSN 2617-409X Матеріали та методи Проведений
дискфорез
в
поліакриламідному
гелі
цитокінів
сенсибілізованих Т-лімфоцитів у 105 дітей,
хворих на БА та 20 здорових дітей у віці від
5 до 14 років зі специфічними антигенами
бронхів і легеневої тканини, а також з
неспецифічним
мітогеном
ФГА
фірми
«Reanal». Діагноз БА встановлювали згідно з
рекомендаціями
GINA
та
наказу
МОЗ
України № 2856 від 23.12.2021 р. Обстежені
групи
були
репрезентативними,
рандомізовані по віку, полу, формам та
ступеням тяжкості перебігу захворювання, в
періоді
загострення
захворювання
до
проведення терапії рекомендованої GINA та 23 Actual problems of modern medicine. Issue 11, 2023 ISSN 2617-409X МОЗ України. В ході роботи визначали
розподіл
та
якісний
склад
цитокінів
Т- лімфоцитів в культуральній рідині № 199
методом
дискелетрофореза
в
поліакриламідному гелі під впливом струму
на стовпчики 2-5 мА частина цитокінів, які
мігрують
зі
швидкістю
преальбуміну
відносяться до фракції F1, а зі швидкість
альбуміну F2 - до фракції F2 2. поліакриламідному гелі. Встановлено, що
при БА у дітей більш кількісна наявність
полос і дискфореграмах виявляється в
залежності
від
тяжкості
перебігу
захворювання та характеризується високою
продукцією
цитокінів
F1-фракції
сенсибілізованими Т-лімфоцитами, що дає
підставу судити про їх участь в патогенезі
захворювання (Рис. 1, 2, 3). МОЗ України. В ході роботи визначали
розподіл
та
якісний
склад
цитокінів
Т- лімфоцитів в культуральній рідині № 199
методом
дискелетрофореза
в
поліакриламідному гелі під впливом струму
на стовпчики 2-5 мА частина цитокінів, які
мігрують
зі
швидкістю
преальбуміну
відносяться до фракції F1, а зі швидкість
альбуміну F2 - до фракції F2 2. в поліакриламідному гелі під впливом струму
на стовпчики 2-5 мА частина цитокінів, які
мігрують
зі
швидкістю
преальбуміну
відносяться до фракції F1, а зі швидкість
альбуміну F2 - до фракції F2 2. Структурний
аналіз
дискфореграм
цитокінів
Т-лімфоцитів
отриманих
від
хворих на БА в умовах мітогенної активності
сенсибілізованих Т-лімфоцитів комітованих
з ліпополісахаридними антигенами бронхів
та легеневої тканини, показав їх відмінність:
дискфореграми цитокінів Т-лімфоцитів при
активації антигеном бронхів відрізнялися від
дискфореграм цитокінів Т-лімфоцитів при їх
активації
антигеном
легеневої
тканини
наявністю двох полос, які мають якісні
відмінності визначені товщиною полос, при
загальній
нумерації
від
старту
мають
порядковий номер 8 та 9. Відношення полос
8 та 9 до фракції F1 цитокінів Т-лімфоцитів
вказує на їх участь в імунопатогенезі БА та
обумовлює тяжкість перебігу захворювання
(Рис. 3). Діагностична
значимість
дослідження
полягає у тому, що Т-лімфоцити виділені з
крові дітей, хворих на БА в культуральному
середовище
№199
можуть
активувати
цитотоксичну функцію лише в присутності
специфічних
мітогенів
(антигенів). Матеріали та методи Для
Т-лімфоцитів дітей, хворих на БА, такими
мітогенами є ліпополісахаридні антигени
бронхів і легеневої тканини 2. Результати ті їх обговорення Результати ті їх обговорення При
проведенні
дискелектрофорезу
цитокінів Т-лімфоцитів здорових дітей не
виявлено прозапальних цитокінів фракції F1,
які розганяються в диск електрофорезі
поліакриламідного гелю в преальбуміні,
звертає на себе увагу продукція регуляторних
цитокінів,
які
розганяються
в
поліакриламідному гелі після альбуміну
(Рис. 1, 2, 3 полоси 5, 6, 7 в пробірці 1). в Треба відмітити, що основна фізіологічна
функція цитокінів Т-лімфоцитів, складається
з регуляції імунітету, забезпечується їх
синтезом у такій кількості, що повністю
витрачається на імунологічну реакцію. У разі
імунопатологічної
реакції
Т-лімфоцити
продукуються
в
надмірній
кількості
і
виявляються у вільному або у комплексному
стані в різних рідинах органів і систем та
можуть самостійно проявляти направлену
цитотоксичну дію на клітинно-тканинну
структуру бронхолегеневої системи у дітей,
хворих на БА. При комутації Т-лімфоцитів дітей, хворих
на БА і здорових з неспецифічним мітогеном
фітогемаглютиніном (ФГА) (Рис. 1, 2, 3,
пробірка 3) не виявлений весь спектр
продукції цитокінів Т-лімфоцитів, це вказує
на
те,
що
на
неспецифічний
мітоген
сенсибілізовані Т-лімфоцити, дітей, хворих
на
БА,
відповідають
неспецифічною
продукцією
цитокінів
F1
фракції,
які
розганяються
в
дискелектрофорезі
поліакриламідного
гелю
зі
швидкістю
преальбумінп. Регуляторні
цитокіни
Т-лімфоцитів
під
впливом
мітогенів
в
сироватці крові розганяються зі швидкістю
альбуміну
представлені
лише
однією
полосою (Рис. 1, 2, 3, пробірка 2, полоса 7,
11). При дискелектрофорезі сироватки крові
дітей, хворих БА, виявлено полоси, які
повністю ідентичні полосам дискфореграм
цитокінів сенсибілізованих Т-лімфоцитів у
культуральне середовище №199. При цьому
слід відмітити, що якісні характеристики
виявлених в крові цитокінів Т-лімфоцитів
(товщина полос) і частота їх зустрічі
розрізнялись в залежності від тяжкості
перебігу захворювання. Так, мінімальна Відмічається, що якісний склад цитокінів
Т-лімфоцитів
в
дискфореграмах
дітей,
хворих на БА, суттєво вирізняються по
кількісному складу, за розміром визначених
полос,
рухомістю
в
стовпчиках 24 Актуальні проблеми сучасної медицини. Випуск 11, 2023 ISSN 2617-409X продукцію
цитокінів
сенсибілізованими
Т- лімфоцитами
на
антиген
легеневої
тканини (АГЛ) і в меншій ступені на антиген
бронхів (АГБ), що свідчить про напружений
контроль
зі
сторони
імунної
системи
антигенів легеневої тканини при даній
ступені тяжкості перебігу захворювання
(Рис. 1). товщина і найменша частота полос цитокінів
Т-лімфоцитів фракції F1 встановлена у дітей
з тяжким перебігом БА на специфічний
антиген легеневої тканини (Рис. 1, 3). При легкому перебігу БА у дітей до
проведення протоколу терапії GINA (2022)
дискфореграми цитокінів Т-лімфоцитів зі
специфічними
антигенами
бронхів
і
легеневої
тканини
показують
високу Рис. 1 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, легкий перебіг, період
загострення до проведення протоколу терапії
Fig. Результати ті їх обговорення 1 Spectrum of T-lymphocyte cytokine disc patterns of children with BA, mild course, period of exacerbation
before the therapy protocol Рис. 1 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, легкий перебіг, період
загострення до проведення протоколу терапії
Fig. 1 Spectrum of T-lymphocyte cytokine disc patterns of children with BA, mild course, period of exacerbation
before the therapy protocol 1 – Дискфореграма цитокінів Т-лімфоцитів здорових дітей; 2 – Дискфореграма сироватки крові дітей, хворих на БА, легкий перебіг; 3 – Дискфореграма цитокінів Т-лімфоцитів, культивованих з ФГА;
ф
і і
і ф
і 4 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГБ;
5
Д
ф
і і Т
і ф
і
АГЛ 5 – Дискфореграма цитокінів Т-лімфоцитів кул 6 – Контроль альбумін. від старту під номерами 8 і 9, як в
культуральне середовище № 199, так і в
сироватку крові, що може вказувати на
розвиток
аутоімунного
компоненту
в
структурах бронхолегеневої тканини (Рис. 2). При
середньотяжкому
перебігу
БА
відмічається
підвищення
цитокінової
продукції Т-лімфоцитами на специфічні
ліпополісахаридні
антигени
бронхів
та
легеневої тканини з появою додаткових
полос цитокінів F1 фракції, що розганяються 25 Actual problems of modern medicine. Issue 11, 2023 ISSN 2617-409X Рис. 2 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, середньотяжкий перебіг, період
загострення
Fig. 2 The spectrum of T-lymphocyte cytokine cytokines of children with BA, moderate course, period of exacerbation Рис. 2 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, середньотяжкий перебіг, період
загострення
Fig. 2 The spectrum of T-lymphocyte cytokine cytokines of children with BA, moderate course, period of exacerbation trum of T-lymphocyte cytokine cytokines of children with BA, moderate course, period of exacerbatio 1 – Дискфореграма цитокінів Т-лімфоцитів здорових дітей; – Дискфореграма цитокінів Т-лімфоцитів здорових дітей; – Дискфореграма сироватки крові дітей, хворих на БА, середньотяжкий перебіг; 3 – Дискфореграма цитокінів Т-лімфоцитів, культивованих з ФГА; 4 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГБ; 5 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГЛ; 6 – Контроль альбумін. D4, E4, що обумовлює клінічні прояви
захворювання
у
вигляді
розвитку
бронхоспазму, набряку і гіперсекреції слизу. При
тяжкому
перебігу
БА
у
дітей
відмічається висока продукція цитокінів F1
фракції сенсибілізованими Т-лімфоцитами на
специфічні
ліпополісахаридні
антигени
бронхів і легеневої тканини з гіперпродукцією
8 і 9 як в культуральне середовище № 199, так
і в сироватку крові, що свідчить про
прогресуючий
аутоімунний
процес
в
структурах бронхів та легеневої тканини (Рис. 3). D4, E4, що обумовлює клінічні прояви
захворювання
у
вигляді
розвитку
бронхоспазму, набряку і гіперсекреції слизу.
З проведеного дослідження видно, що
тільки у контрольній групі здорових дітей
(пробірка 1 на рис. 1, 2, 3,) має місце продукція
F2
фракція цитокінів Т-лімфоцитів, яка
розганяється в поліакриламідному гелі зі
швидкістю альбуміну та має регуляторні
властивості,
нормалізує
імунологічний
гомеостаз
організму
дитини.
Регуляторні
цитокіни F2 фракції відсутні у хворих на БА
при середньотяжкому та тяжкому перебігах
БА,
що
обумовлює
хронічний
перебіг
захворювання.
Тому
необхідна подальша
розробка терапевтичних підходів з метою
зняття
патологічного
впливу
АРПТ
на
структури бронхолегеневої системи у дітей
хворих на БА. 4. Honkoop P. Astma. TvPO. 2021;16(6):41-43. DOI:
https://doi.org/10.1007/s12503-021-0905-z
5. Patel SJ, Teach SJ. Asthma. Pediatrics in Review.
2019;40(11):549-567.
DOI:
https://doi.org/10.1542/pir.2018-0282
6. Umanets TR, Kreposniak AA. Asthma Combined With
Eosinophilic Esophagitis In Children: Modern Condition Of
The Problem. Asthma Allergy. 2019;1:36-43. DOI:
https://doi.org/10.31655/2307-3373-2019-1-36-43 Результати ті їх обговорення антигенів бронхолегеневої системи, як в
надосадову рідину, так і в сироватку крові
можна з високою точністю діагностувати
тяжкість перебігу захворювання. антигенів бронхолегеневої системи, як в
надосадову рідину, так і в сироватку крові
можна з високою точністю діагностувати
тяжкість перебігу захворювання. Результати ті їх обговорення Згідно сучасній концепції патогенезу БА у
дітей в його розвитку приймають участь
ефекторні
клітини
(тучні,
макрофаги,
еозинофіли, тромбоцити), які продукують
хемоатрактанти
з
чітко
визначеними
властивостями
бронхоконстрикції,
яка
підтримується та реалізується через цитокіни
F1 фракції сенсибілізованих Т-лімфоцитів та їх
вплив на метаболізм арахідонової кислоти на
ліпоксигеназний шлях синтезу лейкотриєнів C4, захворювання
у
вигляді
розвитку
бронхоспазму, набряку і гіперсекреції слизу. З проведеного дослідження видно, що
тільки у контрольній групі здорових дітей
(пробірка 1 на рис. 1, 2, 3,) має місце продукція
F2
фракція цитокінів Т-лімфоцитів, яка
розганяється в поліакриламідному гелі зі
швидкістю альбуміну та має регуляторні
властивості,
нормалізує
імунологічний
гомеостаз
організму
дитини. Регуляторні
цитокіни F2 фракції відсутні у хворих на БА
при середньотяжкому та тяжкому перебігах
БА,
що
обумовлює
хронічний
перебіг
захворювання. Тому
необхідна подальша
розробка терапевтичних підходів з метою
зняття
патологічного
впливу
АРПТ
на
структури бронхолегеневої системи у дітей
хворих на БА. З проведеного дослідження видно, що
тільки у контрольній групі здорових дітей
(пробірка 1 на рис. 1, 2, 3,) має місце продукція
F2
фракція цитокінів Т-лімфоцитів, яка
розганяється в поліакриламідному гелі зі
швидкістю альбуміну та має регуляторні
властивості,
нормалізує
імунологічний
гомеостаз
організму
дитини. Регуляторні
цитокіни F2 фракції відсутні у хворих на БА
при середньотяжкому та тяжкому перебігах
БА,
що
обумовлює
хронічний
перебіг
захворювання. Тому
необхідна подальша
розробка терапевтичних підходів з метою
зняття
патологічного
впливу
АРПТ
на
структури бронхолегеневої системи у дітей
хворих на БА. 26 Актуальні проблеми сучасної медицини. Випуск 11, 2023 ISSN 2617-409X ISSN 2617-409X Рис. 3 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, тяжкий перебіг, період
загострення
Fig. 3 Spectrum of T-lymphocyte cytokine cytokines of children with BA, severe course, period of exacerbation Рис. 3 Спектр дискфореграм цитокінів Т-лімфоцитів дітей, хворих на БА, тяжкий перебіг, період
загострення
Fig. 3 Spectrum of T-lymphocyte cytokine cytokines of children with BA, severe course, period of exacerbation rum of T-lymphocyte cytokine cytokines of children with BA, severe course, period of exacerbation 1 – Дискфореграма цитокінів Т-лімфоцитів здорових дітей;
2 – Дискфореграма сироватки крові дітей, хворих на БА, тяжкий перебіг
3 – Дискфореграма цитокінів Т-лімфоцитів, культивованих з ФГА;
4 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГБ;
5 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГЛ; 1 – Дискфореграма цитокінів Т-лімфоцитів здорових дітей; 4 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГБ; ф р
р
ф
у
5 – Дискфореграма цитокінів Т-лімфоцитів культивованих з АГЛ; 6 – Контроль альбумін. 6 – Контроль альбумін. Висновки 1.При
застосування
специфічних
ліпополісахаридних
антигенів
бронхів
та
легеневої
тканини
в
дискелектрофорезі
виявлені
цитокіни
сенсибілізованих
Т-лімфоцитів як в надосадовій рідині, так і в
сироватці крові дітей, хворих на БА. 4.Дискфореграми
сенсибілізованих
Т-лімфоцитів дітей, хворих на БА, показали
чітку
спрямованість
продукції
цитокінів
F1 фракції та їх роль в патогенезі та супресію
регуляторних цитокінів F2 фракції, що і
обумовлює хронічний перебіг захворювання. 4.Дискфореграми
сенсибілізованих
Т-лімфоцитів дітей, хворих на БА, показали
чітку
спрямованість
продукції
цитокінів
F1 фракції та їх роль в патогенезі та супресію
регуляторних цитокінів F2 фракції, що і
обумовлює хронічний перебіг захворювання. 2.Тяжкість
перебігу
БА
корелює
з
вираженістю спектру продукції цитокінів
сенсибілізованих Т-лімфоцитів в сироватку
крові у дітей, хворих на БА. 3.По
продукції
цитокінів
Т-лімфоцитами під впливом специфічних Список літератури
1. Бережний ВВ. Бронхіальна астма у дітей (лекція). Сучасна
педіатрія. 2018;(93):128-132. DOI:
https://doi.org/10.15574/sp.2018.93.128
2. Eckert R. Division of cells of the immune system. Immunological methods. World. 1987. 226-254 p. 3. Global Initiative for Asthma. Global Strategy for Asthma
Management
and
Prevention. 2023. URL
:
www.ginasthma.org. Список літератури
1. Бережний ВВ. Бронхіальна астма у дітей (лекція).
Сучасна
педіатрія.
2018;(93):128-132.
DOI:
https://doi.org/10.15574/sp.2018.93.128
2. Eckert R. Division of cells of the immune system.
Immunological methods. World. 1987. 226-254 p.
3. Global Initiative for Asthma. Global Strategy for Asthma
Management
and
Prevention.
2023.
URL
:
www.ginasthma.org. Список літератури 2. Eckert R. Division of cells of the immune system. Immunological methods. World. 1987. 226-254 p. 3. Global Initiative for Asthma. Global Strategy for Asthma
Management
and
Prevention. 2023. URL
:
www.ginasthma.org. Отримано: 11.04.2023 року
Прийнято до друку: 09.06.2023 року 27 Actual problems of modern medicine. Issue 11, 2023 ISSN 2617-409X T. Liadova A, C, D, O. Volobueva A, C, D, V. Chernusky A, C, D,
M. Popov A, C, D, H. Letiaho B, E, K. Pavlikova B, E
chernusky@karazin.ua – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical
vision of the article; F – final approval of the article A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation; D – writing the article; E – critical
evision of the article; F – final approval of the article ABSTRACT. It is known that under the influence of specific mitogens, sensitized T-lymphocytes produce № 199
cytokines in the culture medium, which are divided into two fractions: F1 and F2. The first (F1) of them
determines the pathogenesis of AD in children and the cytokines of this fraction are produced during
immunopathological reactions, and the second (F2) is responsible for the immunological parameters of activation
of the T-system of immunity and their regulation. Goal. To investigate the functional activity of T-lymphocytes activated by bronchopulmonary antigens and its
differential immunodiagnostic value in children with BA. Materials and methods. To determine the cytokines of sensitized T-lymphocytes, the qualitative composition of
cytokines of T-lymphocytes in the culture fluid, discphoresis in polyacrylamide gel was used in 105 children with
asthma and 20 healthy children aged 5 to 14 years with specific antigens of bronchi and lung tissue, as well as
with non-specific myogen FHA from the company «Reanal». p
y g
p
y
Results. In BA in children, the more quantitative presence of bands and discforograms is detected depending on
the severity of the course of the disease and is characterized by high production of F1-fraction cytokines by
sensitized T-lymphocytes. The structural analysis of cytokine disc-phorograms of T-lymphocytes in AD under
the conditions of mitogenic activity of sensitized T-lymphocytes switched with lipopolysaccharide antigens of the
bronchi and lung tissue showed discrepancies in the form of the presence of two bands that have qualitative
differences determined by the thickness, with the total numbering from the start having the serial number 8 and 9. Conclusions. It has been established that the severity of BA correlates with the expression of the spectrum of
cytokine production of sensitized T-lymphocytes in the blood serum of children with BA, and allows diagnosing
the severity of the disease with high accuracy. Key words: bronchial asthma, children, cytokines of sensitized T-lymphocytes, discphoresis, polyacrylamide gel,
hypersensitivity of the field type, bronchopulmonary antigens Ksenia Pavlikova, PhD, associate professor,
department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: k.pavlikova@karazin.ua, ORSID
ID: https://orcid.org/0000-0002-1228-4915 Information about authors
Tetiana Liadova, MD, PhD, full professor,
professor of the department of infectious diseases
and clinical immunology, dean of the medical
faculty, V. N. Karazina Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: t.lyadova@karazin.ua, ORCID ID
https://orcid.org/0000-0002-5892-2599 Mykola Popov, MD, PhD, full professor,
department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022,
e-mail:
mykola.m.popov@karazin.ua,
ORSID ID: https://orcid.org/0000-0002-5759-
9654 Olga Volobueva, PhD, associate professor, head
of department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: o.volobyeva@karazin.ua, ORSID
ID https://orcid.org/0000-0002-5569-1748 Hanna Letiaho, PhD, associate professor,
department of pediatrics, V. N. Karazin Kharkiv
National University, Svobody Square, 6, Kharkiv,
Ukraine, 61022, e-mail: letyago@karazin.ua,
ORSID
ID
https://orcid.org/0000-0002-6327-
1321 For citation: Liadova T, Volobueva O, Chernusky V, Popov M, Letiaho H, Pavlikova K. DETERMINATION OF THE ACTIVITY OF T-LYMPHOCYTES
ACTIVATED BY BRONCHOPULMONARY ANTIGENS AND THEIR IMMUNODIAGNOSTIC SIGNIFICANCE IN CHILDREN WITH
BRONCHIAL ASTHMA. Actual problems of modern medicine. 2023;11:21-28. DOI: https://doi.org/10.26565/2617-409X-2023-11-03 (in Ukrainian) For citation: Liadova T, Volobueva O, Chernusky V, Popov M, Letiaho H, Pavlikova K. DETERMINATION OF THE ACTIVITY OF T-LYMPHOCYTES
ACTIVATED BY BRONCHOPULMONARY ANTIGENS AND THEIR IMMUNODIAGNOSTIC SIGNIFICANCE IN CHILDREN WITH
BRONCHIAL ASTHMA. Actual problems of modern medicine. 2023;11:21-28. DOI: https://doi.org/10.26565/2617-409X-2023-11-03 (in Ukrainian) Hanna Letiaho, PhD, associate professor,
department of pediatrics, V. N. Karazin Kharkiv
National University, Svobody Square, 6, Kharkiv,
Ukraine, 61022, e-mail: letyago@karazin.ua,
ORSID
ID
https://orcid.org/0000-0002-6327-
1321 Viacheslav Chernusky, MD, PhD, full professor,
department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: chernusky@karazin.ua, ORSID
ID: https://orcid.org/0000-0001-5657-9486 Information about authors Information about authors
Tetiana Liadova, MD, PhD, full professor,
professor of the department of infectious diseases
and clinical immunology, dean of the medical
faculty, V. N. Karazina Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: t.lyadova@karazin.ua, ORCID ID
https://orcid.org/0000-0002-5892-2599 Ksenia Pavlikova, PhD, associate professor,
department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: k.pavlikova@karazin.ua, ORSID
ID: https://orcid.org/0000-0002-1228-4915 Olga Volobueva, PhD, associate professor, head
of department of infectious diseases and clinical
immunology, V. N. Karazin Kharkiv National
University, Svobody Square, 6, Kharkiv, Ukraine,
61022, e-mail: o.volobyeva@karazin.ua, ORSID
ID https://orcid.org/0000-0002-5569-1748 28 References
1. Berezhnyi VV. Bronchial asthma in children (a lecture). Sovrem Pediatr.
2018;(5(93)):128-32.
[in
Ukrainian].
DOI: https://doi.org/10.15574/sp.2018.93.128
2. Eckert R. Division of cells of the immune system. Immunological
methods. World. 1987. 226-254.
3. Global Initiative for Asthma. Global Strategy for Asthma Management
and Prevention. 2023. URL : www.ginasthma.org. Conflicts of interest: author has no conflict of interest to declare.
Конфлікт інтересів: відсутній. ISSN 2617-409X References
1. Berezhnyi VV. Bronchial asthma in children (a lecture). Sovrem Pediatr. 2018;(5(93)):128-32. [in
Ukrainian]. DOI: https://doi.org/10.15574/sp.2018.93.128
2. Eckert R. Division of cells of the immune system. Immunological
methods. World. 1987. 226-254. 3. Global Initiative for Asthma. Global Strategy for Asthma Management
and Prevention. 2023. URL : www.ginasthma.org. 4. Honkoop
P. Astma. TvPO. 2021;16(6):41-3. DOI:
https://doi.org/10.1007/s12503-021-0905-z
5. Patel SJ, Teach SJ. Asthma. Pediatrics in Review. 2019;40(11):549-67. DOI: https://doi.org/10.1542/pir.2018-0282
6. Umanets TR, Kreposniak AA. Asthma Combined With Eosinophilic
Esophagitis In Children: Modern Condition Of The Problem. Asthma
Allergy. 2019;2019(1):36-43. [in
Ukrainian]. DOI:
https://doi.org/10.31655/2307-3373-2019-1-36-43 References Received: 11.04.2023 Accepted: 09.06.2023 Accepted: 09.06.2023 Conflicts of interest: author has no conflict of interest to declare. Конфлікт інтересів: відсутній.
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KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC
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Oncogene
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Abstract During the evolution into castration or therapy resistance, prostate cancer cells reprogram the androgen responses to cope
with the diminishing level of androgens, and undergo metabolic adaption to the nutritionally deprived and hypoxia
conditions. AR (androgen receptor) and PKM2 (pyruvate kinase M2) have key roles in these processes. We report in this
study, KDM8/JMJD5, a histone lysine demethylase/dioxygnase, exhibits a novel property as a dual coactivator of AR and
PKM2 and as such, it is a potent inducer of castration and therapy resistance. Previously, we showed that KDM8 is involved
in the regulation of cell cycle and tumor metabolism in breast cancer cells. Its role in prostate cancer has not been explored. Here, we show that KDM8’s oncogenic properties in prostate cancer come from its direct interaction (1) with AR to affect
androgen response and (2) with PKM2 to regulate tumor metabolism. The interaction with AR leads to the elevated
expression of androgen response genes in androgen-deprived conditions. They include ANCCA/ATAD2 and EZH2, which
are directly targeted by KDM8 and involved in sustaining the survival of the cells under hormone-deprived conditions. Notably, in enzalutamide-resistant cells, the expressions of both KDM8 and EZH2 are further elevated, so are
neuroendocrine markers. Consequently, EZH2 inhibitors or KDM8 knockdown both resensitize the cells toward
enzalutamide. In the cytosol, KDM8 associates with PKM2, the gatekeeper of pyruvate flux and translocates PKM2 into the
nucleus, where the KDM8/PKM2 complex serves as a coactivator of HIF-1α to upregulate glycolytic genes. Using shRNA
knockdown, we validate KDM8’s functions as a regulator for both androgen-responsive and metabolic genes. KDM8 thus
presents itself as an ideal therapeutic target for metabolic adaptation and castration-resistance of prostate cancer cells. Oncogene (2019) 38:17–32
https://doi.org/10.1038/s41388-018-0414-x Oncogene (2019) 38:17–32
https://doi.org/10.1038/s41388-018-0414-x ARTICLE ARTICLE KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates
AR/EZH2 network and tumor metabolism in CRPC Hung-Jung Wang1,2
●Mamata Pochampalli3
●Ling-Yu Wang3
●June X Zou3
●Pei-Shan Li2
●Sheng-Chieh Hsu1,4
●
Bi-Juan Wang5
●Shih-Han Huang5
●Ping Yang6,7
●Joy C. Yang6
●Cheng-Ying Chu8
●Chia-Ling Hsieh8
●
Shian-Ying Sung8
●Chien-Feng Li9
●Clifford G. Tepper
6
●David K. Ann8,10
●Allen C. Gao1,6
●
Christopher P. Evans6,11
●Yoshihiro Izumiya11
●Chi-Pin Chuu5
●Wen-Ching Wang12
●Hong-Wu Chen3,11
●
Hsing-Jien Kung2,3,4,8 Received: 2 November 2017 / Revised: 19 May 2018 / Accepted: 21 June 2018 / Published online: 2 August 2018
© The Author(s) 2018. This article is published with open access KDM8 is overexpressed in a subset of prostate
tumors with high Gleason scores and its elevation
drives the development of CRPC We first examined whether KDM8 expression is deregu-
lated in tumors of PCa by immunohistochemistry (IHC)
analysis. Evaluation of anti-KDM8 immunostaining of
prostate specimens from 121 cases revealed that 61% of
malignant tumors showed high levels of KDM8 expression
while the majority of normal or non-malignant prostate
tissues displayed no detectable or low levels of KDM8 (Fig. 1a, b, Table S1). Approximately 80% of tumors with high
Gleason score (7–10) had positive KDM8 staining with
high IHC scores, while about 25% of low (2–6) Gleason
tumors displayed high KDM-IHC scores (Fig. 1b). Similar to the expression pattern seen in prostate tissues,
higher levels of KDM8 protein were detected by immuno-
blotting in PCa cell lines as compared to immortalized
prostate epithelial cell line RWPE1 (Fig. 1c). In agreement
with our own screening results, the TCGA transcriptome
dataset
extracted
from
Oncomine
database
(www. oncomine.com)
also
showed
a
higher
expression
of
KDM8 in malignant PCa (p < 0.0001) (Fig. 1d). These data
suggest KDM8 may be involved in the development of
castration and therapy resistance. KDM8/JMJD5, the newest member of the histone
demethylase family, is involved in embryogenesis [16],
oncogenesis [16, 17], and stem-cell renewal [18]. Over-
expression of KDM8 was observed in a variety of tumor
tissues [17, 19, 20] and knockdown of KDM8 compromised
the growth of cancer cells [16, 17] (and this study). Toge-
ther, they suggest a critical role of KDM8 in development
and cell growth. We and others [16, 17, 21–24] showed that
KDM8
is
associated
with
H3K36me2
demethylation
in vivo. Macron et. al. identified RCCD1, a centromere and
DNA-binding protein, can augment KDM8’s activity [23,
25].Other studies suggest that KDM8 may also function as a
dioxygenase [26], and as an aminopeptidase, digesting
methylated histone tails to modulate chromatin conforma-
tion [27, 28]. Accumulating evidence suggest that KDM8 is
a key cell cycle regulator by upregulating Cyclin A [17, 22],
modulation of the expressions of p53 and p21 [16, 18, 22,
29, 30] and interactions with spindle microtubules [31]. In
addition, it is involved in regulating the fidelity of centro-
some duplication by suppression the expression of Satellite
repeats [23] and DNA recombination [24]. KDM8 shuttles
between cytosol and nucleus with the NLS recently mapped
at the N terminus with binding to importin [32]. Results Lysine demethylases (KDMs) have emerged as an
important class of epigenetic factors involved in carcino-
genesis. Among the eight KDM subfamilies, nearly all (e.g.,
KDM1, KDM2A-C. KDM3A, C, KDM4A-D, KDM5A-C,
KDM6B, C, KDM8 (this study)) have been found to be
overexpessed in PCa, and several of them correlate with
poor prognosis, indicating a critical role of histone deme-
thylation in tumorigenesis [9]. The most well-characterized
are the KDM1 and KDM4 subfamilies. It was found that
KDM1, KDM4A, B and C all associate with AR and serve
as coactivators [10, 11], and their overexpressions predict
poor prognosis of PCa [12, 13]. We recently reported that
genetic and pharmacological inhibitors to KDM4A and 4B
suppress the growth of PCa cells without affecting normal
prostate epithelial cells [14]. Duan et al. [15] reported a
similar finding with another KDM4A inhibitor. These data
suggest that KDMs are potential targets for PCa therapy. Introduction One of the most troubling aspects of prostate cancer (PCa)
progression is the conversion from androgen-dependent to
independent (or castration-resistant) state, which at present
defies any effective treatment. The next-generation anti-
androgens, enzalutamide, and abiraterone have improved
the prospects, but eventually therapy-resistant PCa still
developed. During the evolution into castration or therapy
resistance, the tumor cells need to reprogram the androgen
responses to cope with the diminishing level of androgens,
and to undergo metabolic adaption to the nutritional and
hypoxia conditions during therapy. Increased aerobic gly-
colysis has been shown to be associated with castration or
therapy resistance [1–3]. Nearly all glycolytic genes are
overexpressed in advanced PCa [4], many of which are These authors contributed equally: Hung-Jung Wang, Mamata
Pochampalli, Ling-Yu Wang, June X. Zou. * Hung-Jung Wang
hjwang@nhri.org.tw
* Hsing-Jien Kung
hkung@nhri.org.tw
Extended author information available on the last page of the article. Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41388-018-0414-x) contains supplementary
material, which is available to authorized users. These authors contributed equally: Hung-Jung Wang, Mamata
Pochampalli, Ling-Yu Wang, June X. Zou. These authors contributed equally: Hung-Jung Wang, Mamata
Pochampalli, Ling-Yu Wang, June X. Zou. Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41388-018-0414-x) contains supplementary
material, which is available to authorized users. Electronic supplementary material The online version of this article
(https://doi.org/10.1038/s41388-018-0414-x) contains supplementary
material, which is available to authorized users. * Hung-Jung Wang
hjwang@nhri.org.tw
* Hsing-Jien Kung
hkung@nhri.org.tw Extended author information available on the last page of the article. H-J Wang et al. 18 targets of HIF-1α [5]. One of the key factors regulating
glycolysis in tumor cells is PKM2, a cytosolic enzyme
which controls the level of pyruvate and its flow to lactate
versus mitochondria. Through ligand stimulation and post-
translational modifications, PKM2 can be translocated into
nucleus to become a coactivator for HIF-1α, thereby further
fueling the glycolytic pathway [6, 7]. Not surprisingly,
overexpression and modifications of PKM2 are associated
with PCa progression [8]. In the present study, we describe
a novel lysine demethylase which interacts with both AR
and PKM2 to reprogram androgen responses and tumor
metabolism. translocation of PKM2 (pyruvate kinase M2 isoform), a
critical enzyme involved in tumor metabolism [7]. We
found that KDM8 binds PKM2 and enhances the conver-
sion of cytosolic tetramer form of PKM2 into the dimer or
heterodimer form which enters nucleus. Introduction Together with
KDM8, PKM2 serves as a coactivator of HIF-1α, to upre-
gulate enzymes involved in Warburg effect [7]. All data,
taken together, suggest KMD8 is a multi-functional mole-
cule involved in tumor progression, In this study, we extend
its oncogenic role to PCa. KDM8 is overexpressed in a subset of prostate
tumors with high Gleason scores and its elevation
drives the development of CRPC Interest-
ingly, we reported that KDM8 facilitates the nuclear Consistent with its role in the control of PCa cell pro-
liferation and survival, knockdown of KDM8 inhibited the
proliferation of both LNCaP, C4-2B and C4-2B-MDVR, an
enzalutamide-resistant variant of C4-2B, and, to less extent,
the AR negative PC3 cells. Importantly, KDM8 knockdown
had little effect on the non-malignant prostate cell line
RWPE1 (Fig. 2a and Figure S2). By contrast, over-
expression of KDM8 in RWPE1 cells stimulates the growth
of this cell line (Figure S1). These data suggest that KDM8
is related to malignant cell growth. We then asked whether
overexpression of KDM8 can convert an androgen-sensitive
LNCaP into androgen-independent. Accordingly, KDM8
was overexpressed in LNCaP cells via lentivirus infection
and cultured in androgen-deprived conditions. As shown in KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 19 Fig. 1 KDM8 is overexpressed in malignant prostate tissues and
cancer cell lines. a Representative images of anti-KDM8 immuno-
histochemical (IHC) staining of prostate normal and malignant tissue
sections. b Percentage of prostate cancer tumors with different Gleason
scores that are scored as high or low (or negative) in the anti-KDM
IHC analysis. c Western blot analysis of KDM8 in non-malignant
RWPE1 cell line and cancerous prostate epithelial cell lines C4-2B,
LNCaP, PC3, CWR22rv1, and DU145. GAPDH was used as a loading
control. d KDM8 gene expression analysis in prostate cancer (TCGA
dataset extracted from Oncomine database). Box plot derived from
gene expression data in Oncomine database comparing the KDM8
expression in normal (n = 184) and in malignant cancer tissues (n =
141) LNCaP, PC3, CWR22rv1, and DU145. GAPDH was used as a loading
control. d KDM8 gene expression analysis in prostate cancer (TCGA
dataset extracted from Oncomine database). Box plot derived from
gene expression data in Oncomine database comparing the KDM8
expression in normal (n = 184) and in malignant cancer tissues (n =
141) Fig. 1 KDM8 is overexpressed in malignant prostate tissues and
cancer cell lines. a Representative images of anti-KDM8 immuno-
histochemical (IHC) staining of prostate normal and malignant tissue
sections. b Percentage of prostate cancer tumors with different Gleason
scores that are scored as high or low (or negative) in the anti-KDM
IHC analysis. c Western blot analysis of KDM8 in non-malignant
RWPE1 cell line and cancerous prostate epithelial cell lines C4-2B, LNCaP, PC3, CWR22rv1, and DU145. KDM8 is overexpressed in a subset of prostate
tumors with high Gleason scores and its elevation
drives the development of CRPC GAPDH was used as a loading
control. d KDM8 gene expression analysis in prostate cancer (TCGA
dataset extracted from Oncomine database). Box plot derived from
gene expression data in Oncomine database comparing the KDM8
expression in normal (n = 184) and in malignant cancer tissues (n =
141) Fig. 2b and Figure S3a, KDM8 overexpression markedly
stimulated
androgen-independent
cell
proliferation
of
LNCaP cells. breast cancer, a novel function of KDM8 is its association
with PKM2 and its ability to translocate PKM2 into nucleus
to become a coactivator of HIF-1α to transcriptionally
activate glycolytic genes in favor of Warburg effects [7]. We therefore asked whether KDM8 is able to modulate the
tumor metabolism in PCa cells. First, in a reciprocal
immunoprecipitation analysis, we showed that KDM8 and
PKM2 associate with each other in LNCaP cells (Fig. 3a). Furthermore,
in
both
cell
fractionation
and
confocal
microscopy analyses, KDM8 overexpression enhances the
translocation of PKM2 into the nucleus (Fig. 3b, c). Con-
versely, knockdown of KDM8 reduces PKM2 translocation
(Fig. 3b, c). The nuclear translocation studies were aided by
confocal microscopy (Fig. 3c) where the fluorescent inten-
sity of PKM2 across the nucleus was traced as illustrated on
the right panel and the average intensity of counting 10
nuclei for KDM8 overexpressing cells measured. These studies were then extended to in vivo tumorigen-
esis assay. KDM8 overexpressing and vector control
LNCaP cells (Figure S3b) were injected into athymic nu/nu
mice and the tumor growth was monitored. In intact ani-
mals, the KDM8-overexpressing LNCaP grew slightly
faster than vector- infected LNCaP (LNCaP-LKO). Upon
castration,
LNCaP-KDM8
tumors
continued
to
grow
whereas LNCaP-LKO ceased to do so (Fig. 2c). Together,
these data suggest that elevated KDM8 expression is related
to malignant transformation of PCa cells and has the
potential to cause castration-resistance. KDM8 translocates PKM2 into nucleus 2 KDM8 is required for AR-positive prostate cancer cell pro-
liferation and survival, and promotes androgen-independent cell pro-
liferation and tumor growth. a Growth curves of non-malignant cell
line RWPE1 and prostate cancer cell lines LNCaP, PC3, C4-2B, and
C4-2B-MDVR, an enzalutamide-resistant cell line. Cells were trans-
fected with small interfering (si)-RNAs targeting KDM8 or si-non-
targeting (NT) control. Every 2 days after transfection, cell prolifera-
tion was measured by MTT assay. b Growth curves of LNCaP cells
infected with KDM8 overexpressing (KDM8) or control (LKO) len-
tiviruses followed by maintaining in androgen-deprived media. Two
days later infection, cell proliferation was measured by MTT assay at Fig. 2 KDM8 is required for AR-positive prostate cancer cell pro-
liferation and survival, and promotes androgen-independent cell pro-
liferation and tumor growth. a Growth curves of non-malignant cell
line RWPE1 and prostate cancer cell lines LNCaP, PC3, C4-2B, and
C4-2B-MDVR, an enzalutamide-resistant cell line. Cells were trans-
fected with small interfering (si)-RNAs targeting KDM8 or si-non-
targeting (NT) control. Every 2 days after transfection, cell prolifera-
tion was measured by MTT assay. b Growth curves of LNCaP cells
infected with KDM8 overexpressing (KDM8) or control (LKO) len-
tiviruses followed by maintaining in androgen-deprived media. Two
days later infection, cell proliferation was measured by MTT assay at indicated times. c Tumor growth curves of KDM8 overexpressing and
LKO control LNCaP cells in xenografting mouse model. Cells were
injected subcutaneously into the dorsal flanks of athymic nude mice (8
mice per group) and tumor volumes were measured every week by
using calipers. Tumor-bearing mice were also castrated at indicated
time point. Insert: photograph of xenograft tumors. The quantitative
data shown in a and b are the mean ± S.D. of three separate experi-
ments. The average tumor volumes are presented as the mean ± S.E.M. *p < 0.05; **p < 0.01, by paired Students’ t (MTT assay) or ANOVA
test (xenografting study) (GLUT1, HK2, PKM2, LDHA, etc.) are all upregulated,
whereas genes involved in mitochondrial pyruvate flow
such as PDHA1 and PDHB1 are down-modulated. These
data are summarized in the bubble diagram of Fig. 4d. The
overall consequence is the switch of mitochondria oxidative
phosphorylation to glycolysis [7]. Indeed, in LNCaP-
KDM8 cells, the uptake of glucose (Fig. 4b) and lactate
production (Fig. 4c) are increased over the control cell lines,
and they are PKM2-dependent, as siRNA targeting PKM2
reverse the effects. KDM8 translocates PKM2 into nucleus To demonstrate that the heightened
expression of glycolytic genes by KDM8 overexpression is
mediated by PKM2, we knocked down PKM2 in LNCaP-
KDM8, and monitored their expression levels (Fig. 4a, right
panel and Figure S4). Many of the genes overexpressed in
LNCaP-KDM8 are indeed down-modulated in the absence PKM2. Taken together, these data suggest that KDM8 is a
major metabolic regulator, in partnership with PKM2, forms
a complex that translocates into the nucleus to reprogram
gene expression toward aerobic glycolysis (Warburg effect)
(Fig. 4d). KDM8 translocates PKM2 into nucleus A consequence of the KDM8-mediated PKM2 nuclear
translocation is the upregulation of glycolytic and metabolic
genes, the targets of HIF-1α [7]. The genes involved in
glycolysis upon KDM8 overexpression shown in Fig. 4a One of the hallmarks of aggressive PCas including castra-
tion and therapy resistant is the metabolic adaptation, where
aerobic glycolysis dominant over mitochondria oxidative
phosphorylation [1, 2]. Previously, we reported that in H-J Wang et al. 20 Fig. 2 KDM8 is required for AR-positive prostate cancer cell pro-
liferation and survival, and promotes androgen-independent cell pro-
liferation and tumor growth. a Growth curves of non-malignant cell
line RWPE1 and prostate cancer cell lines LNCaP, PC3, C4-2B, and
C4-2B-MDVR, an enzalutamide-resistant cell line. Cells were trans-
fected with small interfering (si)-RNAs targeting KDM8 or si-non-
targeting (NT) control. Every 2 days after transfection, cell prolifera-
tion was measured by MTT assay. b Growth curves of LNCaP cells
infected with KDM8 overexpressing (KDM8) or control (LKO) len-
tiviruses followed by maintaining in androgen-deprived media. Two
days later infection, cell proliferation was measured by MTT assay at
indicated times. c Tumor growth curves of KDM8 overexpressing and
LKO control LNCaP cells in xenografting mouse model. Cells were
injected subcutaneously into the dorsal flanks of athymic nude mice (8
mice per group) and tumor volumes were measured every week by
using calipers. Tumor-bearing mice were also castrated at indicated
time point. Insert: photograph of xenograft tumors. The quantitative
data shown in a and b are the mean ± S.D. of three separate experi-
ments. The average tumor volumes are presented as the mean ± S.E.M. *p < 0.05; **p < 0.01, by paired Students’ t (MTT assay) or ANOVA
test (xenografting study) Fig. 2 KDM8 is required for AR-positive prostate cancer cell pro-
liferation and survival, and promotes androgen-independent cell pro-
liferation and tumor growth. a Growth curves of non-malignant cell
line RWPE1 and prostate cancer cell lines LNCaP, PC3, C4-2B, and
C4-2B-MDVR, an enzalutamide-resistant cell line. Cells were trans-
fected with small interfering (si)-RNAs targeting KDM8 or si-non-
targeting (NT) control. Every 2 days after transfection, cell prolifera-
tion was measured by MTT assay. b Growth curves of LNCaP cells
infected with KDM8 overexpressing (KDM8) or control (LKO) len-
tiviruses followed by maintaining in androgen-deprived media. Two
days later infection, cell proliferation was measured by MTT assay at Fig. KDM8 activates AR response in the absence of androgen In addition to metabolic adaptation, aberrant activation of
AR
responses
is
another
critical
step
of
castration-
resistance. In our transcriptome analysis of KDM8 over-
expressing cells (microarray data were deposited in NCBI KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 21 Fig. 3 KDM8 regulates PKM2 nuclear translocation. a Interaction of
endogenous KDM8 and PKM2 in LNCaP cells. Reciprocal immuno-
precipitation (IP) and immunoblotting (IB) were performed with
PKM2 and KDM8 antibodies as indicated. b Subcellular localization
of KDM8 and PKM2 in LNCaP cells. Nuclear (Nuc) and cytosolic
(Cyto) fractions were prepared from LNCaP cells transfected with si-
NT, si-KDM8, EV, or KDM8-expressing vector, followed by immu-
noblotting analysis with antibodies as indicated. KDM8*, Flag-tagged
KDM8. c Confocal immunomicroscopy analysis of PKM2 nuclear
translocation. Treated cells were fixed and immunostained with anti-
PKM2 (PKM2) and 4′,6-diamidino-2-phenylindole (DAPI, nucleus),
respectively. The framed regions marked in the merged images
(Merge) are zoomed at the next (Zoom). The line profiles of PKM2
and DAPI signals were measured by ZEN 2011 (Carl Zeiss, Germany)
software. Scale bars, 10 µm KDM8. c Confocal immunomicroscopy analysis of PKM2 nuclear
translocation. Treated cells were fixed and immunostained with anti-
PKM2 (PKM2) and 4′,6-diamidino-2-phenylindole (DAPI, nucleus),
respectively. The framed regions marked in the merged images
(Merge) are zoomed at the next (Zoom). The line profiles of PKM2
and DAPI signals were measured by ZEN 2011 (Carl Zeiss, Germany)
software. Scale bars, 10 µm Fig. 3 KDM8 regulates PKM2 nuclear translocation. a Interaction of
endogenous KDM8 and PKM2 in LNCaP cells. Reciprocal immuno-
precipitation (IP) and immunoblotting (IB) were performed with
PKM2 and KDM8 antibodies as indicated. b Subcellular localization
of KDM8 and PKM2 in LNCaP cells. Nuclear (Nuc) and cytosolic
(Cyto) fractions were prepared from LNCaP cells transfected with si-
NT, si-KDM8, EV, or KDM8-expressing vector, followed by immu-
noblotting analysis with antibodies as indicated. KDM8*, Flag-tagged independence, we individually knocked down the upregu-
lated genes (Figure S6) and monitor the growth of LNCaP-
KDM8 in regular and in charcoal-dextran media (Fig. 5d). In regular media, the individual knockdowns had moderate
effects (dark red to light red), while in charcoal-stripped
media, as expected, the LNCaP-EV cells were not growing
well, with individual knockdown of many of the genes
including EZH2 caused severe effects. In LNCaP-KDM8,
the effects of individual gene knockdown of on growth are
generally minimal. KDM8 activates AR response in the absence of androgen In charcoal-stripped media, the inhibi-
tory effects on growth of individual knockdown of genes
such as EZH2 were again detected. These results suggest
that KDM8 induced genes (in the absence of androgen)
were indeed relevant to androgen-independent growth, and
they jointly contribute to the growth and survival of LNCaP
under androgen-deprived conditions. GEO
database
accession
number:
GSE56908),
unsu-
pervised clustering analysis (Fig. 5a) indicated that among
the genes modulated by KDM8 (in the absence of DHT), a
significant fraction corresponds to androgen response genes,
which include AMACR, EZH2, ANCCA, KLK3, and
NSD2/WHSC1, suggesting KDM8 overexpression leads to
aberrant activation of AR. Real-time RT-PCR validated the
microarray results (Fig. 5b). As shown in the Venn diagram
(Fig. 5c), a significant fraction of genes (27% in the absence
of DHT and 37% in the presence of DHT) with altered
expression (2×) elicited by elevated KDM8 overlap with
androgen-responsive genes (ARG). Genes involved in androgen-independent growth To demonstrate that the KDM8-upregulated genes are
functionally
relevant
in
the
induction
of
androgen- 22 H-J Wang et al. Fig. 4 Overexpression of KDM8 reprograms glucose metabolism in
prostate cancer cells. a Heat map of metabolic gene expression in
KDM8-overexpressed LNCaP cells (left panel) and the KDM8-
overexpressed LNCaP cells knocking down with siRNA targeting
PKM2 (right panel). The gene expression levels were measured by
qRT-PCR. Data were analyzed as fold change as compared to the EV
control (left panel) or si-NT after normalizing to internal control, 16S
rRNA. b Measurements of the levels of extracellular lactate and c
glucose update in EV control and KDM8-overexpressed LNCaP cells. Data are expressed as mean ± S.D. with three separate experiments. *p
< 0.05. d A schematic diagram of glucose metabolic flux. Over-
expression of KDM8 induces redirection of metabolic flux into the
biomass synthesis pathways. The affected genes are marked in red
(upregulated) or in blue (downregulated). The sizes of the circles are
proportional to the expression levels of the genes indicated metabolism in
expression in
the KDM8-
NA targeting
measured by
ed to the EV
control, 16S
actate and c
glucose update in EV control and KDM8-overexpressed LNCaP cells. Data are expressed as mean ± S.D. with three separate experiments. *p
< 0.05. d A schematic diagram of glucose metabolic flux. Over-
expression of KDM8 induces redirection of metabolic flux into the
biomass synthesis pathways. The affected genes are marked in red
(upregulated) or in blue (downregulated). The sizes of the circles are
proportional to the expression levels of the genes indicated glucose update in EV control and KDM8-overexpressed LNCaP cells. Data are expressed as mean ± S.D. with three separate experiments. *p
< 0.05. d A schematic diagram of glucose metabolic flux. Over-
expression of KDM8 induces redirection of metabolic flux into the
biomass synthesis pathways. The affected genes are marked in red
(upregulated) or in blue (downregulated). The sizes of the circles are
proportional to the expression levels of the genes indicated Fig. 4 Overexpression of KDM8 reprograms glucose metabolism in
prostate cancer cells. a Heat map of metabolic gene expression in
KDM8-overexpressed LNCaP cells (left panel) and the KDM8-
overexpressed LNCaP cells knocking down with siRNA targeting
PKM2 (right panel). The gene expression levels were measured by
qRT-PCR. Genes involved in androgen-independent growth Data were analyzed as fold change as compared to the EV
control (left panel) or si-NT after normalizing to internal control, 16S
rRNA. b Measurements of the levels of extracellular lactate and c KDM8 associates with AR and acts as a novel AR
coactivator KDM8 with DBD-LBD but not with the NTD (Fig. 6b). The mapping data suggest that the interaction domain of AR
resides in the ligand-binding domain. KDM8 associates with and transactivates AR To determine whether KDM8’s interaction with AR
affects the transcriptional regulation activity of AR, we
performed reporter gene assays with PSA gene enhancer
and promoter-linked luciferase. Figure 6c showed that
although KDM8 alone slightly induced the reporter activity,
co-expression of KDM8 and AR markedly increased the
reporter activity from 4-fold (AR alone) to 15-fold (AR +
KDM8) and from 15-fold (AR alone) to 38-fold (AR +
KDM8) in the absence and presence of DHT, respectively. Interestingly, changes in the amino acid sequence in the
Jumonji domain (H321A), which inactivates its demethy-
lase activity, only partially reduced the coactivation of AR,
indicating the KDM8 behaves in both demethylase depen-
dent and independent manner. Given that a majority of ARGs were regulated by KDM8,
we asked whether KDM8 physically associates with AR
and serves as a coactivator [33, 34]. We first performed co-
immunoprecipitation assay. As shown in Fig. 6a, anti-AR
antibody, but not the control IgG, effectively co-precipitated
KDM8, demonstrating that indeed KDM8 specifically
formed complexes with AR. To further characterize the
interaction, we expressed Flag-tagged KDM8, together with
Myc-tagged AR full-length (FL) or its deletion mutants: N
(N-terminal domain, NTD), ND (NTD plus DNA-binding
domain, DBD), or DL (DBD plus ligand-binding domain
LBD) in 293T cells and performed immunoprecipitation
with Flag-tagged KDM8, and detected strong association of KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 23 Fig. 5 KDM8 controls specific subsets of androgen-responsive pro-
gram. a Heat map of hierarchical clustering of androgen-regulated
genes (ARGs). LNCaP-overexpressed KDM8 and EV control cells
were cultured in the presence or absence of 1 nM DHT followed by
gene expression profiling with microarray analysis. The ARGs that
differentially expressed by KDM8 (≥1.5-fold) and the positions for
well-known ARGs are indicated. Two genes EZH2 and ANCCA
selected for ChIP assay are marked in red. b qRT-PCR analysis of the
selected ARDs. LNCaP cells (KDM8 and EV) were cultured in the
androgen-deprived media for 3 days followed by qRT-PCR analysis. Triplicate experimental data were expressed as fold change as com-
pared to EV control after normalizing to internal control 16S rRNA. c
Venn diagram showing the overlap of KDM8-regulated genes and
ARDs. d Heat map showing cell viability of LNCaP cells (KDM8 and
EV) by knocking down the indicated ARGs in the absence of andro-
gen. KDM8 associates with and transactivates AR LNCaP cells infected with the shRNAs targeting the ARGs as
indicated were then cultured in media containing normal FBS or
charcoal-stripped FBS (CS-FBS) for 3 days. Cell viability was
assessed by MTT assay. EZH2 and ANCCA selected for chromatin
immunoprecipitation (ChIP) assay are marked in red Fig. 5 KDM8 controls specific subsets of androgen-responsive pro-
gram. a Heat map of hierarchical clustering of androgen-regulated
genes (ARGs). LNCaP-overexpressed KDM8 and EV control cells
were cultured in the presence or absence of 1 nM DHT followed by
gene expression profiling with microarray analysis. The ARGs that
differentially expressed by KDM8 (≥1.5-fold) and the positions for
well-known ARGs are indicated. Two genes EZH2 and ANCCA
selected for ChIP assay are marked in red. b qRT-PCR analysis of the
selected ARDs. LNCaP cells (KDM8 and EV) were cultured in the
androgen-deprived media for 3 days followed by qRT-PCR analysis. Triplicate experimental data were expressed as fold change as com-
pared to EV control after normalizing to internal control 16S rRNA. c
Venn diagram showing the overlap of KDM8-regulated genes and
ARDs. d Heat map showing cell viability of LNCaP cells (KDM8 and
EV) by knocking down the indicated ARGs in the absence of andro-
gen. LNCaP cells infected with the shRNAs targeting the ARGs as
indicated were then cultured in media containing normal FBS or
charcoal-stripped FBS (CS-FBS) for 3 days. Cell viability was
assessed by MTT assay. EZH2 and ANCCA selected for chromatin
immunoprecipitation (ChIP) assay are marked in red KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth The relative luciferase activity (RLU, relative light
unit) was calculated by their normalized luciferase activity to the EV
control with (red bar) or without DHT (blue bar) stimulation. Error
bars represent as mean ± S.D. from three different experiments. #, *p <
0.05, ##, **p < 0.01, by paired Students’ t-test. e ChIP assay of KDM8
and AR binding to the PSA enhancer/promoter regions in LNCaP
cells. (Upper) Schematic diagram of the positions of PSA enhancer
and promoter. The distances of the enhancer and promoter from
transcription starting site (+1) as well as AR and KDM8 are presented. Primer sets used for qPCR are indicated. (Lower) ChIP-qPCR analysis
of the occupancy of AR and KDM8 at PSA promoter and enhancer
regions. LNCaP cells maintained in androgen-deprived media were
used for the preparation of immunoprecipitated genomic DNA with
AR or KDM8 antibody as indicated. Data were expressed relative to
relevant IgG control. Data are shown as mean ± S.D. of three inde-
pendent experiments. *p < 0.05, by paired Students’ t-test
24
H-J Wang et al. luciferase activity. The relative luciferase activity (RLU, relative light
unit) was calculated by their normalized luciferase activity to the EV
control with (red bar) or without DHT (blue bar) stimulation. Error
bars represent as mean ± S.D. from three different experiments. #, *p <
0.05, ##, **p < 0.01, by paired Students’ t-test. e ChIP assay of KDM8
and AR binding to the PSA enhancer/promoter regions in LNCaP
cells. (Upper) Schematic diagram of the positions of PSA enhancer
and promoter. The distances of the enhancer and promoter from
transcription starting site (+1) as well as AR and KDM8 are presented. Primer sets used for qPCR are indicated. (Lower) ChIP-qPCR analysis
of the occupancy of AR and KDM8 at PSA promoter and enhancer
regions. LNCaP cells maintained in androgen-deprived media were
used for the preparation of immunoprecipitated genomic DNA with
AR or KDM8 antibody as indicated. Data were expressed relative to
relevant IgG control. Data are shown as mean ± S.D. of three inde-
pendent experiments. *p < 0.05, by paired Students’ t-test Fig. 6 KDM8 acts as a novel coactivator of AR. a Interaction of
endogenous KDM8 and AR in LNCaP cells. Reciprocal immunopre-
cipitation (IP) and immunoblotting (IB) were performed with PKM2
and
AR
antibodies
as
indicated. Input
indicates
non-
immunoprecipitated cell lysates. b Schematic diagram of different
MYC-AR expression vectors. KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth To further validate KDM8 as a critical coactivator of AR in
prostate carcinogenesis, we wish to identify the recruitment
of KDM8 to the promoters of AR target genes involved in
tumor progression. We have selected EZH2 pathway to
illustrate the role of KDM8. EZH2 is among the genes
whose knockdown have the most severe effects on
androgen-independent growth and that there is strong evi-
dence that EZH2 is involved in PCa progression (see Dis-
cussion). We previously showed that EZH2 is activated by
AR via ANCCA, a chromatin remodeling ATPase and a
coactivator of E2F1 [35], which is also upregulated by
KDM8 overexpression (Fig. 7a) (Supplementary Informa-
tion Figure S1b). Here, we show that KDM8 is directly To study whether KDM8 functions as an AR coactivator
in vivo, we studied the co-recruitment of KDM8 and AR at
the enhancer and promoter regions of PSA by ChIP assays
(Fig. 6d). In parental LNCaP, KDM8 was seen to occupy
both the enhancer and promoter region of PSA locus under
androgen-deprived conditions. In KDM8 overexpressing
LNCaP, increased occupancy of KDM8 was detected. The
level of AR also increased under these conditions, sug-
gesting that KDM8 facilitates the recruitment of AR to the
target sites. Together, these results demonstrate that KDM8
can act as a novel AR coactivator through its direct inter-
action with AR. H-J Wang et al. 24 Fig. 6 KDM8 acts as a novel coactivator of AR. a Interaction of
endogenous KDM8 and AR in LNCaP cells. Reciprocal immunopre-
cipitation (IP) and immunoblotting (IB) were performed with PKM2
and
AR
antibodies
as
indicated. Input
indicates
non-
immunoprecipitated cell lysates. b Schematic diagram of different
MYC-AR expression vectors. NTD N-terminal domain, DBD DNA-
binding domain, LBD ligand-binding domain, MYC-AR-FL (full
length), MYC-AR-ND, NTD + DBD; MYC-AR-N, NTD; MYC-AR-
DL, DBD + LBD. c Co-IP and IB of FLAG-KDM8 with different
Myc-AR constructions with anti-M2-Flag and Myc antibodies as
indicated. Non-immunoprecipitated samples are indicated as lysate. d
PSA promoter activity in prostate cancer cell line PC3. Dual luciferase
assays were performed in PC-3 cells transfected with PSA-luc/TK-
rellina (20:1) combining with the expression vectors of AR and KDM8
(wild-type and mutant H321A) as indicated. At 24 h after transfection,
the cells were cultured in RPMI-1640 medium with CS-FBS for 6 h
prior to treatment with 10 nM DHT for 8 h before measurement of the
luciferase activity. KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth NTD N-terminal domain, DBD DNA-
binding domain, LBD ligand-binding domain, MYC-AR-FL (full
length), MYC-AR-ND, NTD + DBD; MYC-AR-N, NTD; MYC-AR-
DL, DBD + LBD. c Co-IP and IB of FLAG-KDM8 with different
Myc-AR constructions with anti-M2-Flag and Myc antibodies as
indicated. Non-immunoprecipitated samples are indicated as lysate. d
PSA promoter activity in prostate cancer cell line PC3. Dual luciferase
assays were performed in PC-3 cells transfected with PSA-luc/TK-
rellina (20:1) combining with the expression vectors of AR and KDM8
(wild-type and mutant H321A) as indicated. At 24 h after transfection,
the cells were cultured in RPMI-1640 medium with CS-FBS for 6 h
prior to treatment with 10 nM DHT for 8 h before measurement of the luciferase activity. The relative luciferase activity (RLU, relative light
unit) was calculated by their normalized luciferase activity to the EV
control with (red bar) or without DHT (blue bar) stimulation. Error
bars represent as mean ± S.D. from three different experiments. #, *p <
0.05, ##, **p < 0.01, by paired Students’ t-test. e ChIP assay of KDM8
and AR binding to the PSA enhancer/promoter regions in LNCaP
cells. (Upper) Schematic diagram of the positions of PSA enhancer
and promoter. The distances of the enhancer and promoter from
transcription starting site (+1) as well as AR and KDM8 are presented. Primer sets used for qPCR are indicated. (Lower) ChIP-qPCR analysis
of the occupancy of AR and KDM8 at PSA promoter and enhancer
regions. LNCaP cells maintained in androgen-deprived media were
used for the preparation of immunoprecipitated genomic DNA with
AR or KDM8 antibody as indicated. Data were expressed relative to
relevant IgG control. Data are shown as mean ± S.D. of three inde-
pendent experiments. *p < 0.05, by paired Students’ t-test involved in both ANCCA and EZH2 activation in LNCaP
cells. We first demonstrated that KDM8 was corecruited
with AR to the enhancer region of ANCCA locus in both
the control (EV) and KDM8 overexpressor (KDM8) (Fig. 7b, upper panel). The amount of AR recruitment increases
with KDM8 overexpression, suggesting that KDM8 as a
coactivator facilitates AR targeting to chromatin. Further-
more, the co-recruitment of KDM8 and AR were induced involved in both ANCCA and EZH2 activation in LNCaP
cells. We first demonstrated that KDM8 was corecruited
with AR to the enhancer region of ANCCA locus in both
the control (EV) and KDM8 overexpressor (KDM8) (Fig. 7b, upper panel). KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth The amount of AR recruitment increases
with KDM8 overexpression, suggesting that KDM8 as a
coactivator facilitates AR targeting to chromatin. Further-
more, the co-recruitment of KDM8 and AR were induced by androgen in a time-dependent manner (Fig. 7b, lower
panel), followed by the recruitment of RNA polymerase II
(Pol II) for transcription. The elevated expression of
ANCCA lead to an increased accumulation of ANCCA and
its transcriptional partner E2F1 near the promoter of EZH2
[35], as seen in KDM8 overexpressing cells (Fig. 7c, upper
panel). Importantly, we found KDM8 is also recruited to
this site in a time-dependent manner (Fig. 7c, lower panel). KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 25 Fig. 7 KDM8 facilitates AR and E2F1 recruitment to the ANCCA and
EZH2 promoters and upregulation of ANCCA and EZH2 gene
expressions. a Immunoblotting assay of EZH2 and ANCCA in the
KDM8-overexpressing LNCaP cells. Cells transfected with FLAG-
tagged wild-type KDM8 or mutant KDM8-H321A were analyzed by
immunoblotting (IB) with the indicated antibodies. GAPDH was used
as a loading control. b ChIP analysis of the association of KDM8, AR,
and E2F1 binding to ANCCA enhancer/promoter in LNCaP cells. (Upper) Schematic diagram of the positions of ANCCA enhancer and
promoter. The transcription starting site is marked as +1. (Lower)
ChIP-qPCR analysis of the occupancy of AR/KDM8 and KDM8/E2F1
at ANCCA enhancer and promoter, respectively. The immunopreci-
pitated chromatins were prepared from LNCaP cells maintained in
androgen-deprived media followed by immunoprecipitation with
indicated antibodies. Data were expressed as percent immunoprecipi-
tation relative to input chromatin. Data are shown as mean ± S.D. of
triplicate experiments. *p < 0.05, Student’s t-test. c ChIP analysis of
the association of KDM8, E2F1, and ANCCA binding to EZH2 pro-
moter in LNCaP cells. (Upper) Schematic diagram of the position of
EZH2 promoter. The transcription starting site is marked as +1. (Lower) ChIP-qPCR analysis of the occupancy of KDM8/E2F1/
ANCCA at EZH2. The immunoprecipitated chromatin was prepared
from LNCaP cells maintained in androgen-deprived media followed
by immunoprecipitation with the indicated antibodies. Pol II, RNA
polymerase II. Data were expressed as percent immunoprecipitation
relative to input chromatin. Data are shown as mean ± S.D. of triplicate
experiments. *p < 0.05, Student’s t-test Fig. 7 KDM8 facilitates AR and E2F1 recruitment to the ANCCA and
EZH2 promoters and upregulation of ANCCA and EZH2 gene
expressions. KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth c Cell survival
curves of C4-2B and C4-2B-MDVR cells treated with EZH2 inhibi-
tors. Cells were exposed to the EZH2 inhibitors GSK343 and GSK126
in different doses from 0 to 50 μg/ml for 72 h followed by MTT assay. Data were expressed as the mean ± S.D. of triplicate experiments. The
values of IC50 were calculated and shown. The regression lines
represent the fit to a non-linear regression model using GraphPad
Prism. d qRT-PCR analysis of KDM8, EZH2, AR, and neuroendo-
crine markers mRNA expression. Parental cell lines (C4-2B) and the
enzalutamide-resistant derivative (C4-2B-MDVR) were transfected
with si-RNAs targeting KDM8 (si-KDM8) or non-targeting control
(si-NT) for 48 h. The relative mRNA levels of the above genes were
normalized to 18S rRNA. Data were represented as the mean ± S.D. of
triplicate experiments. Values in C4-2B cells transfected with si-NT
were set to 1. *,#p < 0.05, **, ##p < 0.01, by paired Students’ t-test. e
Model of KDM8-driven CRPC and neuroendocrine markers expres-
sion via AR–EZH2 axis Fig. 8 Overexpression of KDM8 confers upregulation of neuroendo-
crine markers and renders prostate cancer cells more sensitive to
killing by EZH2 inhibitors. a Immunobloting assay of KDM8
expression in C4-2B cell line and its enzalutamide-resistant derivative,
C4-2B-MDVR. α-tubulin and GAPDH were used as a loading control. b Correlation analysis between gene expression levels of KDM8 and
EZH2 in prostate cancer patients (right panel) and normal controls (left
panel) from TCGA dataset extracted from Oncomine database. Pear-
son’s correlation (r) values are shown in each graph. c Cell survival
curves of C4-2B and C4-2B-MDVR cells treated with EZH2 inhibi-
tors. Cells were exposed to the EZH2 inhibitors GSK343 and GSK126
in different doses from 0 to 50 μg/ml for 72 h followed by MTT assay. Data were expressed as the mean ± S.D. of triplicate experiments. The values of IC50 were calculated and shown. The regression lines
represent the fit to a non-linear regression model using GraphPad
Prism. d qRT-PCR analysis of KDM8, EZH2, AR, and neuroendo-
crine markers mRNA expression. Parental cell lines (C4-2B) and the
enzalutamide-resistant derivative (C4-2B-MDVR) were transfected
with si-RNAs targeting KDM8 (si-KDM8) or non-targeting control
(si-NT) for 48 h. The relative mRNA levels of the above genes were
normalized to 18S rRNA. Data were represented as the mean ± S.D. of
triplicate experiments. Values in C4-2B cells transfected with si-NT
were set to 1. KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth a Immunoblotting assay of EZH2 and ANCCA in the
KDM8-overexpressing LNCaP cells. Cells transfected with FLAG-
tagged wild-type KDM8 or mutant KDM8-H321A were analyzed by
immunoblotting (IB) with the indicated antibodies. GAPDH was used
as a loading control. b ChIP analysis of the association of KDM8, AR,
and E2F1 binding to ANCCA enhancer/promoter in LNCaP cells. (Upper) Schematic diagram of the positions of ANCCA enhancer and
promoter. The transcription starting site is marked as +1. (Lower)
ChIP-qPCR analysis of the occupancy of AR/KDM8 and KDM8/E2F1
at ANCCA enhancer and promoter, respectively. The immunopreci-
pitated chromatins were prepared from LNCaP cells maintained in
androgen-deprived media followed by immunoprecipitation with indicated antibodies. Data were expressed as percent immunoprecipi-
tation relative to input chromatin. Data are shown as mean ± S.D. of
triplicate experiments. *p < 0.05, Student’s t-test. c ChIP analysis of
the association of KDM8, E2F1, and ANCCA binding to EZH2 pro-
moter in LNCaP cells. (Upper) Schematic diagram of the position of
EZH2 promoter. The transcription starting site is marked as +1. (Lower) ChIP-qPCR analysis of the occupancy of KDM8/E2F1/
ANCCA at EZH2. The immunoprecipitated chromatin was prepared
from LNCaP cells maintained in androgen-deprived media followed
by immunoprecipitation with the indicated antibodies. Pol II, RNA
polymerase II. Data were expressed as percent immunoprecipitation
relative to input chromatin. Data are shown as mean ± S.D. of triplicate
experiments. *p < 0.05, Student’s t-test similar to KDM8 knockdown (Supplementary Information
Figure S7b, d). Taken together, these results confirmed
KDM8’s role as a coactivator of AR and is involved in the
activation of EZH2. Finally, to validate the roles of ANCAA and EZH2 in
KDM8-mediated growth, these genes were knocked down
in LNCaP-KDM8 (Supplementary Information Figure S7a,
c), and cell growth was significantly diminished in a manner H-J Wang et al. 26 Targeting KDM8 in therapy resistant PCa cells
MDVR has a heightened expression of KDM8 accom
Fig. 8 Overexpression of KDM8 confers upregulation of neuroendo-
crine markers and renders prostate cancer cells more sensitive to
killing by EZH2 inhibitors. a Immunobloting assay of KDM8
expression in C4-2B cell line and its enzalutamide-resistant derivative,
C4-2B-MDVR. α-tubulin and GAPDH were used as a loading control. b Correlation analysis between gene expression levels of KDM8 and
EZH2 in prostate cancer patients (right panel) and normal controls (left
panel) from TCGA dataset extracted from Oncomine database. Pear-
son’s correlation (r) values are shown in each graph. KDM8 mediates AR activation of EZH2 via ANCCA to
stimulate PCa cell growth *,#p < 0.05, **, ##p < 0.01, by paired Students’ t-test. e
Model of KDM8-driven CRPC and neuroendocrine markers expres-
sion via AR–EZH2 axis KDM8/AR signaling network Overexpression of KDM8 leads to castration-resistance as
determined by in vitro culture and in vivo xenograft model
and the upregulation of a subset of AR responsive genes. Many of these genes have been reported to be important
factors in castration-resistance. We wished to determine
which of them are important downstream effectors of
KDM8-mediated
castration-resistance. Based
on
an Discussion Androgen ablation therapies, especially with the next-
generation anti-androgen ezalutamide and androgen synth-
esis blocker abiraterone, are effective when cells are
dependent on androgen for their growth. Additional thera-
pies and molecular targets are being sought to help targeting
PCa
cells
which
either
harbor
aberrantly
activated,
androgen-independent
AR
or
constitutively
activated
oncogenes bypassing the need of AR signaling. In this
study, we show that KDM8 could be such a target as it is
involved in aberrant AR as well as AR-independent sig-
naling by being a partner for both androgen receptor and
PKM2. Androgen receptor and PKM2 are both engines for
PCa growth, as the former regulates cell cycle, and the
latter, tumor metabolism. Adaptation to tumor metabolism
is also important for tumor cells to escape the nutrition
deprived conditions during therapeutic intervention. Indeed,
Gene set enrichment analysis (GSEA) in KDM8-high
prostate
clinical
samples
several
metabolic
pathways
including those involved in lipid, glycolysis, and pyruvate
are enriched (Figure S9). Thus, targeting KDM8 should not
only thwart castration-resistance but may also reduce ther-
apy resistance. KDM8 as a coactivator of AR KDM8 is overexpressed in nearly all PCa cell lines and in a
significant portion of high-grade PCas. Its overexpression
leads to AR activation under androgen-deprived conditions
and
confers
castration-resistance
in
xenograft
model. Knockdown of KDM8 preferentially affected the growth of
PCa cells with virtually no effects on normal prostate epi-
thelial cell. We showed that KDM8 is a bona fide AR
coactivator by demonstrating its association with AR,
synergistic activation of ARE-driven promoters with AR,
and co-recruitment with AR to the target sites. The exten-
sive overlap of KDM8 and AR-activated genes lends cre-
dence to the notion that KDM8 is a natural coactivator of
AR. At present, we do not know how KDM8 enhances AR
activity. Our data suggest that it facilitate the delivery of AR
to the chromatin targets, likely due to KDM8’s ability to
modulate the chromatin conformation. In this regard, we
found that the demethylase activity for H3K36me3, how-
ever, is not absolutely required for co-activating AR. It is
possible the newly discovered cleavage activity may mod-
ulate the chromatin structure without demethylase activity
[27, 28]. Targeting KDM8 in therapy-resistant PCa cells MDVR has a heightened expression of KDM8, accom-
panied by elevated expression of EZH2, as compared to the
enzalutamide-sensitive C4-2B. TCGA public data analysis
revealed a general positive correlation of KDM8 and EZH2
in PCa cells (Fig. 8b). Both the in vitro (Fig. 2a) and in vivo
(Figure S8) growth of C4-2B-MDVR is inhibited by KDM8 There have been significant interests in developing therapies
overcoming castration as well as enzalutamide resistance. We have developed an enzalutamide-resistant C4-2B cell
line, C4-2B MDVR [36]. As shown in Fig. 8a, C4-2B KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 27 knockdown. We show here that C4-2B-MDVR is sensitive
to cell killing by two EZH2 inhibitors (GSK343 and
GSK126) (Fig. 8c). Interestingly, C4-2B-MDVR is more
sensitive to EZH2 inhibitor than its C4-2B, despite the
higher EZH2 expression level. This suggests that during the
selection of enzalutamide resistance, C4-2B-MDVR has
developed a reliance on the EZH2 pathway. One potential
mechanism is the adaptation of neuroendocrine phenotypes,
caused by EZH2 overexpression [37]. Indeed, in C4-2B-
MDVR cell line, the expressions of neuroendocrine marker
genes (NSE, SYP, and HTRSA) are elevated over C4-2B
(Fig. 8d). Moreover,
siRNA
knockdown
of
KDM8 significantly decreased the expression of neu-
roendocrine genes, suggesting that the KDM8–AR–EZH2
axis may be involved in the generation of neuroendocrine
phenotypes (Fig. 8e), thereby conferring resistance to
enzalutamide. EZH2
inhibitor
or
KDM8
knockdown
reverses this trend and resensitizes these cells toward
enzalutamide. produce sufficient pyruvate, but low enough to accumulate
enough glycolytic precursors for the biosynthesis of mac-
romolecules. PKM’s activity can be modulated by a variety
of ways including metabolite binding [39, 40] and post-
translational modifications [41–44]. An additional way is to
divert some of the cytosolic PKM2 into nucleus where it
serves as a coactivator to transcriptionally activate more
metabolic genes [6, 45, 46]. The mechanisms associated
with PKM2 translocation are multiple, including phos-
phorylation, [47] acetylation [43], and hydroxylation [45]. Here, we describe another way whereby KDM8 binds
PKM2 and transports PKM2 into the nucleus and enhances
metabolic gene expressions. Nearly all the glycolytic
enzymes have higher level of expressions to accelerate the
glycolysis reactions. Many of these genes are targets of
HIF-1a. Their upregulations depend on the presence of both
KDM8 and PKM2, as knockdown of either one diminished
the effects. PSA-luciferase activity assay The PSA-luciferase activity assay in PC-3 cells was as
described previously [60]. Cell lines and cell culture RWPE1, LNCaP, PC3, C4-2B, DU145, CWR22V1, and
HEK293 cells were obtained from the American Type
Culture Collection (ATCC, Manassas, VA). C4-2B cells
resistant to enzalutamide (C4-2B-MDVR) were generated
by culturing C4-2B cells to increasing concentrations of
enzalutamide (5–40 µM) in media for over 12 months and
maintained in complete media supplemented with 20 µM
enzalutamide as described previously [58, 59]. HEK293
cells were cultured in complete DMEM medium. RWPE1,
LNCaP, LNCaP-KDM8 [17], PC3, C4-2B, DU145, and
CWR22RV1 cells were cultured in RPMI-1640 medium
supplied with 10% heat-inactivated FBS, 100 U/ml peni-
cillin, and 0.1 mg/ml streptomycin in a humidified incubator
with 5% CO2. For hormone induction experiments, the
growth media were replaced by RPMI-1640 containing
10% charcoal-dextran-treated (CDT) FBS (CDT-FBS) for
2 days before treatment with 10 nM dihydrotestosterone
(DHT). KDM8/EZH2 and enzalutamide resistance Cells were seeded in 48-well plates in RPMI-1640 complete
medium one day before KDM8 knocking down experi-
ments. After incubation for 24 h, the cells were then trans-
fected with si-RNAs of non-targeting control (si-NT) or
specific targeting KDM8 (si-KDM8#1 and si-KDM8#2). The cell proliferation was measured every 2 days by MTT
colorimetric assay according to the manufacturer’s instruc-
tion (Roche, IN). C4-2B and C4-2B-MDVR cells were
seeded on 96-well plates at a density of 1 × 104 cells per
well in RPMI-1640 media containing 10% FBS and treated
with different concentrations of EZH2 inhibitors (GSK343
and GSK126) for 48 h. Cell viability was determined by the
MTT colorimetric assay and the cell survival rate (%) was
calculated as cell survival rate (%) = (OD570 nm of treat-
ment group/OD570 nm of control group) × 100%. The second-generation anti-androgen enzalutamide has
improved the prospects of castration-resistance patients. However, enzalutamide-resistant tumors eventually emerge,
leading to mortality. Some of the resistant cells, especially
those with RB, p53, and PTEN defects, is marked by an
intermediate phenotype with the expression of both andro-
gen receptor and neuroendocrine markers [37]. EZH2 or
polycomb complex was shown to be an inducer of the
neuroendocrine phenotypes [53, 57]. Inhibition of EZH2
reverses the phenotypes and resensitize the resistant cells
toward enzalutamide [37]. As KDM8 is an upstream reg-
ulator of EZH2 as shown in this study, we checked whether
heightened KDM8 expression is associated with enzaluta-
mide resistance. In the enzalutamide-resistant C4-2B-
MDVR line we developed [1], KDM8 expression was ele-
vated, which is accompanied by upregulation of neu-
roendocrine genes. These clones are more sensitive to
EZH2 inhibitor and KDM8 knockdown reduces neu-
roendocrine markers, overcoming enzalutamide resistance. These data suggest that KDM8 may have a role in therapy
resistance as well. Materials and methods unbiased siRNA screening, we found that all individually
contributed to some extent to the growth and survival
phenotypes under androgen-deprived conditions, with the
knockdown of EZH2, AMACR, NSD2, MCM3, ETV1, and
CD24 giving the most severe consequence. We chose EZH2
to further define the detailed mechanisms of upregulation,
because of its well-recognized role in castration-resistance
[48, 49], EMT/metastasis [50, 51], chemoresistance [52],
and neuroendocrine phenotypes [53] of PCa. EZH2, the
catalytic subunit of the polycomb repressive complex
PRC2, is a general repressor of gene transcription and
shown to be a corepressor of androgen receptor in PCa [54]. Interestingly, in castration-resistant PCa, EZH2 also acts as
a coactivator to modulate genes involved in castration-
resistance [55]. EZH2 is commonly overexpressed in PCas
[48] with the highest expression in lethal-type castration-
resistance PCa [56]. Our studies provide additional insights
into the regulation of the EZH2 in PCa. Our data suggest
that KDM8 and ANNCA are recruited to the promoter of
EZH2 and regulate its expression. Our data confirmed
previous results showing ANCCA is a direct target of AR
and further indicate that KDM8 also participates in the
transcription of this gene. KDM8 as a coactivator of PKM2 PKM2 is a pivotal enzyme in determining the metabolic
flow to lactate, TCA cycle, or biosynthetic pathway [38]. For tumor cells, PKM2 enzymatic activity is required, but
should be maintained at a balanced level, high enough to H-J Wang et al. 28 Acknowledgements This work was supported by grants CA114575,
CA165263,
and
CA206222
from
NIH,
USA;
NHRI03A1- Co-immunoprecipitation and immunoblotting
analysis In summary, we have identified KDM8 as a dual coac-
tivator of AR and PKM2, which drives PCa growth and
provides needed metabolic energy. These properties, toge-
ther with its ability to activate EZH2, a factor known to be
involved in castration and therapy resistance, make KDM8
a promising therapeutic target for PCa. Co-immunoprecipitation was performed using cell lysates
from LNCaP and 293T cells for endogenous and ectopically
expressed
proteins,
respectively,
for
investigating
protein–protein interaction has been described [7]. The KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC 29 immunoblotting assay was performed using the following
antibodies: Flag-M2, Myc-tag (Cell Signaling), KDM8
[17]; β-actin (Sigma-Aldrich); PSA (Santa Cruz Biotech);
ANCCA [61], EZH2 (Cell signaling), and GAPDH (Santa
Cruz Biotech). containing cores from 48 cases, with normal prostate tissues
adjacent to tumors and Gleason scores. The slides were then
incubated with anti-KDM8 rabbit polyclonal antibody
(homemade) at 1:100 dilutions overnight at 4 °C, followed
by incubations with biotinylated secondary antibody and the
ABC reagents in the Vectastain Elite kit and counter-stained
with hematoxylin. The percentage of positive nuclear
staining was scored as follows: 0–<5%, scored as KDM8-
negative or non-detected; 5–<25%, scored as KDM-low;
and >25%, scored as KDM-high. Differences and correla-
tions in immunostaining among groups were analyzed with
χ2 or Fisher’s exact test. Microarray assay Confocal microscopy analysis for assessment of PKM2
nuclear translocation was performed as described pre-
viously [7]. LNCaP cells overexpressing KDM8, KDM8-H321A, or EV
were grown in 10 cm dishes, and total RNAs were extracted
from 80% confluent cell using TRIzol (Invitrogen) extraction
according to the manufacturer’s instructions. Microarray gene
expression profiling was performed by 3′ IVT expression
analysis with Affymetrix GeneChip Human Genome U133
Plus 2.0 Arrays. The KDM8-responsive and androgen-
responsive gene expression changes were identified by pair-
wise comparison analyses (≥1.5-fold threshold), and expres-
sion patterns were analyzed by hierarchical clustering. Statistical analysis Comparisons were performed with a Student’s t-test with p-
values denoted as *p < 0.05 and **p < 0.01 (N.S., not sig-
nificant). Graphpad Prism software (La Jolla, CA, USA)
was used to calculate mean and standard deviation. Glucose uptake assay and lactate production assay Glucose uptake assay and lactate production assay were
described previously [7]. Oncomine data analysis KDM8 and EZH2 expression in PCa patient cohorts was
extracted from Oncomine database (www.oncomine.com)
[63]. In the database, the Cancer Genome Atlas (TCGA)
dataset was extracted and used to compare the differences of
clinical specimens between cancer and normal by using a
threshold of p < 0.05. Statistical analysis was performed
with one-way ANOVA or two-tailed t-tests. Correlation
between EZH2 and KDM8 was assessed by using the
Pearson correlation coefficient. Mouse xenograft tumor study Fractionation of nuclear and cytosolic extracts was per-
formed by using NE-PER® Nuclear and Cytoplasmic
Extraction kit (Thermo Scientific) according to the manu-
facturer’s instruction. Overall, 15 µl of each fraction was
analyzed immunoblotting assay. Xenograft tumor studies were conducted utilizing the 6-
week-old male athymic Nu/Nu mice (Harlan). The total
number of mice (16) was randomly divided into two sets of
8 each. Mice of the two sets were inoculated sub-
cutaneously with one million of LNCaP cells over-
expressing KDM8 or vector control (EV) in 100 μl of 50%
Matrigel (BD Biosciences), respectively. Tumor growth
was monitored and the length (L), width (W), and height
(H) measurements taken every 7 days. The tumor volume
was calculated using the formula (L × W × H) × 0.52. For
castration study, the tumor-bearing mice were castrated
when the tumors reached their, respectively, peak volumes
(6-week post injection). All mice were killed by 10 weeks
post injection. The animal tumor studies were approved by
National Health Research Institutes Institutional Animal
Care
and Use
Committee
(approval number:
NHRI-
IACUC-102087) and carried out under the institutional
guidelines with animal welfare standards. Chromatin immunoprecipitation assay ChIP assay was performed as described previously [61, 62]. The precipitated chromatin DNA was analyzed by real-time
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indicated otherwise in a credit line to the material. If material is not
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oxygen species contributes to cellular antioxidant responses. Science. 2011;334:1278–83. 63. Affiliations Hung-Jung Wang1,2
●Mamata Pochampalli3
●Ling-Yu Wang3
●June X Zou3
●Pei-Shan Li2
●Sheng-Chieh Hsu1,4
●
Bi-Juan Wang5
●Shih-Han Huang5
●Ping Yang6,7
●Joy C. Yang6
●Cheng-Ying Chu8
●Chia-Ling Hsieh8
●
Shian-Ying Sung8
●Chien-Feng Li9
●Clifford G. Tepper
6
●David K. Ann8,10
●Allen C. Gao1,6
●
Christopher P. Evans6,11
●Yoshihiro Izumiya11
●Chi-Pin Chuu5
●Wen-Ching Wang12
●Hong-Wu Chen3,11
●
Hsing-Jien Kung2,3,4,8 Hung-Jung Wang1,2
●Mamata Pochampalli3
●Ling-Yu Wang3
●June X Zou3
●Pei-Shan Li2
●Sheng-Chieh Hsu1,4
●
Bi-Juan Wang5
●Shih-Han Huang5
●Ping Yang6,7
●Joy C. Yang6
●Cheng-Ying Chu8
●Chia-Ling Hsieh8
●
Shian-Ying Sung8
●Chien-Feng Li9
●Clifford G. Tepper
6
●David K. Ann8,10
●Allen C. Gao1,6
●
Christopher P. Evans6,11
●Yoshihiro Izumiya11
●Chi-Pin Chuu5
●Wen-Ching Wang12
●Hong-Wu Chen3,11
●
Hsing-Jien Kung2,3,4,8 Innovation Center for Cancer Medicine, Sun Yat-sen University
Cancer Center, Guangzhou, China Innovation Center for Cancer Medicine, Sun Yat-sen University
Cancer Center, Guangzhou, China 1
Institute of Biotechnology and Pharmaceutical Research, National
Health Research Institutes, 35053 Miaoli County, Taiwan 1
Institute of Biotechnology and Pharmaceutical Research, National
Health Research Institutes, 35053 Miaoli County, Taiwan Institute of Biotechnology and Pharmaceutical Research, National
Health Research Institutes, 35053 Miaoli County, Taiwan
2
Institute of Molecular and Genomic Medicine, National Health
Research Institutes, 35053 Miaoli County, Taiwan
3
Department of Biochemistry and Molecular Medicine, School of
Medicine, University of California, Davis, CA 95817, USA
4
Institute of Biotechnology, National Tsing-Hua University, 30035
Hsinchu, Taiwan
5
Institute of Cellular and System Medicine, National Health
Research Institutes, 35053 Miaoli County, Taiwan
6
Department of Urology, School of Medicine, University of
California, Davis, CA 95817, USA
7
State Key Laboratory of Oncology in South China, Collaborative 8
Ph.D. References Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R,
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integrated data-mining platform. Neoplasia. 2004;6:1–6. H-J Wang et al. 32 Affiliations 7
State Key Laboratory of Oncology in South China, Collaborative Affiliations Program for Translational Medicine, College of Medical
Science and Technology, Taipei Medical University, Taipei City,
Taiwan 2
Institute of Molecular and Genomic Medicine, National Health
Research Institutes, 35053 Miaoli County, Taiwan 3
Department of Biochemistry and Molecular Medicine, School of
Medicine, University of California, Davis, CA 95817, USA 3
Department of Biochemistry and Molecular Medicine, School of
Medicine, University of California, Davis, CA 95817, USA 9
National Institute of Cancer Research, National Health Research
Institutes, 35053 Miaoli County, Taiwan 9
National Institute of Cancer Research, National Health Research
Institutes, 35053 Miaoli County, Taiwan 4
Institute of Biotechnology, National Tsing-Hua University, 30035
Hsinchu, Taiwan 4
Institute of Biotechnology, National Tsing-Hua University, 30035
Hsinchu, Taiwan 10
Department of Molecular Pharmacology, Beckman Research
Institute, City of Hope, Duarte, CA, USA 10
Department of Molecular Pharmacology, Beckman Research
Institute, City of Hope, Duarte, CA, USA 5
Institute of Cellular and System Medicine, National Health
Research Institutes, 35053 Miaoli County, Taiwan 11
Comprehensive Cancer Center, School of Medicine, University of
California, Davis, Sacramento, CA, USA 11
Comprehensive Cancer Center, School of Medicine, University of
California, Davis, Sacramento, CA, USA 6
Department of Urology, School of Medicine, University of
California, Davis, CA 95817, USA 12
Institute of Molecular and Cellular Biology, National Tsing-Hua
University, Hsinchu, Taiwan 12
Institute of Molecular and Cellular Biology, National Tsing-Hua
University, Hsinchu, Taiwan 7
State Key Laboratory of Oncology in South China, Collaborative
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Familial multinodular goiter syndrome with papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1 Chinese family
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Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12406928 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Liao, Shunyao, Wenzhong Song, Yunqiang Liu, Shaoping Deng, Yaming Liang, Zhenlin Tang,
Jiyuan Huang, Dandan Dong, and Gang Xu. 2013. “Familial multinodular goiter syndrome with
papillary thyroid carcinomas: mutational analysis of the associated genes in 5 cases from 1
Chinese family.” BMC Endocrine Disorders 13 (1): 48. doi:10.1186/1472-6823-13-48. http://
dx.doi.org/10.1186/1472-6823-13-48. Published Version
doi:10.1186/1472-6823-13-48 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Open Access * Correspondence: shunyaol@yahoo.com; wz360@hotmail.com
1Diabetes & Endocrinology Center, Sichuan Academy of Medical Science,
Sichuan Provincial People’s Hospital, Chengdu 610072, China
2Department of Thyroid Disease & Nuclear Medicine, Sichuan Academy of
Medical Science, Sichuan Provincial People’s Hospital, Chengdu 610072,
China
F ll li
f
h
i f
i
i
il bl
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d
f h
i l Familial multinodular goiter syndrome with
papillary thyroid carcinomas: mutational analysis
of the associated genes in 5 cases from 1 Chinese
family Shunyao Liao1*, Wenzhong Song2*, Yunqiang Liu3, Shaoping Deng1,4, Yaming Liang1, Zhenlin Tang2,
Jiyuan Huang2, Dandan Dong5 and Gang Xu5 © 2013 Liao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Full list of author information is available at the end of the article Background PTC is the most prevalent malignancy of the thyroid gland. There has been an increasing incidence of PTC worldwide
for the past few decades. The etiology of PTC is related
to environmental, hormonal and genetic factors. About
5-15% of PTC patients show a familial occurrence, and
fPTC is recognized as a distinct entity only in recent years
[1,2]. Families with accumulation of PTCs show an
inherited trait of the disease and patients with fPTC often
have early age at disease onset and increased severity in
successive generations, also, fPTC patients frequently
present more aggressive tumors with increased incidence
of multifocality, local invasion, lymph node metastases than
the sporadic PTC [2,3]. Generally, fPTC is diagnosed when
three or more family members have PTC and in the ab-
sence of other known associated syndromes [1,2]. PTC has
a significant gender bias with much more women affected
than men; it is especially suggestive for the familial predis-
position when men or children were diagnosed with PTC
[1,4]. While, because families share the same environment
and a common genetic background, it is difficult to distin-
guish between environmental and genetic contributing fac-
tors, and also because the majority of fPTC pedigrees are
small in size and may present with a variety of additional
benign thyroid nodules, the genetic predisposition to fPTC
is unknown and the molecular alterations at the origin of
the pathology are only now beginning to emerge [1,5,6]. Sporadic PTC is known to be associated with point
mutation of the BRAF genes and chromosomal rear-
rangements of RET/PTC. The BRAF encodes a serine/
threonine-protein kinase which plays a role in regulat-
ing the MAP kinase/ERKs signaling pathway and affects
cell division, differentiation and secretion; point muta-
tions in BRAF are found in up to 45% PTC cases [7]. The RET protooncogene is one of the receptor tyrosine
kinases, cell-surface molecules that transduce signals
for cell growth and differentiation; rearrangements of
the RET are found in about 35% of sporadic PTC [7]. Abstract Background: Familial papillary thyroid cancer (fPTC) is recognized as a distinct entity only recently and no fPTC
predisposing genes have been identified. Several potential regions and susceptibility loci for sporadic PTC have
been reported. We aimed to evaluate the role of the reported susceptibility loci and potential risk genomic region
in a Chinese familial multinodular goiter (fMNG) with PTC family. Methods: We sequenced the related risk genomic regions and analyzed the known PTC susceptibility loci in the
Chinese family members who consented to join the study. These loci included (1) the point mutations of the BRAF
and RET; (2) the possible susceptibility loci to sporadic PTC; and (3) the suggested potential fMNG syndrome with
PTC risk region. Results: The members showed no mutations in the common susceptible BRAF and RET genomic region, although
contained several different heterozygous alleles in the RET introns. All the members were homozygous for PTC risk
alleles of rs966423 (C) at chromosome 2q35, rs2910164 (C) at chromosome 5q24 and rs2439302 (G) at chromosome
8p12; while carried no risk allele of rs4733616 (T) at chromosome 8q24, rs965513 (A) or rs1867277 (A) at
chromosome 9q22 which were associated with radiation-related PTC. The frequency of the risk allele of rs944289 (T)
but not that of rs116909374 (T) at chromosome 14q13 was increased in the MNG or PTC family members. Conclusions: Our work provided additional evidence to the genetic predisposition to a Chinese familial form of
MNG with PTC. The family members carried quite a few risk alleles found in sporadic PTC; particularly, homozygous
rs944289 (T) at chromosome 14q13 which was previously shown to be linked to a form of fMNG with PTC. Moreover, the genetic determinants of radiation-related PTC were not presented in this family. Keywords: Familial papillary thyroid carcinomas, Multinodular goiter syndrome, Mutational analysis, Genetic
association, Risk alleles Keywords: Familial papillary thyroid carcinomas, Multinodular goiter syndrome, Mutational analysis, Genetic
association, Risk alleles © 2013 Liao et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Page 2 of 11 wide studies [11]. Abstract DIRC3 predicted a non coding RNA
transcript with unknown function, the first 2 exons of
DIRC3 replaced exon 1 of HSPBAP1 and formed a
DIRC3-HSPBAP1 fusion transcript, which are associated
with chromatin remodeling and stress response; (2) It was
reported that the heterozygosity G/C of SNP rs2910164
at 5q24 within the precursor of microRNA-146a pre-
disposed to PTC by altering expression of miR146a
target genes in the Toll-like receptor and cytokine sig-
naling pathway [12,13]; (3) The genome-wide study also
identified that chromosomal 8q24 was associated with
the risk of various cancers, particularly, rs4733616 at
8q24 was founded to be possibly associated with PTC
risk in 26 European families [14-16]; (4) The rs2439302,
located in the intron of HRG-beta1c at 8p12,was
reported to be associated with neuregulin 1 (NRG1) and
confer risk of thyroid cancer [11]. HRG-beta1c is one of
the NRG1 isoforms and interacts with tyrosine kinase to
increase
its
phosphorylation
on
tyrosine
residues,
playing critical roles in the growth and development of
multiple organ system; (5) It was repeatedly observed
that the rs965513 at 9q22.33 were the strong association
signal for NMTC in European people [16-19] and it was
proposed that the rs965513 might linked to the nearest
thyroid transcription factor of forkhead family (FOXE1)
gene, which likely plays a crucial role in thyroid morpho-
genesis;
furthermore,
some
research
indicated
that
rs1867277 within the FOXE1 5′ UTR is also a causal vari-
ant in thyroid cancer susceptibility [16,20]; (6) Finally,
both rs944289 and rs116909374 on 14q13.3 were ob-
served to be strongly associated with NMTC in European
people [11,16-19,21]. Nonetheless, all these genetic asso-
ciations found by the genome-wide association studies
have not been investigated in a family based study. Methods
Patients The fMNG with PTC pedigree is reported in Figure 1. The clinical and pathological findings are summarized in
Table 1. The study protocol was approved by the Review Board
of Clinical Research of the Sichuan Provincial hospital,
and by the Research & Ethics Committee of Sichuan
Medical Research Institution. The blood samples were
collected from the proband (III2), proband’s parents (II1
& 2) and maternal aunts (II5, 8, & 10) with their written
informed consent. Background Al-
though somatic mutations of the genes like BRAF and
RET exclusively play a causative role in sporadic thyroid
cancer development, germline mutations of single nu-
cleotide polymorphisms (SNPs) in these genes were also
reported to act as modifiers in the cancer process [8,9],
it needs to mention here that in a Chinese population,
SNPs of BRAF were shown to be associated with PTC
[10], and thus it is intriguing to verify these mutations
in fPTC families. In addition, a few potential regions for harboring an
fPTC gene have been reported: chromosomal region
1q21 linked to fPTC with papillary renal neoplasia [22],
2q21 linked to familial NMTC type 1 syndrome [23],
and the telomere abnormalities and chromosome fragil-
ity might display in fPTC family [24]; Specifically, famil-
ial NMTC and its relationship with familial MNG are
recognized as distinct clinical entities, and the molecular
pathophysiology of MNG and PTC is different, indeed
MNG1 is located at 14q [25]; however, one study in a
kindred with MNG and PTC suggested that 14q32
linked to a form of inherited MNG syndrome with a sig-
nificant risk of progression to PTC [26]. In the present report we studied 2 PTCs and 3 MNGs
obtained from members of one Chinese family. This fam-
ily was ascertained through initial identification of the pro-
band, a 35-year-old men (III2, Figure 1). The probands’s
mother, 5 maternal aunts and 1 younger first cousin were
diagnosed with MNG or PTC by different hospitals in
China. The mode of inheritance in the family appeared to Recent studies based on population stratification have
made progresses to identify several single nucleotide poly-
morphisms (SNPs) associated with PTC risk. For exam-
ples, (1) It was discovered that rs966423 at 2q35, locating
into the intron region of the disrupted in renal carcinoma
3 gene (DIRC3), was significantly associate with European
nonmedullary thyroid cancer (NMTC) by the genome- Liao et al. BMC Endocrine Disorders 2013, 13:48
Page 3 of 11
http://www.biomedcentral.com/1472-6823/13/48 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Page 3 of 11 Figure 1 Pedigree of the Chinese fPTC. Circles and squares indicate female and male family members, respectively. The proband is indicated
by an arrow. be autosomal dominant. Background For the purpose to improve our
understanding of the PTC predisposition, based on the re-
cent progresses in genetic studies about PTC, we analyzed
in this Chinese family (1) the point mutations of the BRAF
and RET; (2) the possible susceptibility loci to sporadic
PTC; and (3) the suggested potential fMNG syndrome
with PTC risk region. be autosomal dominant. For the purpose to improve our
understanding of the PTC predisposition, based on the re-
cent progresses in genetic studies about PTC, we analyzed
in this Chinese family (1) the point mutations of the BRAF
and RET; (2) the possible susceptibility loci to sporadic
PTC; and (3) the suggested potential fMNG syndrome
with PTC risk region. thyroidectomy specimen pathologic examinations disclosed
that the architecture and nuclear features of the neoplasm
in his both necks were typical for PTC (Figure 2A) and
immunohistochemical staining confirmed the diagnosis
(Figure 2B, C, D); After the total thyroidectomy and radio-
active iodine treatment, the patient is now doing well. Interestingly, in terms of fMNG with PTC, the patient’s
mother is diagnosed with MNG in bilateral thyroid and
underwent a total thyroidectomy in Chongqing, China
(Figure 1 II2). Both of the patient’s maternal twin aunts
and a younger male cousin were diagnosed with MNG
and PTC by different hospitals in Beijing and Chongqing,
China, respectively; the other two maternal aunts were di-
agnosed with MNG by different hospitals in Chengdu and
Dazhou, China, respectively (Figure 1 II8&10). DNA extraction
h
h l
bl The whole blood was collected from the medial cubital
vein into heparin anticoagulant tubes. The total DNA
was purified using the spin protocol of QIAamp DNA
Blood Mini Kit according to the manufacturer’s direc-
tions (Qiagen, Hilden, Germany). The purified DNA was
resuspended in TE buffer and stored at 4 °C. Gel electro-
phoresis and spectrophotometric determination were
used to DNA quantification and quality analysis. The
OD260/OD280 ratio of DNA samples were between 1.8-
2.0 and concentration was more than 100 ng/ml. A 35-year-old man (Figure 1 III2) came to our observa-
tion: the man complained both his lymph nodes containing
palpable lump for more than 10 days, initial ultrasound ex-
aminations revealed an 1.9 × 1.4 cm solid mass with irregu-
lar & indefinite border, sand calcification and blood flow in
his right neck, and also 2 small nodule goiters in his left
neck; The thyroid function tests showed the man was
euthyroid; both the fine needle aspiration cytological and Table 1 Clinical and pathological study of the collected samples
Members
Sex
Age at diagnosis
Histology
sizes for PTCs and MNGs
Surgical treatment
II1
male
64
normal
II2
female
62
bilateral MNG
MNG (1.2 cm), suspicious lesion
completion thyroidectomy
III2
male
35
bilateral MNG with PTC
PTC in MNG, PTC (1.6 cm)
completion thyroidectomy
II5
female
56
bilateral MNG with PTC
PTC in MNG, PTC (1.5 cm)
completion thyroidectomy
II8
female
45
bilateral MNG
MNG (0.3 cm)
II10
female
41
MNG in right thyroid
MNG (0.6 cm) Table 1 Clinical and pathological study of the collected samples Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Page 4 of 11 Figure 2 The histological features of the proband’s papillary carcinoma. A: The cytological feature: crowded oval nuclei, nuclear grooves,
clearing, elongation and overlapping (HE × 400). B: Galectin-3 showed predominantly cytoplasmic staining with occasional nuclear staining
(×200). C: HBME1 showed positive diffuse membrane (×200). D: Cytokeratin 19 showed strong, predominantly cytoplasmic staining (×200). ma. A: The cytological feature: crowded oval nuclei, nuclear grooves,
dominantly cytoplasmic staining with occasional nuclear staining
n 19 showed strong, predominantly cytoplasmic staining (×200). Figure 2 The histological features of the proband’s papillary carcinoma. A: The cytological feature: crowded oval nuclei, nuclear grooves,
clearing, elongation and overlapping (HE × 400). B: Galectin-3 showed predominantly cytoplasmic staining with occasional nuclear staining
(×200). C: HBME1 showed positive diffuse membrane (×200). DNA extraction
h
h l
bl D: Cytokeratin 19 showed strong, predominantly cytoplasmic staining (×200). Genetic mutational analysis gel stained with ethidium bromide and purifed using the
QIAquick PCR purification kit (Qiagen). Purified PCR
products were sequenced directly in both orientations
using standard procedures with an ABI PRISM 3100 Gen-
etic Analyzer (ABI, CA). The sequences were confirmed
with two independent PCRs from two independent DNA
samples. The potential regions and susceptibility loci investigated
in the study were listed in Table 2. Sequencing was per-
formed on PCR-amplified products using primers (Table 2)
according to the published sequences or self-designed
with Primer Premier 6.1 (PREMIER Biosoft, Palo Alto
CA). The PCR amplifications were performed using ABI
GeneAmp PCR System 9700 (Applied Biosystems, Foster
City, CA). The PCR reaction system included 2U Pfu
DNA polymerase (Thermo Fisher Scientific Inc, USA),
50pmol of each sense and antisense primers, 1 × reaction
buffer (20 mM Tris–HCl pH8.8, 10 mM KCl, 10 mM
(NH4)2SO4, 1% (v/v) Triton X-100), 250 μM dNTP,
2.0 mM MgCl2 and 200 ng genomic DNA in a total vol-
ume of 50 μl. The PCR cycling parameters were followed
the recommendations for Pfu DNA polymerase according
to the manufacturer. Precautions were taken to prevent
PCR contamination, and indeed, in each experiment DNA
template negative samples were run in parallel. The PCR
products were resolved by electrophoresis in a 2% agarose Chr10: 43,601,341-43,602, 077 (737 bp) intron: 33,
825–34,307&34,504-34,561 exon5: 34,308-34,503
(196 bp) STS: 33,825-34,398
Chr10: 43,607,577-43,608,444 (868 bp) exon8:
40,061-40,155 (96 bp) intron: 40,156-40,784&40,896-
40,928 exon9: 40,785-40,895 (111 bp)
Chr10: 43,608,459-43,609,249 (791 bp) intron:
40,943-41,487&41,608-41,557 exon10: 41,488-41,607
(120 bp) STS: 41558-41733 5′-CCAGTGGCCCCGCAGGTT-3′5′-
GAAAAGCACGTCTCCCCACAGTCC-3′
Chr14: 36, 5′-TGTAATGGCAGCTCTTGACCTT-3′ 5′-
ACCTTTGATTGCCCTTAGTTTGA-3′
Chr14: 36,738,229-36,738,674(446 bp) rs116909374: 36,738,361 rs116909374: 36,738,361 susceptibility loci of 9q22. Noticeably, all the family mem-
bers including the proband’s father without thyroid disease
were homozygous for the risk alleles of (1) rs966423 (CC)
in DIRC3, (2) rs2910164 (CC) in Pre-miR-146a and (3)
rs2439302 (GG) in HRG-beta1c; While all these members
from the Chinese family contained no risk allele of (4)
rs4733616 at 8q24, (5) rs965513 and rs1867277 at 9q22. and III3), 35 and 25 years old respectively, were diag-
nosed with MNG and PTC (Figure 1); As the family
members resided in different cities and denied radiation
exposure, no other neoplasia syndromes or somatic gen-
etic
alterations in
the
tumor
DNA
was
observed,
according to diagnostic criteria of familial MNG with
PTC [6], we considered the Chinese family presented
hereditary predisposition to PTC. and III3), 35 and 25 years old respectively, were diag-
nosed with MNG and PTC (Figure 1); As the family
members resided in different cities and denied radiation
exposure, no other neoplasia syndromes or somatic gen-
etic
alterations in
the
tumor
DNA
was
observed,
according to diagnostic criteria of familial MNG with
PTC [6], we considered the Chinese family presented
hereditary predisposition to PTC. For the susceptibility loci of 14q13.3, as 14q was
reported to be specifically linked with MNG1 and a form
of MNG with PTC [25,26], it is worth to mention that
the risk T allele of rs944289 was presented in the se-
quences of the most family members affected with thy-
roid disease (II2 & II8, MNG; II5 & III2, MNG with
PTC; Table 1). The sequence result in Table 3 showed
that both MNG with PTC family members II5 and III2
were heterozygous (CT) and the 2 MNG family mem-
bers II2 and II10 were homozygous (TT) at rs944289
locus (Table 3). While for another susceptibility locus of
rs116909674 at 14q13.3 which we checked, none of the
studied Chinese family members carried the risk alleles. The comparison of the susceptibility loci In the current study, we investigated the exon 15 of
BRAF, since several SNPs in the genomic region were
reported to contribute to PTC in a Chinese population
[8] and the transversions in exon 15 are the common
morphotype-specific mutation in adult sporadic PTC. The results were shown in Table 3: the examined BRAF
sequences involved these susceptibility loci carried no
risk alleles and were the same as common TT at
BRAFT1799A and AA at BRAFA1801G. No any other gen-
etic mutation was found in the family members. We also investigated all the known RET susceptibility
loci to family thyroid diseases in this Chinese family. Ei-
ther, no known RET susceptibility loci was mutational in
the family members. However, it needs to mention that in
the genomic regions which we sequenced, the RET introns
contained certain differences among the family members,
such as introns between exon 4 and 5 (rs35800403 &
rs2742243), between 11 and 12 (rs2256550), between 14
and 15 (rs11238441 & rs2472737) (Table 3), and also, there
was a new C to T heterozygous allele in the upstream of
rs111306965 in the genome of memberII8 andII10 by re-
peatedly sequencing. Additionally, rs1800863 in exon 15
contained variants of synonymous code substitution in the
genome of several family members (II2, II5 & II10). The identification for the fMNG with PTC The histological features of the proband’ papillary car-
cinoma were shown in Figure 2. The members of the
Chinese family were diagnosed with MNG and PTC by
different hospitals in China; the affected individuals
showed typical MNG or MNG with PTC, bilateral and
multicentric nodes. In this Chinese family, there were 2
first-degree blood relatives were diagnosed with bilateral
MNG and PTC, 5 second-degree blood relatives includ-
ing a pair of twin sisters were diagnosed with MNG or
PTC; Also among these family members, 2 men (III2 Page 5 of 11 Page 5 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Localization & product 5′-TGCTTGCTCTGATAGGAAAATG-3′ 5′-
CCACAAAATGGATCCAGACA-3′
Chr7:140,453,250-140,453,078 (173 bp) intron:
176,315-176,371 exon15:176,372-176,487 (116 bp) RET at Chr10q11.2: 43,572,517-43,625,799
exon5: 34,308-34,503 (196 bp) R313Q: 34,378(G →A)
rs77702891: 43,601,894 R330Q: 34,429(G →A)
rs80236571: 43,601,945 Chr10: 43,601,341-43,602, 077 (737 bp) intron: 33,
825–34,307&34,504-34,561 exon5: 34,308-34,503
(196 bp) STS: 33,825-34,398
Chr10: 43,607,577-43,608,444 (868 bp) exon8:
40,061-40,155 (96 bp) intron: 40,156-40,784&40,896-
40,928 exon9: 40,785-40,895 (111 bp)
Chr10: 43,608,459-43,609,249 (791 bp) intron:
40,943-41,487&41,608-41,557 exon10: 41,488-41,607
(120 bp) STS: 41558-41733 Chr10: 43,608,459-43,609,249 (791 bp) intron:
40,943-41,487&41,608-41,557 exon10: 41,488-41,607
(120 bp) STS: 41558-41733 5′-GTGGGCCCAATGTGTGGATA −3′ 5′-
CTCTTCAGGGTCCCATGCTG-3′ Chr10: 43,611,512-43,612,272 (761 bp) intron:
43,996-44,515&44,664-44,756 exon10: 44,516-44,663
(148 bp) exon12: 44,516-44,663 (148 bp) exon13: 46, 305–46,412 (108 bp) S765P: 46,313(T →C)
rs75075748: 43,613,829 E768E: 46,324(G →A/C)
rs78014899: 43,613,840 V778I: 46,352(G →A)
rs75686697: 43,613,868 L790F: 46,390(G →C)
rs75030001: 43,613,906 Y791F: 46,392(A →T)
rs77724903: 43,613,908
5′-CGGGGAATTTCTGTGGACGA-3′ 5′-
ATGGCAGTGTCACACCAGAG-3′ Chr10: 43,613,496-43,614,200 (705 bp) intron:
45,980-46,304&46,413-46,684 exon13: 46, 305–
46,412 (108 bp) misc_difference: 46,327 Chr10: 43,614,767-43,615,517 (751 bp) intron:
47,251-47, 462&47,678-48,001 exon14: 47, 463–
47,677 (215 bp)
Chr10: 43,615,159-43,615,837 (679 bp) exon14:
47,643-47,677 (35 bp) intron: 47,678-48,012&48,136-
48,321 exon15: 48,013-48,135 (123 bp) Chr10: 43,615,159-43,615,837 (679 bp) exon14:
47,643-47,677 (35 bp) intron: 47,678-48,012&48,136-
48,321 exon15: 48,013-48,135 (123 bp) Chr10: 43,617,229-43,617,941 (713 bp) intron:
49,713-49, 877&49, 949–50,425 exon16: 49,878-
49,948 (71 bp) STS: 49,832-50,007
Chr10: 43,618,871-43,619,601 (713 bp) intron:
51,355-51, 602&51,741-52,085 exon17: 51, 603–
51,740 (138 bp) 5′-CTCTGATGGGAGTGGCTTGG-3′5′-
CCACTCAGGCACCCCTTAAC-3′ 2q35
DIRC3 (noncoding RNA):218,148,746-218,621,316
(472571 bp)rs966423:218,310,340
5q24
Pre-miR-146a: 159,912,359-159,912,457(99 bp)
rs2910164: 159,912,418
8q24
rs4733616: 128,662,095
8p12
NRG1 transcript variant HRG-beta1c: 31,496,820-
32,622,558(1,125,738 bp) rs2439302: 32,432,369 5′-CGGCCTCGACCAACACTTAT-3′
5′-ACTGGGCGTCTCAACTACAATCTG −3′
Chr2: 218,310,115-218,310,537(423 bp) located in
the intron region of DIRC3, 5′-ATTTTACAGGGCTGGGACAG-3′ 5′-
TCTTCCAAGCTCTTCAGCAG-3′
Chr5: 159,912,297-159,912,523(227 bp) 5′-CACCGGGGATTGGAAGAGATAAG-3′
5′- TGAAGCCACAGGGGAGAAAAGT −3′
Chr8:128,661,750-128,662,159(410 bp) 5′-AATGCAAGAATGGCCTAACACAAT-3′
5′-AACCTGGGGSSSSSTCTGAAGC-3′
Chr8: 32,432,326-32,432,660(334 bp) located in
intron of NRG1 Page 6 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′
5′-CCAGGCTCAGGTTATGTCTTTGTT-3′
Chr9: 100,555,758-100,556,177(420 bp) 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′
5′-CCAGGCTCAGGTTATGTCTTTGTT-3′
Chr9: 100,555,758-100,556,177(420 bp) 5′-CCGGCTTGAGTTCAGGTATGTAGT-3′
5′-CCAGGCTCAGGTTATGTCTTTGTT-3′
Chr9: 100,555,758-100,556,177(420 bp) 9q22
FoxE1: 100,615,537-100,618,997(3,460 bp)rs1867277:
100,615,914
14q13.3
rs944289: 36,649,246 5′-AGACCAGCTGCAGCCACCCCAACC-3′
5′-GTCTCGCCGCGCTCTTCCTTCACG-3′
Chr9: 100,615,806-100,616,270(465 bp)located in
the STS of FoxE1 Discussion The Chinese family presented hereditary predisposition
to PTC, but currently the genetic incline to fPTC is un-
known. With the aim of understanding the involvement
of genetic factors underlying fPTC, we analyzed the
reported possible PTC susceptibility genetic regions by
sequence in the Chinese family members who consented
to join the study. First, it is worthy to mention that no
risk allele of rs965513 (A) or rs1867277 (A) at 9q22 was
observed among the Chinese family members. These
susceptibility loci of FOXE1 at 9q22 were related to
radiation-induced PTC [19], hence it may be reasonable
that the FOXE1 risk alleles were not presented in the fa-
milial form of MNG with PTC, as the members denied
radiation exposure and resided in quite different envir-
onment. Either, the Chinese family members carried no
risk allele of rs4733616 (T) at 8q24 which has been shown
to be associated with sporadic PTC in Europeans [14-16],
but the pathogenic role of the allele is currently unknown. Discussion With respect to the other susceptibility loci identified, as
shown in Table 3, all the members from the Chinese fam-
ily had equal sequences in the (1) DIRC3 susceptibility
locus at 2q35, (2) Pre-miR-146a susceptibility locus at
5q24, (3) NRG1 transcript variant HRG-beta1c susceptibil-
ity locus at 8p12, (4) susceptibility loci of 8q24, and (5) p y members
Chr10q11.2: 43,572,517-43,625,799
403
rs2742243
rs77702891
rs80236571
rs75873440
rs77558292
rs77939446
rs80069458
15
43,601,749 intron
43,601,894
exon5
43,601,945
exon5
43,607,621
exon8&9
43,609,069
exon10
43,609,070
exon10
43,609,077 exon10
T/C
A:germline
G:germline
G →A
missense
A:germline
G:germline
G →A
missense
G:germline
T:germline
G →T
missense
T:germline
C:germline
T →C
missense
G:germline A:
germline G →A
missense
C:germline
G:germline
C →G
missense
TC
GG
GG
GG
TT
GG
CC
TT
GG
GG
GG
TT
GG
CC
TC
GG
GG
GG
TT
GG
CC
TT
GG
GG
GG
TT
GG
CC
TC
GG
GG
GG
TT
GG
CC
TT
GG
GG
GG
TT
GG
CC
810
rs77503355
rs79890926
rs121913313 rs2256550
rs75075748
rs78014899
rs75686697
rs75030001
02
43,609,103 exon10
43,609,104
exon10
43,609,104
exon10
43,611,865
exon12
43,613,829
exon13
43,613,840
exon13
43,613,868
exon13
43,613,906
exon13
ne
ne
e
A:germline C:germline
G:germline T:germline
G →A& G →C& G →T
missense
C:germline
G:germline
C →G
missense
not
availiable
cds-indel
T/C intron
C:germline
T:germline
T →C
missense
A:unkown C:
somatic G:
germline G →A&
G →C cds-synon
A:germline
G:germline
G →A
missense
C:unkown
G:germline
G →C
missense
GG
CC
no del
TC
TT
GG
GG
GG
GG
CC
no del
TT
TT
GG
GG
GG
GG
CC
no del
TC
TT
GG
GG
GG
GG
CC
no del
TT
TT
GG
GG
GG
GG
CC
no del
TC
TT
GG
GG
GG
GG
CC
no del
TT
TT
GG
GG
GG
441
new
rs2472737
rs121913306 rs75234356
rs76087194
rs121913309
rs1800863
rs78347871
82
43,615,404 intron
43,615,505
intron
43,615,567
exon15
43,615,592
exon15
43,615,611
exon15
43,615,613
exon15
43,615,633
exon15
43,617,398
exon16
C/T
G/A
AGC:
germline
TTT:somatic
cds-indel
G:germline
T:germline
T →G
missense
A:germline
G:germline
G →A
missense
not availiable cds-
indel
not
availiable C/
G cds-synon
C:germline
G:germline
G →C
missense CC
GG
CG
GG
CC
GG
CG
GG
CC
GG
CG
GG
nted in the family members,
http://www.biomedcentral.com/1472-6823/13/48 p Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 Page 9 of 11 Liao et al. Discussion All
these susceptibility loci have been reported to associate
with sporadic PTC [11,13], but currently the pathogenic
functions of these alleles are not known well. We think all
these risk alleles might contribute jointly to the develop-
ment of MNG and PTC in the Chinese family members;
while considering the risk alleles also presented in the pro-
band’s father with normal thyroid, it is possible that differ-
ent pathogenic mechanisms exist to activate the tumor
transformation in the family members with thyroid disease. Interestingly, we observed that the frequency of T risk
allele of rs944289 at 14q13.3 locus was increased in these
MNG and PTC Chinese family members (C: T = 0.4:0.6 vs
0.571:0.429 in normal people). Several studies suggested
the possible genetic predisposition of 14q to familial PTC
[25] while no association between the radiation-related
PTC and 14q13.3 [19]. Also, family nontoxic MNG locus
maps to chromosome 14q [24]. Further research suggested
that rs944289 was located in a CEBP-alpha/CEBP-beta
binding element in the 5-prime UTR of a thyroid-specific
lincRNA gene, papillary thyroid carcinoma susceptibility
candidate 3 (PTCSC3), PTCSC3 had the characteristics of
a tumor suppressor, the rs944289 T risk allele reduced
PTCSC3 promoter activation and thereby predisposes to
PTC [21]. Nevertheless, the tumor suppression mechan-
ism of PTCSC3 is currently unknown. In addition, the thy-
roid transcription factor of NK2 homeobox 1, NKX2-1, is
also located in the 14q13.3; NKX2-1 regulates the expres-
sion of thyroid-specific genes involved in morphogenesis. But how rs944289 was associated with NKX2-1 remains to
be investigated. Also, we investigated PTC susceptibility
locus of rs116909374 (T) locating between PTCSC3 and
NKX2-1 at the same 14q, the family members carried no
risk allele at all. Hence, our current work implied the pos-
sible role of rs944289 in familial MNG with PTC. Whereas, it is surprise that heterozygosity as CT rather
than homozygosity as TT presented in the fPTC family
members; the same phenomenon was once suggested as a
possible special form of genetic epistasis in the rs2910164
allele of pre-miR-146a gene [12], which may also contrib-
uted to this Chinese fMNG with PTC as shown by the
study. Briefly, our results in the Chinese family agreed that
rs944289 but not rs116909374 at 14q13.3 locus might be
associated with genetic predisposition to familial form of
MNG with PTC; it will be intriguing to further analyze the
pathogenic link between rs944289 and the disease. Discussion BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 As we failed to detect somatic genetic alterations in
the tumor DNA, such as the BRAF and RET proto-
oncogene in the Chinese family members, in the current
study, we investigated the genomic region containing the
BRAF susceptible variants in sporadic PTC, and also all
the known RET susceptibility loci to thyroid diseases
(Tables 2 and 3). Our sequencing results confirmed that
the BRAF and RET mutations were not germline muta-
tions or susceptibility genetic events in this Chinese fam-
ily. However, we noticed that in the sequenced RET
genetic region, several different heterozygous alleles
were presented among the Chinese family members, and
most alleles were in the intron region. Recently, the
chromosomal fragile sites breakage was proposed to
cause PTC by forming chromosome rearrangement [26]. The chromosomal fragile sites are regions of the genome
with a high susceptibility to forming DNA breaks and
are often associated with cancer. Exposure to a variety of
external factors such as chemotherapeutic, dietary and
environmental compounds can induce and accelerate
the fragile site breakage. Several intron regions of RET
were identified as DNA breakage region. Hence, we are
wondering if it is possible that the polymorphisms of in-
trons could link to the structural difference in the RET
region and could impact the related chromosome archi-
tecture and thyroid gene expression, albeit there was no
RET mutation in the cancerous thyroid. Interestingly,
there were 2 related facts to be considered: (1) it was
shown that transfecting thyroid cells with RET produced
morphological changes in nuclei that mimicked those
seen in PTC [27]; (2) it is curious that the RET gene is
not expressed in the thyroid follicular cells from which
PTC develops, but rearrangements of the RET are found
in PTC cases [28]. Hence, we think it will be intriguing
to investigate the association between the genomic
structural of RET region and the regulation mechanism
of RET. Our results verified that, for the predisposition to familial
form of PTC and radiation-related PTC, their mechanism
of PTC susceptibility did not completely overlap each
other, since the genetic determinants associated with
radiation-related PTC were not presented in the Chinese
family members with PTC and MNG. It is also noticeable that all the family members were
homozygous for the risk alleles of rs966423 (CC) at 2q35,
rs2910164 (CC) at 5q24 and rs2439302 (GG) at 8p12. Discussion Our work may provide additional evidence to the gen-
etic predisposition to familial form of MNG with PTC. Due to unavailability of samples and the complex of
pathogenesis, the current studied Chinese family was
small and limited. Nonetheless, for complex diseases like
PTC, there may be many genes influencing risk as well
as the effects of environment, also, it is much more diffi-
cult to collect pedigrees with multiple affected relatives
and there is no guarantee of the same (or any) gene
(SNP) segregating in these family. To provide insights
into the genetic risk factors for familiar PTC, more re-
searches are needed. Competing interests Competing interests
The authors have non-financial competing interests. Funding
h
d This study was supported by the research grants (to Wenzhong Song and to
Shunyao Liao) from Sichuan Provincial Health Department, China
(100450,120074). 17. Gudmundsson J, Sulem P, Gudbjartsson DF, Jonasson JG, Sigurdsson A,
Bergthorsson JT, et al: Common variants on 9q22.33 and 14q13.3 predispose
to thyroid cancer in European populations. Nat Genet 2009, 41(4):460–464. Received: 4 June 2013 Accepted: 17 October 2013
Published: 21 October 2013 20. Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F,
Leskelä S, et al: The variant rs1867277 in FOXE1 gene confers thyroid
cancer susceptibility through the recruitment of USF1/USF2 transcription
factors. PLoS Genet 2009, 5(9):e1000637. Authors’ contributions 13. Jazdzewski K, Liyanarachchi S, Swierniak M, Pachucki J, Ringel MD, Jarzab B,
de la Chapelle A: Polymorphic mature microRNAs from passenger strand
of pre-miR-146a contribute to thyroid cancer. Proc Natl Acad Sci USA
2009, 106(5):1502–1505. SY L, YQ L and WZ S designed the molecular genetic studies, participated in
the sequence alignment and drafted the manuscript. DD D and G X carried
out the immunohistochemical assay. SP D and YM L have been involved in
revising the manuscript critically. ZL T, JY H participated in data acquisition
and helped to draft the manuscript. All authors read and approved the final
manuscript. 14. Neta G, Yu CL, Brenner A, Gu F, Hutchinson A, Pfeiffer R, Sturgis EM, Xu L,
Linet MS, Alexander BH, Chanock S, Sigurdson AJ: Common genetic
variants in the 8q24 region and risk of papillary thyroid cancer. Laryngoscope 2012, 122(5):1040–1042. Acknowledgments 15. He H, Nagy R, Liyanarachchi S, Jiao H, Li W, Suster S, Kere J, de la Chapelle
A: A susceptibility locus for papillary thyroid carcinoma on chromosome
8q24. Cancer Res 2009, 69(2):625–631. g
We thank the members of the Chinese fMNG with PTC family for their
essential contribution to scientific research. We thank Dr. Hongji Yang and
the colleagues in Department of General Surgery, Sichuan Academy of
Medical Science, Sichuan Provincial People’s Hospital, for their support and
collaboration. 16. Jones AM, Howarth KM, Martin L, Gorman M, Mihai R, Moss L, Auton A,
Lemon C, Mehanna H, Mohan H, Clarke SE, Wadsley J, Macias E,
Coatesworth A, Beasley M, Roques T, Martin C, Ryan P, Gerrard G, Power D,
Bremmer C, Consortium TCUKIN, Tomlinson I, Carvajal-Carmona LG: Thyroid
cancer susceptibility polymorphisms: confirmation of loci on
chromosomes 9q22 and 14q13, validation of a recessive 8q24 locus and
failure to replicate a locus on 5q24. J Med Genet 2012, 49(3):158–163. Medical Science, Sichuan Provincial People’s Hospital, for their support and
collaboration. Author details
1 18. Matsuse M, Takahashi M, Mitsutake N, Nishihara E, Hirokawa M, Kawaguchi T,
Rogounovitch T, Saenko V, Bychkov A, Suzuki K, Matsuo K, Tajima K, Miyauchi A,
Yamada R, Matsuda F, Yamashita S: The FOXE1 and NKX2-1 loci are associated
with susceptibility to papillary thyroid carcinoma in the Japanese
population. J Med Genet 2011, 48(9):645–648. 1Diabetes & Endocrinology Center, Sichuan Academy of Medical Science,
Sichuan Provincial People’s Hospital, Chengdu 610072, China. 2Department
of Thyroid Disease & Nuclear Medicine, Sichuan Academy of Medical Science,
Sichuan Provincial People’s Hospital, Chengdu 610072, China. 3Department
of Medical Genetics and Division of Morbid Genomics, State Key Laboratory
of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041,
China. 4Department of Surgery, Harvard Medical School, Massachusetts
General Hospital, Boston, MA, USA. 5Department of Pathology, Sichuan
Academy of Medical Science, Sichuan Provincial People’s Hospital, Chengdu
610072, China. 19. Takahashi M, Saenko VA, Rogounovitch TI, Kawaguchi T, Drozd VM,
Takigawa-Imamura H, Akulevich NM, Ratanajaraya C, Mitsutake N, Takamura N,
Danilova LI, Lushchik ML, Demidchik YE, Heath S, Yamada R, Lathrop M,
Matsuda F, Yamashita S: The FOXE1 locus is a major genetic determinant for
radiation-related thyroid carcinoma in Chernobyl. Hum Mol Genet 2010,
19(12):2516–2523. Received: 4 June 2013 Accepted: 17 October 2013
Published: 21 October 2013 Conclusions Based on our current investigation in the Chinese fMNG
with PTC, the risk allele homozygote of rs966423 (CC)
at 2q35, rs2910164 (CC) at 5q24 and rs2439302 (GG) at Page 10 of 11 Liao et al. BMC Endocrine Disorders 2013, 13:48
http://www.biomedcentral.com/1472-6823/13/48 8p12 could contribute to the fMNG with PTC, while the
other
identified
risk
alleles
for
sporadic
PTC
or
radiation-related PTC might not be involved. Also, cor-
responding to the previous studies on the association be-
tween chromosome 14q and fMNG with PTC, our work
approved that rs944289 but not rs116909374 at 14q13
locus might be associated with genetic predisposition to
a Chinese family MNG with PTC. Though several differ-
ent heterozygous alleles in the RET introns presented,
the common BRAF and RET mutations were not suscep-
tibility genetic events in this Chinese family. 8. Shifrin AL, Ogilvie JB, Stang MT, Fay AM, Kuo YH, Matulewicz T, Xenachis CZ,
Vernick JJ: Single nucleotide polymorphisms act as modifiers and correlate
with the development of medullary and simultaneous medullary/papillary
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proto-oncogene mutation. Surgery 2010, 148(6):1274–1280. 9. Shifrin AL, Fay A, Kuo YH, Ogilvie J: Response to “Single nucleotide
polymorphisms and development of hereditary medullary thyroid
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disequilibrium? ”. Surgery 2012, 151(6):902–903. y
10. Zhang Q, Song F, Zheng H, Zhu X, Song F, Yao X, Zhang L, Chen K: Association
between single-nucleotide polymorphisms of BRAF and papillary thyroid
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Jonasdottir A, et al: Discovery of common variants associated with low
TSH levels and thyroid cancer risk. Nat Genet 2012, 44(3):319–322. 12. Jazdzewski K, Murray EL, Franssila K, Jarzab B, Schoenberg DR, de la Chapelle A:
Common SNP in pre-miR-146a decreases mature miR expression and
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noncoding RNA gene of tumor suppressor type. Proc Natl Acad Sci USA
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1999, 44(2):96–102. doi:10.1186/1472-6823-13-48
Cite this article as: Liao et al.: Familial multinodular goiter syndrome
with papillary thyroid carcinomas: mutational analysis of the associated
genes in 5 cases from 1 Chinese family. BMC Endocrine Disorders
2013 13:48. 25. Bignell GR, Canzian F, Shayeghi M, Stark M, Shugart YY, Biggs P, et al:
Familial nontoxic multinodular thyroid goiter locus maps to
chromosome 14q but does not account for familial nonmedullary
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https://www.nature.com/articles/s41477-023-01556-0.pdf
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English
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Evolution of a plant growth-regulatory protein interaction specificity
|
Nature Plants
| 2,023
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cc-by
| 15,565
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Article https://doi.org/10.1038/s41477-023-01556-0 1Department of Biology, University of Oxford, Oxford, UK. 2State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China. 3National Key Laboratory of Crop Genetics & Germplasm
Enhancement and Utilization, Nanjing Agricultural University, Nanjing, PR China. 4Department of Biochemistry, University of Oxford, Oxford, UK.
5College of Life Sciences, University of Chinese Academy of Sciences, Beijing, P. R. China. 6New Cornerstone Science Laboratory, Beijing, P. R. China.
e-mail: nicholas.harberd@biology.ox.ac.uk Evolution of a plant growth-regulatory
protein interaction specificity Zhe Ji
1,2, Eric J. Belfield
1, Siyu Zhang
3, Jacques Bouvier
1, Shan Li
2,3,
Jason Schnell
4, Xiangdong Fu
2,5,6 & Nicholas P. Harberd
1 Specific protein–protein interactions (PPIs) enable biological regulation. However, the evolution of PPI specificity is little understood. Here we
trace the evolution of the land-plant growth-regulatory DELLA–SLY1/
GID2 PPI, revealing progressive increase in specificity of affinity of SLY1/
GID2 for a particular DELLA form. While early-diverging SLY1s display
relatively broad-range DELLA affinity, later-diverging SLY1s tend towards
increasingly stringent affinity for a specific DELLA A’ form generated by the
growth-promoting phytohormone gibberellin (GA). Our novel mutational
strategy reveals amino acid substitutions contributing to the evolution
of Arabidopsis thaliana SLY1 A’ specificity, also showing that routes
permitting reversion to broader affinity became increasingly constrained
over evolutionary time. We suggest that progressive affinity narrowing may
be an important evolutionary driver of PPI specificity and that increase
in SLY1/GID2-DELLA specificity enabled the enhanced flexibility of plant
physiological environmental adaptation conferred by the GA-DELLA
growth-regulatory mechanism. Nature Plants | Volume 9 | December 2023 | 2059–2070
The DELLA-SLY1/GID2 (refs. 1–6) PPI regulates plant growth, survival
of environmental adversity7 and resource assimilation that under-
pins terrestrial ecosystems and agriculture8–10. SLY1/GID2 is the
F-box DELLA-specificity component of growth-promoting SCFSLY1/
GID2 E3 ubiquitin ligase3–6. In angiosperms, gibberellin (GA) promotes
DELLA–SCFSLY1/GID2 binding and resultant destruction of DELLA growth
repressors, as follows. First, the GA-bound GID1 (refs. 11–13) GA recep-
tor binds the DELLA N terminus. While the unbound N terminus is
intrinsically unstructured, GA–GID1 binding induces folding14 and pre-
sumed conversion of the native (here called A) form of the C-terminal
DELLA GRAS domain into an A’ alternative2. Although the molecular
distinction between A and A’ is unclear, phosphorylation4,5 and/or
reconfiguration of the GRAS domain structure may be causal. Nev-
ertheless, the A to A’ transition is an important switch because SLY1/
GID2 specifically binds the A’ GRAS domain, thus promoting DELLA
destruction and growth. Angiosperm
recently, although
LAs probably evo
GRAS protein of b
tors are exclusive
GID1-related prot
(encoding AtSLY1)
MpSLY1 (ref. 19)) e
(perhaps through
less, the lack of fu
GID1-mediated D
arose after bryoph
bryophytes lack th
To understan
lysed variants with
our analyses of SL
1Department of Biology, University of Oxford, Oxford, UK. 2State Key Laboratory of Plant Cell and
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China. Evolution of a plant growth-regulatory
protein interaction specificity 3Natio
Enhancement and Utilization, Nanjing Agricultural University, Nanjing, PR China. 4Department of
5College of Life Sciences, University of Chinese Academy of Sciences, Beijing, P. R. China. 6New
e-mail: nicholas.harberd@biology.ox.ac.uk The DELLA-SLY1/GID2 (refs. 1–6) PPI regulates plant growth, survival
of environmental adversity7 and resource assimilation that under-
pins terrestrial ecosystems and agriculture8–10. SLY1/GID2 is the
F-box DELLA-specificity component of growth-promoting SCFSLY1/
GID2 E3 ubiquitin ligase3–6. In angiosperms, gibberellin (GA) promotes
DELLA–SCFSLY1/GID2 binding and resultant destruction of DELLA growth
repressors, as follows. First, the GA-bound GID1 (refs. 11–13) GA recep-
tor binds the DELLA N terminus. While the unbound N terminus is
intrinsically unstructured, GA–GID1 binding induces folding14 and pre-
sumed conversion of the native (here called A) form of the C-terminal
DELLA GRAS domain into an A’ alternative2. Although the molecular
distinction between A and A’ is unclear, phosphorylation4,5 and/or
reconfiguration of the GRAS domain structure may be causal. Nev-
ertheless, the A to A’ transition is an important switch because SLY1/
GID2 specifically binds the A’ GRAS domain, thus promoting DELLA
destruction and growth. Angiosperms and GA-promoted growth both arose relatively
recently, although at different times2,15–18 (Extended Data Fig. 1a). DEL-
LAs probably evolved in the land-plant common ancestor2,19 from a
GRAS protein of bacterial origin20, while functional GID1 GA recep-
tors are exclusive to vascular plants (although some bryophytes have
GID1-related proteins)16–19. Intriguingly, Arabidopsis thaliana AtSLY1
(encoding AtSLY1) orthologues (for example, Marchantia polymorpha
MpSLY1 (ref. 19)) exist in liverworts, but not in mosses or hornworts
(perhaps through gene loss during bryophyte evolution21). Neverthe-
less, the lack of functional bryophyte GID1s suggests that both GA–
GID1-mediated DELLA destruction and SCFSLY1/GID2 A’ specificity first
arose after bryophyte divergence (Extended Data Fig. 1a). Accordingly,
bryophytes lack the active GA species that GID1 binds2,22. To understand the origin of SLY1/GID2 A’ specificity, we first ana-
lysed variants with enhanced A affinity (reduced A’ specificity). Second,
our analyses of SLY1s and DELLAs23 from early-diverging land plants Nature Plants | Volume 9 | December 2023 | 2059–2070 Nature Plants | Volume 9 | December 2023 | 2059–2070 2059 Article https://doi.org/10.1038/s41477-023-01556-0 the yeast-selected Atsly1 variants promoted gai degradation and dem-
onstrating their biological (in planta) relevance. revealed strong SLY1–DELLA A interactions. Together, our findings sug-
gest that while ancestral SLY1 had dual A + A’ affinity, this affinity was pro-
gressively narrowed towards A’ specificity during land-plant evolution. mino acid substitutions enhance AtSLY1 A affinity y
The A. thaliana mutant gai protein lacks the GAI DELLA domain1, does
not bind GA–GID1 (ref. 2) and is therefore in the A form. Because AtSLY1
has low A affinity5,6, gai causes GA-insensitive dwarfism1,24 (Fig. 1a), a
property enabling discovery of enhanced A affinity Atsly1 variants. For
example, the E138 to K (E138K) amino acid substitution in Atsly1gar2-1
(encoded by Atsly1gar2-1) enhances A affinity, promotes gai destruction
and suppresses gai-conferred dwarfism (Fig. 1a)5,6. Further Atsly1 alleles
(Fig. 1a) suppressed gai less (Atsly1gar2-2) or more (Atsly1gar2-3) than Ats-
ly1gar2-1 (Fig. 1a,b and Extended Data Fig. 1b,c) due to G84D (Atsly1gar2-2)
and P114L (Atsly1gar2-3) substitutions (Fig. 1c). Yeast 2-hybrid experi-
ments next revealed the height gradient (Fig. 1a,b) to correspond with a
gai affinity gradient (Atsly1gar2-3 > Atsly1gar2-1 > Atsly1gar2-2; Fig. 1d,e; AtSLY1
exhibits baseline gai affinity) also detected in vitro, with His-tagged
gai pulling down increasing MBP-tagged Atsly1 amounts (Atsly1gar2-3 >
Atsly1gar2-1 > Atsly1gar2-2; Fig. 1f) and partially reflected in plant extract
gai destruction rates (Fig. 1g; although the Atsly1gar2-1/Atsly1gar2-3 dif-
ferential was less clear than in Fig. 1e,f). In planta immuno-detected
gai abundances were correspondingly reduced (Fig. 1h). Thus, E138K,
G84D and P114L differentially enhance AtSLY1 affinity for gai DELLA
A, in turn causing the height gradient (Fig. 1a,b). Furthermore, E138K,
G84D and P114L exemplify distinct routes to enhancing AtSLY1 gai (A)
affinity (reducing A’ specificity). Atsly1 substitutions alter the DELLA-interacting region
AtSLY1 has N-terminal F-box (SCF-tethering) and C-terminal
DELLA-interacting GGF and LSL domains5,6. AlphaFold25,26 predicts a
core GGF region of three α-helices, a ~9-residue linker and C-terminal
LSL helical regions (Fig. 3a and Extended Data Fig. 3a), with RoseT-
TAfold27 and ESMfold28 predictions (Extended Data Fig. 3b,c) broadly
agreeing. Interestingly, predicted LSL helix and GGF domain alignment
errors are large, and predicted LSL helix structures and positions rela-
tive to the GGF domain differ (Extended Data Fig. 3a–c). Nevertheless,
all models predict an outward-facing LSL helix, consistent with a likely
role in direct AtSLY1–DELLA interactions. Evolution of a plant growth-regulatory
protein interaction specificity Importantly, Atsly1 variant A affinity enhancement is not restricted
to GAI (or gai). Of the 5 A. thaliana DELLAs2, AtSLY1 displays baseline
GAI, RGL1 and RGL3, but not RGA or RGL2 affinity (Extended Data Fig. 2i). Interestingly, an Atsly1 RGA A affinity gradient (Fig. 2f and Extended
Data Fig. 2j) essentially replicates (despite quantitative reduction) the
GAI gradient (Fig. 2b), suggesting general DELLA A affinity enhance-
ment. Furthermore, increased A affinity is not detectably associated
with reduced A’ affinity. DELLAs retain GA responsivity5,6 in sly1gar2-1,
indicating that E138K enhances A affinity without reducing affinity for
the GA-promoted A’ form. We showed similar retention of A’ affinity in
additional Atsly1 variants. Employing a yeast 3-hybrid approach with
GAI (prey), SLY1/sly1 (bait) and GID1C (bridge) partners, we expected
GA to cause GAI binding to GA-GID1C, thus generating A’ and (because
SLY1 A’ affinity is strong) detectably increasing SLY1-GAI interactions (as
seen in Fig. 2g). In contrast, while reduced A’ affinity (if any) would be
expected to reduce GAI–Atsly1 interactions in response to GA, this was
detected neither in yeast (Fig. 2g) nor in complementary ‘pull-down’
experiments (Fig. 2h). Thus, the enhanced A affinity of Atsly1 vari-
ants is not detectably associated with reduced A’ affinity. Rather, the
high-specificity A’ affinity of AtSLY1 is transformed in Atsly1 variants
into broader A + A’ affinity. Yeast-based discovery of Atsly1 variants y
y
Next, error-prone PCR (EP-PCR)-generated Atsly1 variants were
screened in yeast for enhanced GAI affinity (Methods and Extended
Data Fig. 2a,b). GAI is a yeast proxy for in planta A because, in the
absence of GA or GID1, A’ cannot arise. The screen revealed 9 substitu-
tions (Fig. 2a and Supplementary Table 1), 2 of which (E138K and P114L)
replicate Atsly1gar2-1 and Atsly1gar2-3 substitutions (Figs. 1a–c and 2a), thus
validating yeast AtSLY1–GAI interactions as in planta proxy. The remain-
ing 7 yeast-selected Atsly1 variants are novel, and their yeast-reported
enhanced A affinities (Fig. 2b and Extended Data Fig. 2c) are unlikely
due to increased accumulation (Extended Data Fig. 2d) or non-specific
binding (Extended Data Fig. 2e,f). All Atsly1 substitutions alter GGF/LSL residues (Figs. 1c,2a and 3a). Our probabilistic considerations next determined whether they alter
AtSLY1–DELLA interactions via function-critical residue loss, or via
function-altering replacement. For example, we recovered 8 inde-
pendent H104Y substitutions (Supplementary Table 1). However,
single-nucleotide mutation of the H104 codon can theoretically cause
1 of 7 substitutions (H to D, L, N, P, Q, R or Y). Using EP-PCR-generated
mutation frequencies (Extended Data Fig. 4a), we determined expected
amino acid substitution frequencies, finding that without selection,
H104R should predominate (Extended Data Fig. 4b) and that recov-
ery of 8 H104Y substitutions suggests selection (χ2 = 36.8, P < 0.01;
Supplementary Table 2). Presumably, H104Y enhances AtSLY1–GAI
interactions, while other substitutions do not (see also below). K126
(χ2 = 39.7, P < 0.01; Supplementary Table 2) and E138 (χ2 = 26.9, P < 0.01; Yeast-reported and in vitro Atsly1 variant A affinity enhancement
ranges from weak (E138K; Atsly1gar2-1) to strong (Atsly1H104Y; Fig. 2b,c
and Extended Data Fig. 2c). Constructs expressing Atsly1 variants
(Extended Data Fig. 2g) conferred corresponding graduated sup-
pression of gai-conferred dwarfism: Atsly1G84D caused mild height
increase, while Atsly1T128A and Atsly1H104Y conferred tall (similar to wild
type (WT)) phenotypes (Fig. 2d,e). This height gradient was reflected
in gai abundance reductions (Extended Data Fig. 2h), confirming that between E. coli-expressed His-tagged gai and MBP-tagged Atsly1 variants. Anti-His serves as control and confirms that similar amounts of gai protein were
used to ‘pull down’ a SLY1 or sly1 variant protein in each immunoprecipitation
(IP) reaction, while anti-MBP shows how much SLY1 (quantified against anti-His,
arbitrarily set at 1.00) or sly1 variant was pulled down. Article https://doi.org/10.1038/s41477-023-01556-0 Supplementary Table 2) substitutions also indicate selection, with
possible weak preference at T128 (χ2 = 9.44, P < 0.05; Supplementary
Table 2) and no preference detectable at P114 (Supplementary Table 2). We suggest that P114 substitutions cause function-critical residue loss,
while function-altering replacements at H104, K126, E138 and possibly
T128 increase AtSLY1–DELLA interactions. Testing these suggestions, we showed that expected (unselected)
predominant substitutions at H104, K126, T128 or E138 did not enhance
baseline AtSLY1–GAI interactions and indeed reduced (Atsly1K126R) or abol-
ished (Atsly1H104R, Atsly1T128P and Atsly1E138G; Fig. 3b and Extended Data Fig. 4c) them. Yeast-based discovery of Atsly1 variants The increasing amounts
of MBP-SLY1, MBP-sly1gar2-2, MBP-sly1gar2-1 and MBP-sly1gar2-3 detected suggests
that the height (b) and yeast-based interaction (e) gradients reflect a gradient
of increasing affinity. g, Destruction rates of E. coli-expressed His-gai in plant
extracts quantified against Actin control (arbitrarily set at 1.00 for timepoint
0), genotypes as shown. While His-gai is appreciably degraded in the gai (SLY1)
control by 60 min, gar2 variant alleles confer faster degradation, with gar2-3
being the fastest, consistent with the affinity gradient (f). h, Abundance of
immuno-detectable gai (quantified against Actin, arbitrarily set at 1.00) or GAI
and sly1 (or SLY1) in plant extracts (genotypes as shown). Anti-Actin and Ponceau
S staining serve as loading controls. Fig. 1 | Mutant gar2 alleles suppress the gai phenotype. a, gar2 alleles variably
suppress gai-conferred dwarfism. gai (gai GAR2) on far left, WT (GAI GAR2) on far
right. Bolt stems cut from vegetative rosette. Scale bar, 5 cm. b, Mean (±s.d.) plant
heights, genotypes as in a; red dots indicate individual heights (n = 10), different
letters (a–e) indicate significant differences (one-way ANOVA with Tukey’s test). c, Amino acid substitutions encoded by gar-2 alleles. Conserved F-box, GGF and
LSL domains (positions 1–151) are indicated. d, Yeast 2-hybrid analysis of gai–
Atsly1 interactions. Top line (SD/-LW) confirms double transformation (bait and
prey constructs), bottom line (SD/-LWAH/X) indicates interaction: no detectable
interaction in the absence of bait (empty vector), baseline gai-SLY1 interaction
(medium blue), stronger gai–Atsly1gar2 interactions. e, Mean (±s.d.) yeast 2-hybrid
interaction strengths, mutants as in d; note gradient of increasing interaction
strength correlating with increase in plant height (b). ND, not detected; red
dots indicate individual values (n = 3), different letters (a–d) indicate significant
differences (one-way ANOVA with Tukey’s test). f, In vitro analysis of interactions Nature Plants | Volume 9 | December 2023 | 2059–2070 2060 Article In contrast, all possible P114 substitutions enhanced AtSLY1–
GAI interactions (Fig. 3c and Extended Data Fig. 4d), again indicating that
loss of function-critical P114 confers phenotypic change. reduced subset, AtSLY1 selectively interacts with MpDELLA, GAI (not
RGA) and SLR1, while GID2 exhibits weak interaction with MpDELLA
only (Fig. 4b,c). We conclude that basal SLY1s (MpSLY1; SmSLY1) have
broad-ranging affinity for various DELLAs (perhaps reflecting ances-
tral SLY1) and that affinity became more stringent, more species and
A’-specific during evolution. In addition, because GID2 interacts weakly
with MpDELLA, DELLA-SLY1 co-evolution is a possibility, although this
was not investigated further. reduced subset, AtSLY1 selectively interacts with MpDELLA, GAI (not
RGA) and SLR1, while GID2 exhibits weak interaction with MpDELLA
only (Fig. 4b,c). We conclude that basal SLY1s (MpSLY1; SmSLY1) have
broad-ranging affinity for various DELLAs (perhaps reflecting ances-
tral SLY1) and that affinity became more stringent, more species and
A’-specific during evolution. In addition, because GID2 interacts weakly
with MpDELLA, DELLA-SLY1 co-evolution is a possibility, although this
was not investigated further. Both AtSLY1 intramolecular and AtSLY1–GAI intermolecular inter-
actions probably influence AtSLY1–GAI affinity. An AlphaFold-multimer
AtSLY1–GAI complex model (Fig. 3d) predicts that although the GRAS
domain directly interacts with the LSL helix, some residues targeted
in Atsly1 variants are buried within AtSLY1, distant from the GAI inter-
face. For example, H104, buried within the GGF domain, has predicted
intramolecular AtSLY1 contacts but no predicted GAI intermolecular
contacts (Fig. 3e). H104Y therefore probably enhances AtSLY1–GAI
interactions via internal effects, perhaps changing the relative orien-
tation or stability of the GGF/LSL interface. Accordingly, the variation
in predicted LSL structure (Extended Data Fig. 3a–c; see above) may
reflect functionally relevant conformational dynamics. The model
(Fig. 3d) further indicates that the LSL helix K126 (Extended Data
Fig. 4e) and E138 (Extended Data Fig. 4f) substitutions directly affect
interatomic electrostatic contacts at the AtSLY1–GAI interface, whereas
T128 points away from it and towards the GGF/LSL domain interface. Of
the two remaining substituted positions, G84 is on the surface of the
GGF region and close to both the intermolecular GAI interface and the
intramolecular LSL helix interface, suggesting both direct and indirect
effects, while P114 is in an unstructured loop connecting the GGF and
LSL helices in predicted structures of AtSLY1 alone, but terminates
the third GGF helix in the predicted AtSLY1–GAI complex. Article This confirmed the specificity of the selected H104, K126, T128
and E138 substitutions and the importance of these sites to AtSLY1–GAI ants | Volume 9 | December 2023 | 2059 2070
0 1 2 3 4 5 30
40
50
60
β-galactosidase units
c
d
ND
ND
ND
ND
ND
Vector
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
Bait
Prey
Vector
gai
b
a
gai
gai gar2-2
gai gar2-1
gai gar2-3
WT
0
4
8
12
16
20
24
28
32
Height (cm)
d
e
c
b
a
0
15
30
45
60
Min
a
gai
gai
gar2-2
gai
gar2-1
gai
gar2-3
WT
b
F-box
GGF
LSL
1
78
116
151
gar2-1
E138K
gar2-3
P114L
gar2-2
G84D
c
SD/-LW
SD/-LWAH/X
Prey
gai
Bait
Vector
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
anti-
GAI/gai
anti-Actin
anti-
SLY1/sly1
Ponceau S
d
e
g
anti-His
anti-Actin
gai
1.00
1.09
0.74
0.61
0.68
gai
gar2-2
anti-His
anti-Actin
1.00
0.79
0.68
0.67
0.52
gai
gar2-1
anti-His
anti-Actin
1.00
0.73
0.27
0.30
0.05
gai
gar2-3
anti-His
anti-Actin
1.00
0.43
0.29
0.25
0.02
IP: His
anti-MBP
anti-His
MBP
His-gai
Input
anti-MBP
anti-His
f
h
kDa
70
130
70
130
kDa
130
55
130
55
130
55
130
55
kDa
64
19
51
51
51
14
39
1.00
1.61
2.67
4.59
1.00
0.97
0.79
0.64
0.12
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
gai gar2-2
gai
gai gar2-1
gai gar2-3
WT gai
gai gar2-2
gai gar2-1
gai gar2-3
WT
0
4
8
12
16
20
24
28
32
Height (cm)
d
e
c
b
a
b b a
gai
gai
gar2-2
gai
gar2-1
gai
gar2-3
WT g
g
ga
SD/-LW
SD/-LWAH/X
Prey
gai
Bait
Vector
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
d 0 1 2 3 4 5 30
40
50
60
β-galactosidase units
c
d
ND
ND
ND
ND
ND
Vector
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3
Bait
Prey
Vector
gai
b
a
F-box
GGF
LSL
1
78
116
151
gar2-1
E138K
gar2-3
P114L
gar2-2
G84D
c
e d c IP: His
anti-MBP
anti-His
MBP
His-gai
Input
anti-MBP
anti-His
f
kDa
70
130
70
130
1.00
1.61
2.67
4.59
SLY1
sly1gar2-2
sly1gar2-1
sly1gar2-3 f f 0
15
30
45
60
Min
g
anti-His
anti-Actin
gai
1.00
1.09
0.74
0.61
0.68
gai
gar2-2
anti-His
anti-Actin
1.00
0.79
0.68
0.67
0.52
gai
gar2-1
anti-His
anti-Actin
1.00
0.73
0.27
0.30
0.05
gai
gar2-3
anti-His
anti-Actin
1.00
0.43
0.29
0.25
0.02
kDa
130
55
130
55
130
55
130
55 g anti-
GAI/gai
anti-Actin
anti-
SLY1/sly1
Ponceau S
h
kDa
64
19
51
51
51
14
39
1.00
0.97
0.79
0.64
0.12
gai gar2-2
gai
gai gar2-1
gai gar2-3
WT h Nature Plants | Volume 9 | December 2023 | 2059–2070 2061 https://doi.org/10.1038/s41477-023-01556-0 Article interactions. Evolutionarily revertant Atsly1 variant substitutions Evolutionarily revertant Atsly1 variant substitutions
Although within or close to the conserved GGF/LSL domains (Fig. 5a
and Extended Data Fig. 7), Atsly1 substitutions often target recently
acquired residues, such as K126 (which is unique to AtSLY1; Extended
Data Fig. 7). Similarly, the recent T128 is S in almost all (including basal)
sequences (Extended Data Fig. 7) and is restored in Atsly1T128S. While
P114R strongly enhances A affinity, R is at the equivalent position in
basal SLY1s (Extended Data Fig. 7). Finally, H104 was probably substi-
tuted before angiosperm divergence (104 or equivalent position is
H in all angiosperms, including the basal Amborella; Extended Data
Fig. 7). Thus, the phenotype-changing substitutions at K126, T128,
P114 and H104 are all evolutionarily revertant substitutions. Quantita-
tively, H104Y confers an A affinity (Fig. 2b) roughly equivalent to that of
MpSLY1 (Fig. 4c), indicating a major role for H104Y during GA-DELLA
signalling evolution. We suggest that AtSLY1 evolved by suppressing
ancestral A affinity, with some Atsly1 substitutions partially restoring
A affinity by reversing evolutionary substitutions. Basal SLY1s exhibit broad-range DELLA affinity g
y
We next determined whether Atsly1 properties reflect SLY1 evolution. Previous analyses identified duplicate A. thaliana AtSLY1 and AtSLY2
genes, with encoded AtSLY1 dominating AtSLY2 in GA signalling5,29. Our
SLY1 phylogeny reflected land-plant evolution, revealing a bryophyte
(liverwort) SLY1 clade and two more-recent SLY1/GID2 (containing
AtSLY1) and SLY2 (containing AtSLY2) clades, which separated before
lycophyte divergence (Fig. 4a and Extended Data Fig. 5). Broadly,
while the liverwort Marchantia polymorpha genome encodes MpSLY1,
MpDELLA, but no functional GID1 (GA receptor orthologue), the lyco-
phyte Selaginella moellendorfii genome encodes SmSLY1, SmSLY2,
SmDELLA1, SmDELLA2 and SmGID1 representatives2,16–19 (Extended
Data Fig. 1a). Comparing the yeast DELLA A form affinities of MpSLY1,
SmSLY1, SmSLY2, AtSLY1, AtSLY2 and GID2 (rice (monocot angiosperm
Oryza sativa) AtSLY1 orthologue), we first found that SLY2 clade rep-
resentatives (SmSLY2, AtSLY2) lacked detectable DELLA interactions
(Extended Data Fig. 6a) and excluded them from further analysis. Next,
we detected progressively reduced SLY1 A affinity: MpSLY1-MpDELL
A > SmSLY1-SmDELLA1/SmDELLA2 > AtSLY1–GAI/RGA > GID2-SLR1
(Fig. 4b,c; SLR1 is rice DELLA), suggesting progressive evolutionary
reduction of strong ancestral SLY1 A affinity. Furthermore, while
MpSLY1 interacts with all DELLAs tested, and SmSLY1 interacts with a The H104Y charged to hydrophobic side-chain substitution has
the greatest observed effect on AtSLY1 A affinity (Fig. 2b f). We found
that Mpsly1Y108H (MpSLY1 108 is equivalent to AtSLY1 104) abolished
MpSLY1-MpDELLA interactions (Fig. 5b), suggesting that loss of Y108
(substitution with H) contributed to the reduced angiosperm SLY1 A
affinity. Conversely, gid2H145Y (GID2 145 is equivalent to AtSLY1 104)
exhibited weakly but detectably enhanced GID2 A affinity (for SLR1;
Fig. 5b; the weak effect is probably because OsGID2 is more diver-
gent, see below; Fig. 4a and Extended Data Fig. 5). Intriguingly, F (also
hydrophobic side chain) occupies the SmSLY1 equivalent position and,
while Atsly1H104F displays enhanced GAI affinity (enhanced similarly to
Atsly1H104Y), Mpsly1Y108F exhibits reduced interaction (compared with
MpSLY1; Fig. 5c and Extended Data Fig. 8a). We conclude that Y > H with non-transgenic WT and gai. Red dots indicate individual heights (n = 10),
different letters (a–c) indicate significant differences (one-way ANOVA with
Tukey’s test). f, Mean (±s.d.) yeast 2-hybrid AtSLY1–RGA interaction strengths,
Atsly1 variants as in a. Red dots indicate individual values (n = 3), different letters
(a–d) indicate significant differences (one-way ANOVA with Tukey’s test). g, Yeast
3-hybrid analysis of affinities for GAI A and A’ forms. with non-transgenic WT and gai. Red dots indicate individual heights (n = 10),
different letters (a–c) indicate significant differences (one-way ANOVA with
Tukey’s test). f, Mean (±s.d.) yeast 2-hybrid AtSLY1–RGA interaction strengths,
Atsly1 variants as in a. Red dots indicate individual values (n = 3), different letters
(a–d) indicate significant differences (one-way ANOVA with Tukey’s test). g, Yeast
3-hybrid analysis of affinities for GAI A and A’ forms. GA promotes GAI–GID1C
binding, converting GAI from A to A’. h, In vitro analysis of interactions between
E. coli-expressed His-tagged GAI and MBP-tagged Atsly1 variants in the presence
and absence of GST-GID1C and GA3. Anti-His confirms that similar amounts of
GAI protein were used in each IP reaction, while anti-MBP and anti-GST show
how much SLY1 (quantified against anti-His, arbitrarily set at 1.00) or sly1 variant
and GID1C (if present) were pulled down. GA promotes GAI–GID1C binding,
converting GAI from A to A’. Article Prolines
are uniquely potent in terminating helices, perhaps explaining why
all observed P114 substitutions enhance GAI affinity. In further experiments, MpSLY1 exhibited no detectable differ-
ential A vs A’ affinity, SmSLY1 exhibited mild preference for A’, while A’
preference was yet stronger in angiosperm AtSLY1 and GID2 (Fig. 4d and
Extended Data Fig. 6b–d). Because transgenic expression of MpSLY1
suppresses Arabidopsis gai phenotype (Extended Data Fig. 6e), these
observations are biologically relevant, suggesting that ancestral SLY1 had
strong A + A’ affinity (despite the ancestral absence of A’) and that while A
affinity declined during evolution, A’ affinity was retained. Thus, while the
relative A’ specificity of angiosperm SLY1s is probably due to evolutionary
narrowing of broad-range A + A’ affinity, the partially restored A + A’ affini-
ties of Atsly1 variants exemplify ‘evolutionary revertant’ phenotypes. Article SLY1
sly1G84D
sly1E138K
sly1K126E
sly1K23+T
sly1P114L
sly1P114S
sly1T128S
sly1K126N
sly1T128A
sly1H104Y
SD/-LWAHM/X/GA3
GAI in A+ A’ Forms
Prey
g
IP: His
anti-MBP
anti-His
Input
anti-MBP
anti-His
h
IP: His
anti-MBP
anti-GST
MBP
His-GAI
anti-His
GST-GID1C
GA3
–
+
–
+
–
+
–
+
–
–
–
–
+
+
+
+
–
–
+
+
Bait
kDa
75
140
1.00
1.41
2.36
3.05
kDa
– 75 –
60
100
75
140
60
100
– 75 –
– 60 –
– 140 –
– 100 –
1.00
1.86
1.89
2.27
5.65
2.41
3.88
7.38
9.37
11.85
GAI in A Form
GAI in A + A’ Forms
GAI
Prey
SLY1
sly1P114L
sly1E138K
sly1T128A
sly1H104Y
SLY1
sly1P114L
sly1E138K
sly1T128A
sly1H104Y 0
1 10
30
50
70
90 110 130
β-galactosidase units
Vector
SLY1
sly1E138K
sly1K126E
sly1K126T
sly1P114L
sly1T128S
sly1K126N
sly1T128A
sly1H104Y
d
d
e
ef
f
g
ND
sly1P114S
d
c
b
a
Bait
b
GAI
Prey a b c MBP
His-GAI
SLY1
sly1G84D
sly1T128A
sly1H104Y
c
IP: His
anti-MBP
anti-His
Input
anti-MBP
anti-His
kDa
75
140
1.00
1.41
2.36
3.05
60
100
75
140
60
100 F-box
GGF
LSL
AtSLY1
1
78
116
151
E138K
P114L
P114S
G84D
H104Y
T128A
T128S
K126E
K126N
K126T 0
1
2
3
4
5 20 30 40 50 60
β-galactosidase units
Vector
SLY1
sly1K126E
sly1K126T
sly1T128S
sly1P114L
sly1E138K
sly1K126N
sly1T128A
sly1H104Y
RGA
ND
ND
ND
b
c
c
d
d
d
sly1P114S
c
a
Bait
Prey
WT
gai pSLY1::sly1H104Y
gai pSLY1::sly1T128A
gai pSLY1::sly1G84D
gai pSLY1::SLY1
gai
0
10
20
30
40
50
Height (cm)
a
a
a
b
c
c
β galactosidase units
e
f RGA
Prey
WT
gai pSLY1::sly1H104Y
gai pSLY1::sly1T128A
gai pSLY1::sly1G84D
gai pSLY1::SLY1
gai
0
10
20
30
40
50
Height (cm)
a
a
a
b
c
c
β galactos dase u
ts
e
f f d e e WT
gai pSLY1::sly1H104Y
gai pSLY1::sly1T128A
gai pSLY1::sly1G84D
gai pSLY1::SLY1
gai β-galactosidase units ga
ga
ga
h
IP: His
anti-MBP
anti-GST
MBP
His-GAI
Input
anti-His
anti-MBP
anti-GST
GST-GID1C
GA3
–
+
–
+
–
+
–
+
anti-His
–
–
–
–
+
+
+
+
–
–
+
+
kDa
– 75 –
– 75 –
– 60 –
– 140 –
– 100 –
– 75 –
– 75 –
– 60 –
– 140 –
– 100 –
1.00
1.86
1.89
2.27
5.65
2.41
3.88
7.38
9.37
11.85
GAI in A Form
GAI in A + A’ Forms
SLY1
sly1P114L
sly1E138K
sly1T128A
sly1H104Y
SLY1
sly1P114L
sly1E138K
sly1T128A
sly1H104Y GID1C
GAI
SD/-LWM
SD/-LWAHM/X
GAI in A Form
Bridge
Bait
N.A. Article https://doi.org/10.1038/s41477-023-01556-0 Systematic replacement of MpSLY1 residues (in equivalent posi-
tions) with the AtSLY1 residues substituted in the remaining Atsly1
variants (P114, K126 and T128, but not E138 because the MpSLY1 equiva-
lent is also E) revealed all resultant variants (Mpsly1R118P, Mpsly1H147K and Y > F substitutions at what became AtSLY1 position 104 (possibly
by altering internal SLY1 GGF and LSL domain structural relations),
contributed to the evolution of A’ specificity, and that Atsly1H104Y is
indeed evolutionarily revertant. and Y > F substitutions at what became AtSLY1 position 104 (possibly
by altering internal SLY1 GGF and LSL domain structural relations),
contributed to the evolution of A’ specificity, and that Atsly1H104Y is
indeed evolutionarily revertant. 0
1
2
3
4
5 20 30 40 50 60
β-galactosidase units
Vector
SLY1
sly1K126E
sly1K126T
sly1T128S
sly1P114L
sly1E138K
sly1K126N
sly1T128A
sly1H104Y
RGA
ND
ND
ND
b
c
c
d
d
d
sly1P114S
c
a
Bait
Prey
WT
WT
gai pSLY1::sly1H104Y
gai pSLY1::sly1H104Y
gai pSLY1::sly1T128A
gai pSLY1::sly1T128A
gai pSLY1::sly1G84D
gai pSLY1::sly1G84D
gai pSLY1::SLY1
gai pSLY1::SLY1
gai
gai
0
10
20
30
40
50
Height (cm)
a
a
a
b
c
c
0
1 10
30
50
70
90 110 130
β-galactosidase units
Vector
SLY1
sly1E138K
sly1K126E
sly1K126T
sly1P114L
sly1T128S
sly1K126N
sly1T128A
sly1H104Y
d
d
e
ef
f
g
ND
sly1P114S
d
c
b
a
Bait
MBP
His-GAI
SLY1
sly1G84D
sly1T128A
sly1H104Y
F-box
GGF
LSL
AtSLY1
1
78
116
151
E138K
P114L
P114S
G84D
H104Y
T128A
T128S
K126E
K126N
K126T
a
b
c
d
e
f
GID1C
GAI
SD/-LWM
SD/-LWAHM/X
GAI in A Form
Bridge
Bait
N.A. Basal SLY1s exhibit broad-range DELLA affinity GA promotes GAI–GID1C
binding, converting GAI from A to A’. h, In vitro analysis of interactions between
E. coli-expressed His-tagged GAI and MBP-tagged Atsly1 variants in the presence
and absence of GST-GID1C and GA3. Anti-His confirms that similar amounts of
GAI protein were used in each IP reaction, while anti-MBP and anti-GST show
how much SLY1 (quantified against anti-His, arbitrarily set at 1.00) or sly1 variant
and GID1C (if present) were pulled down. GA promotes GAI–GID1C binding,
converting GAI from A to A’. Fig. 2 | Yeast-based discovery of novel Atsly1 mutant proteins. a, Atsly1
substitutions shown in red were detected in yeast-based screens only, those
in blue in both yeast-based and in planta screens. G84D (for reference) was
detected in in planta screens only (see also Fig. 1c). b, Quantified (mean ± s.d.)
yeast 2-hybrid AtSLY1–GAI interactions, Atsly1 variants as in a, arranged in a
gradient of increasing interactions. Red dots indicate individual values (n = 3),
different letters (a–g) indicate significant differences (one-way ANOVA with
Tukey’s test). c, In vitro interactions between E. coli-expressed His-tagged GAI
and MBP-tagged Atsly1 variants. Anti-His confirms similar amounts of GAI in
each IP reaction, while anti-MBP shows how much SLY1 (quantified against
anti-His, arbitrarily set at 1.00) or sly1 variant was pulled down. d, Transgenic
expression of Atsly1 variants suppresses gai phenotype. Genotypes as shown,
with gai (far left) and WT (far right) (for equivalence of expression, see Extended
Data Fig. 2g). Scale bar, 5 cm. e, Mean (±s.d.) plant height, genotypes as in d, Nature Plants | Volume 9 | December 2023 | 2059–2070 2062 Article All residue contacts ≤5 Å are within SLY1 and, apart
from the F-box residue A49, all are within the GGF region and include W72, I75
and C76. A ‘bulge’ visible at Y107 in the helix is due to formation of a π-helix turn
starting at H104. Fig. 3 | Structural and selective consideration of Atsly1 mutant proteins. a, AlphaFold AtSLY1 structure prediction shown in 90o rotation and with
helical F-box (cyan), core helical GGF (brown) and outward-facing helical LSL
domains (purple) (see also Extended Data Fig. 3a–c). Amino-acid side chains
of residues targeted by substitutions in Atsly1 mutant proteins (for example,
H104) are shown as sticks and labelled. The position of G84 is indicated by a
sphere. The N-terminal 25 residues, which are predicted to be unstructured,
are not shown. b, Mean (±s.d.) strengths of yeast 2-hybrid interactions between
AtSLY1 (or indicated variants) and GAI. Red dots indicate individual values
(n = 3), different letters (a, b) indicate significant difference (two-sided Student’s
t-test). P = 0.0044. c, Mean (±s.d.) strengths of yeast 2-hybrid interactions
between AtSLY1 (or indicated variants) and GAI. Red dots indicate individual and Mpsly1S149T) to exhibit reduced MpDELLA affinity (Extended Data
Fig. 8b,c); this suggests that the respective evolutionary substitu-
tions (such as at the H104 equivalent) all contributed to evolutionary
decline in A affinity. Further experiments testing selected Arabidopsis
non-DELLA GRAS proteins showed that AtSLY1 interacts with AtSCR
alone (Extended Data Fig. 8d). Conversely, MpSLY1 interacts with
AtSCL4 alone (Extended Data Fig. 8d). Next, while no Atsly1 variants
exhibited detectably enhanced AtSCR interactions, several of them
(Atsly1H104Y, Atsly1P114L, Atsly1P114S, Atsly1T128A and Atsly1T128S) exhibited
novel AtSCL4 interactions (Extended Data Fig. 8e,f). Thus, these vari-
ants exhibit a broadened affinity mimicking that of MpSLY1, again
suggesting them to be evolutionarily revertant. very few OsGID2 (highly diverged; Fig. 4a) sites (3) were targeted (with
some targeted sites in equivalent positions; for example, SmSLY1S129,
AtSLY1T128 and OsGID2S180; Fig. 5e). We also replicated 9 Atsly1 substi-
tutions (Fig. 2a) at equivalent GID2 positions, finding that only H145Y
(as above, interaction was too weak for detection in the screen) and
S180A (recovered in the screen) enhanced SLR1 interactions (Fig. 5f). However, combining H145Y and R155L (GID2 R155 is equivalent to AtSLY1
P114), or R155L and Q190K (GID2 Q190 is equivalent to AtSLY1 E138)
significantly increased gid2H145Y/R155L and gid2R155L/Q190K SLR1 interac-
tions (Fig. 5f). Article SLY1
sly1G84D
sly1E138K
sly1K126E
sly1K23+T
Y
SD/-LWAHM/X/GA3
GAI in A+ A’ Forms
Prey
g h g GAI in A + A’ Forms sly1P114L
sly1P114S
sly1T128S
sly1K126N
sly1T128A
sly1H104Y
Input
SD/-LWM
Bait
SD/-LWAHM/X
GAI in A Form
SD/-LWAHM/X/GA3
GAI in A + A’ Forms Nature Plants | Volume 9 | December 2023 | 2059–2070 2063 https://doi.org/10.1038/s41477-023-01556-0 Article 0
1 20
40
60
80 100
β-galactosidase units
Bait
GAI
Prey
SLY1
sly1P114T
sly1P114S
sly1P114L
sly1P114H
sly1P114A
sly1P114R
a
b
c
d
e
f
g
0
0.1 0.2 0.3 0.4 0.5
β-galactosidase units
sly1H104R
Bait
SLY1
GAI
Prey
sly1T128P
sly1E138G
sly1K126R
a
b
ND
ND
ND
a
P114
K126
H104
N
C
G84
T128
N
C
90°
b
c
d
e
G84
P114
H104
K126
T128
E138
E138
H104
A49
I75
C76
W72
Y107
K126
G84
T128
H104
P114
E138
F-box
GGF
LSL
F-box
GGF
LSL
ig. 3 | Structural and selective consideration of Atsly1 mutant proteins. , AlphaFold AtSLY1 structure prediction shown in 90o rotation and with
elical F-box (cyan), core helical GGF (brown) and outward-facing helical LSL
omains (purple) (see also Extended Data Fig. 3a–c). Amino-acid side chains
f residues targeted by substitutions in Atsly1 mutant proteins (for example,
H104) are shown as sticks and labelled. The position of G84 is indicated by a
phere. The N-terminal 25 residues, which are predicted to be unstructured,
re not shown. b, Mean (±s.d.) strengths of yeast 2-hybrid interactions between
AtSLY1 (or indicated variants) and GAI. Red dots indicate individual values
n = 3), different letters (a, b) indicate significant difference (two-sided Student’s
test). P = 0.0044. c, Mean (±s.d.) strengths of yeast 2-hybrid interactions
etween AtSLY1 (or indicated variants) and GAI. Red dots indicate individual
values (n = 3), different letters (a–g) indicate significant differences (one-way
ANOVA with Tukey’s test). d, AlphaFold prediction of the AtSLY1–GAI complex
structure. Amino-acid residues targeted by substitutions in Atsly1 mutant
proteins are shown as spheres and labelled (see also Extended Data Fig. 4e,f). N-terminal residues of SLY1 and GAI predicted to be unstructured are not shown. e, AlphaFold AtSLY1–GAI structural prediction showing H104 intramolecular
contacts within AtSLY1. The H104 side chain is in van der Waals spheres and side
chains for residues with atoms ≤5 Å from any H104 atom are shown as sticks. GAI
is shown as a grey ribbon. Article We conclude that GID2 is so locked into A’ specificity
that reverting it typically requires multiple substitutions. Conversely,
because AtSLY1 and SmSLY1 are less locked, single substitutions can
detectably increase A affinity. Article All residue contacts ≤5 Å are within SLY1 and, apart
from the F-box residue A49, all are within the GGF region and include W72, I75
and C76. A ‘bulge’ visible at Y107 in the helix is due to formation of a π-helix turn
starting at H104. 0
0.1 0.2 0.3 0.4 0.5
β-galactosidase units
sly1H104R
Bait
SLY1
GAI
Prey
sly1T128P
sly1E138G
sly1K126R
a
b
ND
ND
ND
b b a C
d
K126
G84
T128
H104
P114
E138 0
1 20
40
60
80 100
β-galactosidase units
Bait
GAI
Prey
SLY1
sly1P114T
sly1P114S
sly1P114L
sly1P114H
sly1P114A
sly1P114R
a
b
c
d
e
f
g
c d e
H104
A49
I75
C76
W72
Y107 e 0
1 20
40
60
80 100
β-galactosidase units Fig. 3 | Structural and selective consideration of Atsly1 mutant proteins. values (n = 3), different letters (a–g) indicate significant differences (one-way
ANOVA with Tukey’s test). d, AlphaFold prediction of the AtSLY1–GAI complex
structure. Amino-acid residues targeted by substitutions in Atsly1 mutant
proteins are shown as spheres and labelled (see also Extended Data Fig. 4e,f). N-terminal residues of SLY1 and GAI predicted to be unstructured are not shown. e, AlphaFold AtSLY1–GAI structural prediction showing H104 intramolecular
contacts within AtSLY1. The H104 side chain is in van der Waals spheres and side
chains for residues with atoms ≤5 Å from any H104 atom are shown as sticks. GAI
is shown as a grey ribbon. All residue contacts ≤5 Å are within SLY1 and, apart
from the F-box residue A49, all are within the GGF region and include W72, I75
and C76. A ‘bulge’ visible at Y107 in the helix is due to formation of a π-helix turn
starting at H104. values (n = 3), different letters (a–g) indicate significant differences (one-way
ANOVA with Tukey’s test). d, AlphaFold prediction of the AtSLY1–GAI complex
structure. Amino-acid residues targeted by substitutions in Atsly1 mutant
proteins are shown as spheres and labelled (see also Extended Data Fig. 4e,f). N-terminal residues of SLY1 and GAI predicted to be unstructured are not shown. e, AlphaFold AtSLY1–GAI structural prediction showing H104 intramolecular
contacts within AtSLY1. The H104 side chain is in van der Waals spheres and side
chains for residues with atoms ≤5 Å from any H104 atom are shown as sticks. GAI
is shown as a grey ribbon. Nature Plants | Volume 9 | December 2023 | 2059–2070 Discussion sativa; OsGID2) with MpSLY1L
(MpSLY1-like) outgroup. The tree was constructed in MEGA11 using the
maximum-likelihood method and the JTT matrix-based model. The percentage of
trees in which the associated taxa clustered together (bootstrap value) is shown
next to the branches (out of 500 bootstrap replicates). The tree is drawn to scale,
with branch lengths measured as the number of substitutions per site. For a more Fig. 4 | Basal SLY1s exhibit broader DELLA affinities. a, Phylogenetic tree comprehensive phylogeny, see Extended Data Fig. 5. b, Interactions between
SLY1 and DELLA orthologues. c, Mean (±s.d.) yeast 2-hybrid SLY1 orthologue–GAI
interaction strengths; note gradient of increasing interaction strength ranging
from OsGID2 to MpSLY1. Red dots indicate individual values (n = 3), different
letters (a–i) indicate significant differences (one-way ANOVA with Tukey’s test). d, Yeast 3-hybrid determination of relative affinities of SLY1 orthologues for GAI
A and A’ forms. (MpSLY1-like) outgroup. The tree was constructed in MEGA11 using the
maximum-likelihood method and the JTT matrix-based model. The percentage of
trees in which the associated taxa clustered together (bootstrap value) is shown
next to the branches (out of 500 bootstrap replicates). The tree is drawn to scale,
with branch lengths measured as the number of substitutions per site. For a more NOXA proteins). Progressive A’ refinement also incurs ‘locking’. GID2
has so many substitutions (for example, relative to MpSLY1) that
it is effectively almost entirely locked into A’ specificity: relatively
few single substitutions enable reversion to dual A + A’ affinity. In
a comparable study, negative interaction ‘locking’ elements have
been shown to maintain insulation between two paralogous bacterial
toxin–antitoxin systems33. growth-regulatory GA–GID1–DELLA complex formation. Because such
complex formation can directly influence DELLA function (via a mecha-
nism not involving destruction2), recruitment of SLY1-mediated A vs A’
differential proteasome-dependent DELLA destruction may have been
a further step in the evolution of GA–GID1–DELLA signalling. While our
observations suggest continued refinement of A’ specificity post vas-
cularization, this likelihood would benefit from further investigation. Alternatively, because SLY1 is present in the bryophyte lineage19 and
because MpSLY1 binds DELLA, it is possible that SLY-mediated DELLA
regulation was established before GID1 recruitment22. Despite recent advances in understanding of PPI34, relatively few
studies address the evolution of PPI specificity32,35–41. While the extent
to which specificity enhancement is a general driver of PPI evolution is
debated42, our study suggests the importance of affinity narrowing and
provides a specific example. Discussion The partially increased A affinity of gid2H145Y (Fig. 5b) suggests the
sometimes-limited effects of single-residue substitutions. Accord-
ingly, pairwise AtSLY1 substitutions additively increase A affinity
(Fig. 5d and Extended Data Fig. 8g). For example, an H104Y/E138K com-
bination enhances affinity close to the ‘maximum’ MpSLY1-MpDELLA
affinity (Figs. 4c and 5d), indicating that evolutionary enhancement
of A’ specificity (reducing A affinity) probably involved multiple sub-
stitutions. Further experiments showed that evolution of SLY1 away
from A + A’ affinity increasingly ‘locked’ A’ specificity (reduced the
number of positions where single substitutions could restore A affinity;
Fig. 5e and Extended Data Fig. 8h). Multiple SmSLY1 (least diverged;
Fig. 4a) sites (14), fewer AtSLY1 (medium divergence; Fig. 4a) sites (5) and DELLAs regulate plant biology via interaction with multiple transcrip-
tion factors2. Post-translational modifications (for example, phospho-
rylation, SUMOylation, glycosylation) influence these interactions, thus
modulating DELLA activity30. In contrast, how the DELLA A to A’ transi-
tion increases DELLA–SCFSLY1/GID2 interactions, and how A’ specificity
evolved, was hitherto little understood. Collectively, our findings reveal
progressive refinement of ancestral SLY1 dual A + A’ affinity towards A’
specificity during land-plant evolution. DELLA function predated GA signalling2,31. Furthermore, the
N-terminal DELLA domain probably had an initial transcriptional
transactivation function19, implying its subsequent recruitment for Nature Plants | Volume 9 | December 2023 | 2059–2070 2064 https://doi.org/10.1038/s41477-023-01556-0 Article 0
1
3 6 9 20 40 60 80 100 120 140 160 180
β-galactosidase units
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpSLY1
SmSLY1
AtSLY1
OsGID2
a
f
i
d
hi
e
b
g
c
hi
i
ND
h
ND
ND
i
ND
i
i
ND
ND
ND
ND
ND
Bait
Prey
a
b
c
d
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
OsGID2
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
AtSLY1
SmSLY1
MpSLY1
MpSLY1L
0.50
86
97
Bait
SD/-LW
AtGID1C
AtGAI
SD/-LWM
Bridge
Bait
Prey
SD/-LWAH/X
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
Bait
SD/-LWAHM/X
GAI in A Form
SD/-LWAHM/X/GA3
GAI in A + A’ Forms
MpSLY1
SmSLY1
AtSLY1
OsGID2
ig. 4 | Basal SLY1s exhibit broader DELLA affinities. a, Phylogenetic tree
howing SLY1/GID2 orthologues from M. polymorpha (MpSLY1), S. moellendorfii
SmSLY1), A. thaliana (AtSLY1) and rice (O. sativa; OsGID2) with MpSLY1L
MpSLY1-like) outgroup. The tree was constructed in MEGA11 using the
maximum-likelihood method and the JTT matrix-based model. Nature Plants | Volume 9 | December 2023 | 2059–2070 Discussion The percentage of
ees in which the associated taxa clustered together (bootstrap value) is shown
ext to the branches (out of 500 bootstrap replicates). The tree is drawn to scale,
ith branch lengths measured as the number of substitutions per site. For a more
comprehensive phylogeny, see Extended Data Fig. 5. b, Interactions between
SLY1 and DELLA orthologues. c, Mean (±s.d.) yeast 2-hybrid SLY1 orthologue–GAI
interaction strengths; note gradient of increasing interaction strength ranging
from OsGID2 to MpSLY1. Red dots indicate individual values (n = 3), different
letters (a–i) indicate significant differences (one-way ANOVA with Tukey’s test). d, Yeast 3-hybrid determination of relative affinities of SLY1 orthologues for GAI
A and A’ forms. b
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
Bait
SD/-LW
SD/-LWAH/X
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
Bait b
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
Bait
SD/-LW a
OsGID2
AtSLY1
SmSLY1
MpSLY1
MpSLY1L
0.50
86
97 b a MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
SD/-LW
SD/-LWAH/X
MpSLY1
SmSLY1
AtSLY1
OsGID2
Prey
Bait 0
1
3 6 9 20 40 60 80 100 120 140 160 180
β-galactosidase units
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpDELLA
SmDELLA1
SmDELLA2
AtGAI
AtRGA
OsSLR1
MpSLY1
SmSLY1
AtSLY1
OsGID2
a
f
i
d
hi
e
b
g
c
hi
i
ND
h
ND
ND
i
ND
i
i
ND
ND
ND
ND
ND
Bait
Prey
c c d
AtGID1C
AtGAI
SD/-LWM
Bridge
Bait
Prey
SD/-LWAHM/X
GAI in A Form
SD/-LWAHM/X/GA3
GAI in A + A’ Forms
MpSLY1
SmSLY1
AtSLY1
OsGID2 d β-galactosidase units Fig. 4 | Basal SLY1s exhibit broader DELLA affinities. a, Phylogenetic tree
showing SLY1/GID2 orthologues from M. polymorpha (MpSLY1), S. moellendorfii
(SmSLY1), A. thaliana (AtSLY1) and rice (O. sativa; OsGID2) with MpSLY1L
(MpSLY1-like) outgroup. The tree was constructed in MEGA11 using the
maximum-likelihood method and the JTT matrix-based model. The percentage of
trees in which the associated taxa clustered together (bootstrap value) is shown
next to the branches (out of 500 bootstrap replicates). The tree is drawn to scale,
with branch lengths measured as the number of substitutions per site. For a more Fig. 4 | Basal SLY1s exhibit broader DELLA affinities. a, Phylogenetic tree
showing SLY1/GID2 orthologues from M. polymorpha (MpSLY1), S. moellendorfii
(SmSLY1), A. thaliana (AtSLY1) and rice (O. Discussion First, a change in SLY1 core conformation
(Y to H substitution at the buried site equivalent to AtSLY1 104) some-
time before angiosperm divergence reduced A (vs A’) affinity (perhaps
by a change in LSL accessibility). Second, mutation accumulation
during angiosperm evolution (for example, at sites AtSLY1 126/138)
reduced electrostatic interactions with A at the interaction interface. The resultant trend towards SLY1/GID2 A’ specificity was probably
driven by selective advantage: consequent GA regulation enhanced Interestingly, enhanced A affinity Atsly1 variants frequently revert
evolutionary substitutions that were probably causal of A’ refinement. For example, AtslyH104Y restores the Y of basal SLY1s. This predictability
contrasts with findings that narrowing of initial animal dual B-cell
lymphoma-2 (BCL-2) affinity for BID1 and NOXA partners to specificity
for BID1 was achieved experimentally via substitutions at sites often
not evolutionarily targeted32. Perhaps our study reveals greater pre-
dictability because it concerns evolving affinities for distinct (A/A’)
conformations of the same protein (rather than different BID1 and 2065 https://doi.org/10.1038/s41477-023-01556-0 Article 1
78
116
151
1
119
168
217
1
52
117
158
0
1 10
40
70
100 130 160
β-galactosidase units
Vector
SLY1
sly1E138K
sly1T128A
sly1P114L
GAI
sly1H104Y/E138K
Bait
sly1T128A/E138K
sly1P114L/E138K
sly1H104Y
a
b
d
c
d
e
f
g
ND
Bait
Prey
0
1 10 40 70 100130
β-galactosidase units
Atsly1H104F
Bait
AtSLY1
Atsly1H104Y
MpSLY1
Mpsly1Y108F
Mpsly1Y108H
GAI
Prey
a
a
b
c
d
ND
c
e
SD/-LW
SD/-LWAH/X
Prey
Bait
MpDELLA
OsSLR1
b
d
SD/-LW
SD/-LWAH/X
Prey: SLR1
Bait
GID2
gid2H145Y
gid2R155L
gid2R155S
gid2Q178E
gid2G125D
gid2Q178N
gid2S180A
gid2Q190K
gid2H145Y/R155L
gid2R155L/Q190K
gid2Q178T
SD/-LW
SD/-LWAH/X
Bait
F-box
GGF
LSL
AtSLY1
E138K
P114L
P114S
H104Y
T128A
T128S
K126E
K126N
K126T
F-box
GGF
LSL
SmSLY1
I51L
L52H
S81T
C80R
E91G
E91K
F101L
F101Y
L102H
H103Y
M107I
Q114R
S129A
I135V
C137Y
L139M
F-box
GGF
LSL
OsGID2
V147A
V147D
L136R
S180A
f
a
MpSLY1
Mpsly1Y108H
OsGID2
OsGID2H145Y
Evolutionary constraints on SLY1 reversion routes. a, Alignment
no acid sequences of SLY1 orthologues. Black highlights identity, grey
ghts residue similarity, red arrows indicate sites in AtSLY1 targeted by
ed amino acid substitutions (and equivalent sites in MpSLY1, SmSLY1 and
D2). For more comprehensive alignment, see Extended Data Fig. 7. ractions between MpSLY1 or OsGID2 (or indicated variants) and MpDELLA
SLR1. c, Mean (±s.d.) strengths of yeast 2-hybrid interactions between
1 (or indicated variants) or MpSLY1 (or indicated variants) and GAI. Discussion GAI
Prey
0
1 10 40 70 100130
β-galactosidase units
Atsly1H104F
Bait
AtSLY1
Atsly1H104Y
MpSLY1
Mpsly1Y108F
Mpsly1Y108H
GAI
Prey
a
a
b
c
d
ND
c
d
135V SD/-LW
SD/-LWAH/X
Prey
Bait
MpDELLA
OsSLR1
b
MpSLY1
Mpsly1Y108H
OsGID2
OsGID2H145Y 0
1 10
40
70
100 130 160
β galactosidase units
Vector
SLY1
sly1E138K
sly1T128A
sly1P114L
sly1H104Y/E138K
Bait
sly1T128A/E138K
sly1P114L/E138K
sly1H104Y
a
b
d
c
d
e
f
g
ND
Bait b d c 1
78
116
151
1
119
168
217
1
52
117
158
0
β-ga
e
F-box
GGF
LSL
AtSLY1
E138K
P114L
P114S
H104Y
T128A
T128S
K126E
K126N
K126T
F-box
GGF
LSL
SmSLY1
I51L
L52H
S81T
C80R
E91G
E91K
F101L
F101Y
L102H
H103Y
M107I
Q114R
S129A
I135V
C137Y
L139M
F-box
GGF
LSL
OsGID2
V147A
V147D
L136R
S180A 1
78
116
151
1
52
117
158
e
F-box
GGF
LSL
AtSLY1
E138K
P114L
P114S
H104Y
T128A
T128S
K126E
K126N
K126T
F-box
GGF
LSL
SmSLY1
I51L
L52H
S81T
C80R
E91G
E91K
F101L
F101Y
L102H
H103Y
M107I
Q114R
S129A
I135V
C137Y
L139M e β-galactosidase units
SD/-LW
SD/-LWAH/X
Prey: SLR1
Bait
GID2
gid2H145Y
gid2R155L
gid2R155S
gid2Q178E
gid2G125D
gid2Q178N
gid2S180A
gid2Q190K
gid2H145Y/R155L
gid2R155L/Q190K
gid2Q178T
SD/-LW
SD/-LWAH/X
Bait
f f Fig. 5 | Evolutionary constraints on SLY1 reversion routes. a, Alignment
of amino acid sequences of SLY1 orthologues. Black highlights identity, grey
highlights residue similarity, red arrows indicate sites in AtSLY1 targeted by
selected amino acid substitutions (and equivalent sites in MpSLY1, SmSLY1 and
OsGID2). For more comprehensive alignment, see Extended Data Fig. 7. b, Interactions between MpSLY1 or OsGID2 (or indicated variants) and MpDELLA
or OsSLR1. c, Mean (±s.d.) strengths of yeast 2-hybrid interactions between
AtSLY1 (or indicated variants) or MpSLY1 (or indicated variants) and GAI. Red
dots indicate individual values (n = 3), different letters (a–d) indicate significant
differences (one-way ANOVA with Tukey’s test). d, Mean (±s.d.) strengths of
yeast 2-hybrid interactions between AtSLY1 (or indicated variants) and GAI. Red
dots indicate individual values (n = 3), different letters (a–g) indicate significant Fig. 5 | Evolutionary constraints on SLY1 reversion routes. a, Alignment differences (one-way ANOVA with Tukey’s test). e, Comparison of sites where
amino acid substitutions confer increased affinity for the DELLA A form. Data
from EP-PCR/yeast 2-hybrid screens. For SmSLY1, mutants were initially selected
for increased affinity for GAI, then validated with SmDELLA1. For AtSLY1,
substitutions were as previously described (Fig. 2a). Discussion Red
ndicate individual values (n = 3), different letters (a–d) indicate significant
ences (one-way ANOVA with Tukey’s test). d, Mean (±s.d.) strengths of
2-hybrid interactions between AtSLY1 (or indicated variants) and GAI. Red
ndicate individual values (n = 3), different letters (a–g) indicate significant
differences (one-way ANOVA with Tukey’s test). e, Comparison of sites whe
amino acid substitutions confer increased affinity for the DELLA A form. Da
from EP-PCR/yeast 2-hybrid screens. For SmSLY1, mutants were initially sel
for increased affinity for GAI, then validated with SmDELLA1. For AtSLY1,
substitutions were as previously described (Fig. 2a). For OsGID2, mutants w
initially selected for increased affinity for GAI, then validated with the rice D
SLR1. Targeted amino acids occupying equivalent positions are indicated w
the same colour. Specifically, SmSLY1 E91 is equivalent to OsGID2 L136 (ora
SmSLY1 L102 is equivalent to OsGID2 V147 (blue); SmSLY1 L139 is equivalen
AtSLY1 E138 (purple); SmSLY1 S129 is equivalent to AtSLY1 T128 and OsGID2
S180 (red). All other targeted positions (black) are unique to SmSLY1 or AtS
f, Interaction of indicated OsGID2 and Osgid2 variants with SLR1. a a 0
1 10
40
70
100 130 160
β-galactosidase units
Vector
SLY1
sly1E138K
sly1T128A
sly1P114L
GAI
sly1H104Y/E138K
Bait
sly1T128A/E138K
sly1P114L/E138K
sly1H104Y
a
b
d
c
d
e
f
g
ND
Bait
Prey
0 40 70 100130
ctosidase units
a
a
b
c
d
SD/-LW
SD/-LWAH/X
Prey: SLR1
Bait
GID2
gid2H145Y
gid2R155L
gid2R155S
gid2Q178E
gid2G125D
gid2Q178N
gid2S180A
gid2Q190K
gid2H145Y/R155L
gid2R155L/Q190K
gid2Q178T
SD/-LW
SD/-LWAH/X
Bait
f
differences (one-way ANOVA with Tukey’s test). e, Comparison of sites where
amino acid substitutions confer increased affinity for the DELLA A form. Data
from EP-PCR/yeast 2-hybrid screens. For SmSLY1, mutants were initially selected
for increased affinity for GAI, then validated with SmDELLA1. For AtSLY1,
substitutions were as previously described (Fig. 2a). For OsGID2, mutants were
initially selected for increased affinity for GAI, then validated with the rice DELLA
SLR1. Targeted amino acids occupying equivalent positions are indicated with
the same colour. Specifically, SmSLY1 E91 is equivalent to OsGID2 L136 (orange);
SmSLY1 L102 is equivalent to OsGID2 V147 (blue); SmSLY1 L139 is equivalent to
AtSLY1 E138 (purple); SmSLY1 S129 is equivalent to AtSLY1 T128 and OsGID2
S180 (red). All other targeted positions (black) are unique to SmSLY1 or AtSLY1. f, Interaction of indicated OsGID2 and Osgid2 variants with SLR1. Discussion For OsGID2, mutants were
initially selected for increased affinity for GAI, then validated with the rice DELLA
SLR1. Targeted amino acids occupying equivalent positions are indicated with
the same colour. Specifically, SmSLY1 E91 is equivalent to OsGID2 L136 (orange);
SmSLY1 L102 is equivalent to OsGID2 V147 (blue); SmSLY1 L139 is equivalent to
AtSLY1 E138 (purple); SmSLY1 S129 is equivalent to AtSLY1 T128 and OsGID2
S180 (red). All other targeted positions (black) are unique to SmSLY1 or AtSLY1. f, Interaction of indicated OsGID2 and Osgid2 variants with SLR1. b, Interactions between MpSLY1 or OsGID2 (or indicated variants) and MpDELLA
or OsSLR1. c, Mean (±s.d.) strengths of yeast 2-hybrid interactions between
AtSLY1 (or indicated variants) or MpSLY1 (or indicated variants) and GAI. Red
dots indicate individual values (n = 3), different letters (a–d) indicate significant
differences (one-way ANOVA with Tukey’s test). d, Mean (±s.d.) strengths of
yeast 2-hybrid interactions between AtSLY1 (or indicated variants) and GAI. Red
dots indicate individual values (n = 3), different letters (a–g) indicate significant a 16 h light/8 h dark photoperiod (irradiance 120 μmol m−2 s−1). Seed-
lings (10-day-old) were transplanted to soil (ICL Levington advanced
F2 compost) and grown in controlled environment rooms (CERs) in the
same environmental conditions as above. M. polymorpha accession
Takaragaike-1 (Tak-1; male) was cultured on half-strength MS medium
(pH 5.6) containing 0.5% sucrose, 0.5 g l−1 MES and 0.8% agar in the same
growth conditions. S. moellendorffii plants were kindly provided by the
University of Bristol Botanic Garden and maintained at 24 °C in a 16 h
light/8 h dark photoperiod (irradiance 90 μmol m−2 s−1) in plant growth the flexibility of adaptational plant growth control in response to
environmental variables. Arabidopsis transformation DNA fragments consisting of the promoter (~2 kbp upstream of the tran-
scription start site) and genomic DNA sequences of SLY1 and sly1G84D
were amplified from gai and gai gar2-2 Arabidopsis plants, respectively,
then cloned into pCAMBIA2300 to make pSLY1::SLY1 and pSLY1::sly1G84D
constructs. pSLY1::sly1H104Y and pSLY1::sly1T128A were generated using
Q5 site-directed mutagenesis (NEB, E0554S) of pSLY1::SLY1. To make
the 35S::HA-MpSLY1 overexpression construct, the coding sequence of
MpSLY1 was amplified from complementary DNA (cDNA) and cloned into
the pEarlyGate201 vector. All constructs were transformed into gai (Col-0
background) using the Agrobacterium (GV3101 strain)-mediated floral dip
method44. Relevant primer sequences are listed in Supplementary Table 3. Yeast 2-hybrid assay and mutant screen Various SLY1 and DELLA coding sequences were amplified from
cDNA and cloned into vectors pGBKT7 and pGADT7 to generate bait
(pGBKT7-SLY1) and prey (pGADT7-DELLA) constructs, respectively. Bait and prey constructs (100 ng) were co-transformed into yeast
strain AH109 (TaKaRa) and selected on the synthetic defined (SD) yeast
leucine and tryptophan dropout medium (SD/-LW) for 3 d at 30 °C. At
least four colonies from each transformation were selected at random
and resuspended in 50 μl of 0.9% NaCl, 5 μl of which was spotted onto
the SD/-LW (for loading control) and the leucine, tryptophan, adenine
and histidine dropout medium supplemented with 40 μg ml−1 X-α-Gal
(SD/-LWAH/X, for assessing interaction strength). The plates were then
incubated for 3–5 d at 30 °C. Relevant primer sequences are listed in
Supplementary Table 3. Leaf chlorophyll measurement Leaf chlorophyll content was measured using a SPAD-502 metre
(Konica-Minolta) as previously described43. Absolute chlorophyll
concentration in nmol mg−1 fresh weight was calculated using the
previously derived equation: For the yeast 2-hybrid mutant screen, AH109 yeast cells were first
transformed with pGADT7-GAI and maintained on leucine dropout
medium (SD/-L), followed by transformation with 100–200 ng of bait
vectors containing EP-PCR-mutagenized SLY1/GID2 libraries. For each
screen, at least 3,000 colonies were plated on SD/-LW medium sup-
plemented with 40 μg ml−1 X-α-Gal (SD/-LW/X). Colonies turning blue
after 3–4 d were selected and cultured overnight in liquid tryptophan
dropout medium (SD/-W) for plasmid extraction with the Zymoprep
yeast plasmid miniprep kit (Zymo Research, D2001). SLY1/GID2 DNA was
then PCR-amplified from the extracted plasmid for Sanger sequencing
(Source BioScience), thus enabling detection of EP-PCR-generated
mutations potentially responsible for the selected enhanced bait–prey
interaction. Likely candidate mutations were first identified as those
recovered at least three times from the screens. These candidate muta-
tions were next reconstructed from the original pGBKT7-SLY1/GID2
vectors using Q5 site-directed mutagenesis (NEB, E0554S) to remove
additional potentially confounding EP-PCR-generated mutations, and
confirmed genuine by performing yeast 2-hybrid assays with their
respective DELLA partners (SmDELLA, AtGAI or OsSLR1). All screens
were performed at least four times using independently mutagenized
libraries to avoid repetitively selecting clonal candidates. y = 0.0007x2 + 0.0230x + 0.0544
(1) (1) y = 0.0007x2 + 0.0230x + 0.0544 y = 0.0007x2 + 0.0230x + 0.0544 where y is the absolute chlorophyll concentration and x is the SPAD
metre reading43. Plant materials and growth conditions Arabidopsis thaliana seeds were sterilized with 75% ethanol for
10 min and germinated on half-strength Murashige and Skoog (MS,
Sigma-Aldrich, M5519) salt medium (pH 5.8) containing 0.5% sucrose,
0.5 g l−1 2-morpholinoethanesulfonic acid (MES) and 1% agar at 22 °C in Nature Plants | Volume 9 | December 2023 | 2059–2070 Nature Plants | Volume 9 | December 2023 | 2059–2070 2066 Article https://doi.org/10.1038/s41477-023-01556-0 Mutagenic EP-PCR incubators (Sanyo, MLR 351). Seeds of O. sativa subspecies japonica
(Nipponbare variety) were peeled and sterilized in 70% ethanol for 30 s,
followed by 15 ml of 10% sodium hypochlorite (NaClO) for 30 min. The
seeds were then thoroughly rinsed with distilled water and stratified at
37 °C in the dark for 3–7 d. Following germination, rice seedlings were
transferred to 1.5 l hydroponic devices containing 1 l of half-strength
MS liquid media supplemented with vitamins (Duchefa Biochemie,
M0222) in plant growth incubators (Sanyo, MLR 351) set at 22 °C in a
16 h light/8 h dark photoperiod (irradiance 200 μmol m2 s−1). Nutrient
solution was replaced every 3 d until the tissue was ready to harvest. g
The EP-PCR reaction mixture consisted of the following: 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 7 mM MgCl2, 0.5 mM MnCl2, 1 mM dCTP, 1 mM
dTTP, 0.2 mM dATP, 0.2 mM dGTP, 2 μM 5’ primer, 2 μM 3’ primer,
0.05 U μl−1 Taq DNA polymerase, 20 pg μl−1 DNA template and if neces-
sary, 3% dimethyl sulfoxide. Thirty-five cycles of PCR were performed
at a Tm of 55 °C. The resultant enhancement of the natural error rate of
the Taq polymerase was due to the elevated MgCl2 concentration, the
presence of MnCl2 (which stabilizes non-complementary nucleotide
pairing) and an uneven ratio of nucleotides in the reaction. The result-
ant mutagenized DNA libraries were gel purified with the QIAquick gel
extraction kit (QIAGEN, 28704), then cloned into pGBKT7 vector using
In-Fusion Snap Assembly (TaKaRa, 638947). Primers used for EP-PCR
are listed in Supplementary Table 3. EP-PCR enables semi-random
mutation generation and is not necessarily a reliable proxy for the
multiple mechanisms via which mutations are generated during bio-
logical evolution. Arabidopsis seed mutagenesis and mutant screen Approximately 50,000 Arabidopsis gai progenitor seeds (La-er back-
ground) were mutagenized by incubating in 0.2% ethyl methane sul-
fonate (EMS, Sigma-Aldrich, M0880) for 15 h, followed by 10 washes
with distilled water. The seeds (M1) were then separated into batches
of ~2,000 and sown on soil. The M1 plants were allowed to self-pollinate
and the resultant M2 seeds were collected for genetic screens. Mutants
that were visibly taller than the gai progenitor were selected for herita-
bility and segregation tests and taken to the subsequent M3 generation. Leaf material from homozygous mutant populations was collected for
DNA extraction and gai was sequenced to eliminate gai loss-of-function
mutations (that is, gai-d mutations1,24). For the remainder, SLY1 gene
sequencing identified sly1 mutations conferring the mutant pheno-
type. Primers used to amplify gai and SLY1 from genomic DNA for
Sanger sequencing are listed in Supplementary Table 3. Nature Plants | Volume 9 | December 2023 | 2059–2070 RNA isolation, cDNA synthesis and quantitative PCR with
reverse transcription (RT–qPCR) analysis Total RNA was extracted from ~100 mg plant material using TRIzol
reagent (ThermoFisher, 15596026) and treated with the DNA-free
DNA removal kit (ThermoFisher, AM1906) following manufacturer
instructions. Full-length cDNA was subsequently reverse transcribed
using SuperScript IV reverse transcriptase (ThermoFisher, 18090010)
before being used for RT–qPCR on an Applied Biosystem StepOne-
Plus real-time PCR system (Thermo Scientific) using the qPCRBIO
SyGreen Mix Hi-Rox reagent (PCR Biosystems, PB20.12). RT–qPCR
assays included three biological replicates, and the results were ana-
lysed using the StepOnePlus software v.2.3 and Microsoft Office Excel
v.16.71. The Arabidopsis Actin2 gene (At3g18780) was used as internal
control. Primers used for RT–qPCR are listed in Supplementary Table 3. β-galactosidase quantification of yeast 2-hybrid interactions
Yeast 2-hybrid quantitative assays were performed with strain Y187
(in liquid culture) using chlorophenol red β-d-galactopyranoside
(CPRG, Roche, 11379119103) as substrate. For each interaction pair,
three colonies were cultured overnight in SD/-LW liquid before
being diluted 5-fold in liquid rich medium, and grown further until
the optical density (OD)600 was within the 0.5–0.8 range. The culture
(1.5 ml) was then pelleted and resuspended in 300 μl buffer 1 (2.38 g
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.9 g
NaCl, 0.065 g l-aspartate, 1 g BSA and 50 μl Tween-20 in 100 ml solu-
tion, pH adjusted to 7.25–7.30, filter sterilized). The cell suspension Nature Plants | Volume 9 | December 2023 | 2059–2070 2067 https://doi.org/10.1038/s41477-023-01556-0 Article was next divided into three 100 μl aliquots and cells were broken
open by repetitively (at least three times) freezing the culture in liquid
nitrogen, followed by immediate rapid thawing in a 37 °C water bath. Buffer 2 (0.7 ml) (2.23 mM CPRG in buffer 1, filter sterilized) was then
added to start the reaction. The reaction was stopped when the colour
of the sample turned orange/red by adding 0.5 ml 3 mM ZnCl2. Cell
debris was removed by spinning and the OD578 of the supernatant was
recorded using the Evolution 260 BIO UV-visible spectrophotometer
with INSIGHT2 software. β-galactosidase activity (units) was calculated
using the following equation: expression. Recombinant His-GAI/gai, MBP-SLY1/sly1 and GST-GID1C
proteins were purified using Ni-NTA agarose (QIAGEN, 30210), amyl-
ose resin (NEB, E8021S) and glutathione beads (Smart-Lifesciences,
SA008100), respectively, according to manufacturer instructions. For the in vitro pull-down assay, 5 μg His-GAI/gai was incubated with
Ni-NTA agarose (QIAGEN, 30210) for 3 h at 4 °C. Reporting summary Further information on research design is available in the Nature Port-
folio Reporting Summary linked to this article. Phylogenetic analysis Protein sequences of SLY1/GID2 orthologues from diverse land plants
were obtained from a variety of sources including Phytozome45,
PLAZA46, OneKP47 and FernBase48, using SmSLY1 and SmSLY2 as queries
for BLASTP with an expected (e-value) threshold of 1 × 10−20. Multiple
sequence alignment was performed using the T-Coffee alignment
server49 and phylogenetic trees were constructed in MEGA11 (ref. 50)
using the maximum-likelihood method and the Jones–Taylor–Thornton
(JTT) matrix-based model. Protein structure predictions Predicted SLY1 structures were obtained from the AlphaFold Protein
Structure Database25,26 (Q9STX3), or predicted by RoseTTAFold27 or
ESMFold28. The structure of the GAI-SLY1 complex was predicted by
AlphaFold2-multimer51. All protein structures were visualized, analysed
and annotated in PyMOL. Western blot analysis Total protein was extracted from ~100 mg of plant material using
extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
0.1% NP-40 detergent, 10% glycerol, 1 mM dithiothreitol and protease
inhibitor cocktail (Roche, 11697498001). Yeast protein was extracted
from liquid overnight culture using YeastBuster (Merck, 71186) supple-
mented with Tris(hydroxypropyl)phosphine solution (Merck, 71194)
and protease inhibitor cocktail (Roche, 11697498001) following manu-
facturer instructions. Protein samples were heated at 70 °C for 10 min
before being subjected to sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose
membrane (VWR, PIER88013). The membrane was stained in Ponceau
S solution (Sigma-Aldrich, P7170), which was subsequently washed off
with 0.1 M NaOH before blocking. GAI, SLY1 and Actin proteins in plant
extracts were detected using AF2 (1:5,000)14, anti-SLY1 (Agrisera, AS13
2638, 1:5,000) and anti-Actin (Agrisera, AS13 2640, 1:5,000), respec-
tively. RPT5 (regulatory particle triple-A ATPase 5), HA-tagged GAI
and Myc-tagged SLY1 proteins in yeast extracts were detected using
anti-RPT5 (abcam, ab22676, 1:10,000), anti-HA (MBL Life science,
M180-7, 1:5,000) and anti-Myc (MBL Life science, M192-7, 1:5,000),
respectively. The membranes were visualized on an iBright FL1500
imaging system (ThermoFisher). Band intensity was quantified using
gel analysis methods (ImageJ). RNA isolation, cDNA synthesis and quantitative PCR with
reverse transcription (RT–qPCR) analysis After centrifugation,
the supernatant was removed and 5 μg MBP-SLY1/sly1 was added to
the pelleted beads. When required, an equal amount of GST-GID1C and
0.1 mM GA3 were added to the reaction. After a further 1 h incubation
at 4 °C, the beads were washed five times before being resuspended
in SDS loading buffer. The proteins were released into the solution by
boiling for 5 min and detected through immunoblots using anti-His
(Santa Cruz, SC-8036, 1:2,000), anti-MBP (NEB, E8032S, 1:10,000)
and anti-GST (MBL, PM013-7, 1:5,000) antibodies. Relevant primer
sequences are listed in Supplementary Table 3. Band intensity was
quantified using gel analysis methods (ImageJ). y = 1, 000 × OD578/(t × V × OD600)
(2) (2) y = 1, 000 × OD578/(t × V × OD600) where y is the β-galactosidase unit; t is the elapsed time (in minutes)
of incubation; V is 0.1 × concentration factor (in this case V = 0.5). An
interaction was deemed ‘not detected’ (ND) if the OD578 was <0.01 after
3 h of colour development. Yeast 3-hybrid assay Total protein was extracted from 14-day-old Arabidopsis seedlings in
lysis buffer (25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 4 mM
phenylmethylsulfonyl fluoride, 5 mM dithiothreitol and 10 mM ATP). Protein concentration was determined using the Bradford protein assay
(ThermoFisher, B6916) and adjusted to 5 μg μl−1. Plant extract (150 μl)
was incubated with 100 ng of recombinant His-gai protein at room
temperature, with samples taken at a series of timepoints. Proteins were
detected through immunoblots using anti-His (Santa Cruz, SC-8036,
1:2,000) and anti-Actin (EASYBIO, BE0021, 1:5,000) antibodies. Band
intensity was quantified using gel analysis methods (ImageJ). SLY1 and GID1 coding sequences were amplified from cDNA and cloned
into the pBridge vector (TaKaRa), with SLY1 fused with the GAL4
DNA-binding domain and GID1 fused with the Met promoter. Prey and
pBridge vectors were co-transformed into strain AH109 that had previ-
ously been streaked three times on methionine dropout media (SD/-M). Transformed colonies were selected on methionine, leucine and tryp-
tophan dropout medium (SD/-LWM) 5–7 d after transformation. At
least five colonies for each interaction pair were selected at random
and resuspended in 50 μl 0.9% NaCl, 5 μl of which was spotted onto
the SD/-LWM (for the loading control), SD/-LWAHM medium supple-
mented with X-α-Gal (SD/-LWAHM/X) and SD/-LWAHM/X medium sup-
plemented with 0.1 mM GA3. The plates were then incubated for 3–5 d at
30 °C. Relevant primer sequences are listed in Supplementary Table 3. Statistics and reproducibility All statistical analyses (Student’s t-test, one-way analysis of variance
(ANOVA) with Tukey’s test and chi-square test) were performed using
GraphPad Prism 9. P < 0.05 was considered to indicate statistical signifi-
cance. Exact P values are provided either in the figure legends, supple-
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proteins, the coding sequences of WT and mutant variants of GAI and
SLY1 were amplified from cDNA or pGBKT7 bait vectors recovered
from yeast 2-hybrid screens and cloned into pCold-TF (TaKaRa) and
pMAL-c2X (NEB), respectively. The coding sequence of GID1C was
amplified from cDNA and cloned into pGEX-4T-1 (GE Healthcare). These constructs were transformed into E. coli strain BL21 for protein All data generated in this study are included in the main text and in the
Supplementary Information. Structural models of AtSLY1 (UniProt
ID: Q9STX3) and AtGAI (UniProt ID: Q9LQT8) were obtained from the
AlphaFold database. All experimental materials constructed in this
study are available from the corresponding author upon request. Source data are provided with this paper. Nature Plants | Volume 9 | December 2023 | 2059–2070 2068 Article https://doi.org/10.1038/s41477-023-01556-0 Nature Plants | Volume 9 | December 2023 | 2059–2070 Reprints and permissions information is available at Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
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as long as you give appropriate credit to the original author(s) and the
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indicated otherwise in a credit line to the material. If material is not
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use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Additional information 48. Li, F. W. et al. Fern genomes elucidate land plant evolution and
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all. Nat. Methods 19, 679–682 (2022). Peer review information Nature Plants thanks Jutarou Fukazawa,
Salomé Prat and Daoxin Xie for their contribution to the peer review of
this work. Peer review information Nature Plants thanks Jutarou Fukazawa,
Salomé Prat and Daoxin Xie for their contribution to the peer review of
this work. Nature Plants | Volume 9 | December 2023 | 2059–2070 Competing interests
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green plant genomics. Nucleic Acids Res. 40, D1178–D1186 (2012). Nature Plants | Volume 9 | December 2023 | 2059–2070 2069 Article https://doi.org/10.1038/s41477-023-01556-0 creative input, and wrote the paper. All authors discussed the results
and contributed to the paper. 46. Van Bel, M. et al. PLAZA 4.0: an integrative resource for functional,
evolutionary and comparative plant genomics. Nucleic Acids Res. 46, D1190–D1196 (2018). Acknowledgements We thank P. Jarvis (University of Oxford) for providing yeast strains
(AH109; Y187) and vectors (pGBKT7; pGADT7); W. T. Jones,
J. Rakonjac, D. J. Sheerin and T. Foster for antibody AF2 (ref. 14);
Z. J. Ding for training in yeast 2-hybrid methods, and Q. Meng for
training in molecular biological techniques; P. Harms and N. Wray
(University of Bristol Botanic Garden, UK) for providing Selaginella
moellendorffii plants. This work was supported by UKRI (BBSRC, BB/
S013741/1 to N.P.H.), the National Natural Science Foundation of
China (grants 32122065 to S.L. and 32020103004 to X.F.), and the
Oxford Interdisciplinary Bioscience Doctoral Training Partnership,
UKRI-BBSRC (BB/M011224/1 to J.B.). J.S. and N.P.H. gratefully
acknowledge research funding support from St John’s College,
Oxford. Author contributions Z.J. designed and performed most of the experiments; E.J.B. designed
experiments, performed some AlphaFold analyses and provided
support for molecular cloning and plant growth; S.Z. and S.L. performed pull-down and cell-free degradation assays; J.B. assisted
with yeast 2-hybrid screens and computational structural analysis;
J.S. designed and conducted the computational structural analysis;
X.F., J.S. and S.L. provided creative input and revised the paper; N.P.H. designed experiments, conceived the project, provided further © The Author(s) 2023 © The Author(s) 2023 2070 https://doi.org/10.1038/s41477-023-01556-0 Article Extended Data Fig. 1 | Land-plant phylogeny summary and suppression of
gai phenotype by mutant gar2 alleles. a, Summary of land-plant phylogeny
showing major clades, origin of DELLA (red) and origin of GA-GID1-mediated
signalling (blue). b, Mean chlorophyll contents, genotypes as shown, red dots
indicate individual contents (n = 10), error bars indicate standard deviation,
different letters (a-c) indicate significant difference (one-way ANOVA with
Tukey’s test). c, Mean shoot fresh weights, genotypes as shown, red dots indicate
individual weights (n = 10), error bars indicate standard deviation, different
letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). different letters (a-c) indicate significant difference (one-way ANOVA with
Tukey’s test). c, Mean shoot fresh weights, genotypes as shown, red dots indicate
individual weights (n = 10), error bars indicate standard deviation, different
letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). different letters (a-c) indicate significant difference (one-way ANOVA with
Tukey’s test). c, Mean shoot fresh weights, genotypes as shown, red dots indicate
individual weights (n = 10), error bars indicate standard deviation, different
letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). different letters (a-c) indicate significant difference (one-way ANOVA with
Tukey’s test). c, Mean shoot fresh weights, genotypes as shown, red dots indicate
individual weights (n = 10), error bars indicate standard deviation, different
letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). Extended Data Fig. 1 | Land-plant phylogeny summary and suppression of
gai phenotype by mutant gar2 alleles. a, Summary of land-plant phylogeny
showing major clades, origin of DELLA (red) and origin of GA-GID1-mediated
signalling (blue). b, Mean chlorophyll contents, genotypes as shown, red dots
indicate individual contents (n = 10), error bars indicate standard deviation, Nature Plants https://doi.org/10.1038/s41477-023-01556-0 https://doi.org/10.1038/s41477-023-01556-0 Article
https://doi.org/10.1038/s41477-023-01556-0
Extended Data Fig. 2 | See next page for caption. Article Extended Data Fig. 2 | See next page for caption. Nature Plants Article https://doi.org/10.1038/s41477-023-01556-0 with Myc. RPT5 and Ponceau S provide loading controls. e, GAI compared with
C-terminally truncated GAI-NT2. f, SLY1 and sly1 variant interactions with GAI and
GAI-NT2 (as shown). AtSLY16 and all Atsly1 variants interact with GAI but not with
GAI-NT2. The increased interaction of Atsly1 variants is thus not due to increased
general ‘stickiness’. g, Mean relative AtSLY1 mRNA abundances in selected
transgenic lines (genotypes as shown), with non-transgenic WT and gai. Red dots
indicate individual values (n = 3), error bars indicate standard deviation, different
letters (a-c) indicate significant difference (one-way ANOVA with Tukey’s test). h, Abundance of immuno-detectable gai (quantified against Actin, arbitrarily
set at 1.00) or GAI and sly1 (or SLY1) in plant extracts (genotypes as shown). Anti-
Actin and Ponceau S staining serve as loading controls. i, Interactions between
AtSLY1 and Arabidopsis DELLAs GAI, RGA, RGL1, RGL2 and RGL3. j, Interactions
between AtSLY1 or Atsly1 variants and RGA. Extended Data Fig. 2 | Yeast-based discovery of novel Atsly1 mutant proteins. a, Combined EP-PCR/yeast-2-hybrid screen for Atsly1 variants having increased
AtGAI affinity. Different Atsly1 amino acid substitution variants (represented by
different colours) are generated via error prone PCR, then used as bait against an
AtGAI prey in a yeast-2-hybrid screen. Colonies exhibiting increased interaction
are blue (rather than white; see also b). DNA sequencing then reveals interaction-
increasing amino acid substitutions (see Methods). b, Example yeast-2-hybrid
screen plate showing a blue colony (also in close-up panel) amongst whites
(also in close-up). c, Yeast-2-hybrid analysis of interactions between GAI and
Atsly1 variants. Top line (SD/-LW) confirms double transformation with bait
and prey constructs, bottom line (SD/-LWAH/X) indicates interaction: baseline
interaction between GAI and SLY1 (medium blue), stronger interactions between
GAI and Atsly1 variants. d, Accumulation of immuno-detectable AtSLY1 and
Atsly1 variants in yeast. GAI is tagged with HA, SLY1 and sly1 variants are tagged Nature Plants Article https://doi.org/10.1038/s41477-023-01556-0 Article
https://doi.org/10.1038/s41477-023-01556-0
Extended Data Fig. 3 | AtSLY1 structural predictions. a, AlphaFold AtSLY1
structural prediction shown in 90o rotation and color-coded according to pLDDT
confidence levels. At right is the predicted aligned error for the structural model. The colour at position (x, y) indicates AlphaFold’s expected position error at
residue x, when the predicted and true structures are aligned on residue y. https://doi.org/10.1038/s41477-023-01556-0 b, AtSLY1 structural alignment between the AlphaFold model (darker shades)
and the top RoseTTAFold (lighter shades) model shown in 90˚ rotation and
color-coded according to region: helical F-box (cyan), GGF (brown), and LSL helix
(purple). The N-terminal 25 residues, which are predicted to be unstructured, are
not shown. c, AtSLY1 structural alignment between the AlphaFold (darker shades)
and ESMfold (lighter shades) models shown in 90˚ rotation and color-coded as
in b. The N-terminal 25 residues, which are predicted to be unstructured, are not
shown. Extended Data Fig. 3 | AtSLY1 structural predictions. a, AlphaFold AtSLY1
structural prediction shown in 90o rotation and color-coded according to pLDDT
confidence levels. At right is the predicted aligned error for the structural model. The colour at position (x, y) indicates AlphaFold’s expected position error at
residue x, when the predicted and true structures are aligned on residue y. b, AtSLY1 structural alignment between the AlphaFold model (darker shades)
and the top RoseTTAFold (lighter shades) model shown in 90˚ rotation and
color-coded according to region: helical F-box (cyan), GGF (b
(purple). The N-terminal 25 residues, which are predicted to
not shown. c, AtSLY1 structural alignment between the Alpha
and ESMfold (lighter shades) models shown in 90˚ rotation a
in b. The N-terminal 25 residues, which are predicted to be un
shown. Extended Data Fig. 3 | AtSLY1 structural predictions. a, AlphaFold AtSLY1
structural prediction shown in 90o rotation and color-coded according to pLDDT
confidence levels. At right is the predicted aligned error for the structural model. The colour at position (x, y) indicates AlphaFold’s expected position error at
residue x, when the predicted and true structures are aligned on residue y. b, AtSLY1 structural alignment between the AlphaFold model (darker shades)
and the top RoseTTAFold (lighter shades) model shown in 90˚ rotation and
color-coded according to region: helical F-box (cyan), GGF (brown), and LSL h
(purple). The N-terminal 25 residues, which are predicted to be unstructured,
not shown. c, AtSLY1 structural alignment between the AlphaFold (darker shad
and ESMfold (lighter shades) models shown in 90˚ rotation and color-coded a
in b. The N-terminal 25 residues, which are predicted to be unstructured, are n
shown. color-coded according to region: helical F-box (cyan), GGF (brown), and LSL helix
(purple). The N-terminal 25 residues, which are predicted to be unstructured, are
not shown. https://doi.org/10.1038/s41477-023-01556-0 c, AtSLY1 structural alignment between the AlphaFold (darker shades)
and ESMfold (lighter shades) models shown in 90˚ rotation and color-coded as
in b. The N-terminal 25 residues, which are predicted to be unstructured, are not
shown. color-coded according to region: helical F-box (cyan), GGF (brown), and LSL helix
(purple). The N-terminal 25 residues, which are predicted to be unstructured, are
not shown. c, AtSLY1 structural alignment between the AlphaFold (darker shades)
and ESMfold (lighter shades) models shown in 90˚ rotation and color-coded as
in b. The N-terminal 25 residues, which are predicted to be unstructured, are not
shown. Extended Data Fig. 3 | AtSLY1 structural predictions. a, AlphaFold AtSLY1 Extended Data Fig. 3 | AtSLY1 structural predictions. a, AlphaFold AtSLY1
structural prediction shown in 90o rotation and color-coded according to pLDDT
confidence levels. At right is the predicted aligned error for the structural model. The colour at position (x, y) indicates AlphaFold’s expected position error at
residue x, when the predicted and true structures are aligned on residue y. b, AtSLY1 structural alignment between the AlphaFold model (darker shades)
and the top RoseTTAFold (lighter shades) model shown in 90˚ rotation and Nature Plants https://doi.org/10.1038/s41477-023-01556-0 Article Extended Data Fig. 4 | Expected frequencies and GAI interactions of Atsly1
variants. a, Frequencies of specific nucleotide substitutions generated by
EP-PCR. b, Example calculation of expected frequencies of EP-PCR generated
amino acid substitutions at position H104 of AtSLY1 in the absence of selection. Individual nucleotide substitutions are shown in red. Resultant expected amino
acid substitutions (percentages) and actual numbers (out of 8 events) are as
shown. Whilst H104R (red) and H104L (orange) are expected most frequently,
H104Y (blue) predominates. c, d, Yeast-2-hybrid analysis of interactions between
GAI and Atsly1 variants (as shown). e, AtSLY1 K126 intermolecular contacts in the
AlphaFold predicted complex with GAI. The K126 sidechain of AtSLY1 (purple)
and the sidechains of all GAI (grey) residues with atoms ≤5 Å from K126 are shown
as sticks. f, As in e, but for E138. shown. Whilst H104R (red) and H104L (orange) are expected most frequently,
H104Y (blue) predominates. c, d, Yeast-2-hybrid analysis of interactions between
GAI and Atsly1 variants (as shown). e, AtSLY1 K126 intermolecular contacts in the
AlphaFold predicted complex with GAI. The K126 sidechain of AtSLY1 (purple)
and the sidechains of all GAI (grey) residues with atoms ≤5 Å from K126 are shown
as sticks. https://doi.org/10.1038/s41477-023-01556-0 f, As in e, but for E138. shown. Whilst H104R (red) and H104L (orange) are expected most frequently,
H104Y (blue) predominates. c, d, Yeast-2-hybrid analysis of interactions between
GAI and Atsly1 variants (as shown). e, AtSLY1 K126 intermolecular contacts in the
AlphaFold predicted complex with GAI. The K126 sidechain of AtSLY1 (purple)
and the sidechains of all GAI (grey) residues with atoms ≤5 Å from K126 are shown
as sticks. f, As in e, but for E138. Extended Data Fig. 4 | Expected frequencies and GAI interactions of Atsly1
variants. a, Frequencies of specific nucleotide substitutions generated by
EP-PCR. b, Example calculation of expected frequencies of EP-PCR generated
amino acid substitutions at position H104 of AtSLY1 in the absence of selection. Individual nucleotide substitutions are shown in red. Resultant expected amino
acid substitutions (percentages) and actual numbers (out of 8 events) are as Extended Data Fig. 4 | Expected frequencies and GAI interactions of Atsly1
variants. a, Frequencies of specific nucleotide substitutions generated by
EP-PCR. b, Example calculation of expected frequencies of EP-PCR generated
amino acid substitutions at position H104 of AtSLY1 in the absence of selection. Individual nucleotide substitutions are shown in red. Resultant expected amino
acid substitutions (percentages) and actual numbers (out of 8 events) are as Fig. 4 | Expected frequencies and GAI interactions of Atsly1 Nature Plants https://doi.org/10.1038/s41477-023-01556-0 Article Article
https://doi.org/10.1038/s41477 023 01556
Extended Data Fig. 5 | SLY1 orthologue phylogenetic tree. Comprehensive
phylogenetic analysis of SLY1 orthologues from diverse species, showing
major SLY1/GID2, SLY2, Lycophyte/Gymnosperm SLY and Liverwort SLY1/GID2
clades. MpSLY1, SmSLY1, AtSLY1 and OsGID2 are highlighted (red arrows). A
related sequence (Mapoly0027s0014/MpSLY1L from M. polymorpha) is used as
outgroup. The tree was constructed in MEGA11 using the Maximum Likelihood
method and JTT matrix-based model. The tree is drawn to scale, with branch
lengths measured in the number of substitutions per site. related sequence (Mapoly0027s0014/MpSLY1L from M. polymorpha) is used as
outgroup. The tree was constructed in MEGA11 using the Maximum Likelihood
method and JTT matrix-based model. The tree is drawn to scale, with branch
lengths measured in the number of substitutions per site. Extended Data Fig. 5 | SLY1 orthologue phylogenetic tree. Comprehensive
phylogenetic analysis of SLY1 orthologues from diverse species, showing
major SLY1/GID2, SLY2, Lycophyte/Gymnosperm SLY and Liverwort SLY1/GID2
clades. MpSLY1, SmSLY1, AtSLY1 and OsGID2 are highlighted (red arrows). A Nature Plants https://doi.org/10.1038/s41477-023-01556-0 Article ature Plants
xtended Data Fig. https://doi.org/10.1038/s41477-023-01556-0 6 | See next page for caption. Extended Data Fig. 6 | See next page for caption. Extended Data Fig. 6 | See next page for caption. Nature Plants Extended Data Fig. 6 | Basal SLY1s exhibit broader DELLA affinities.
a, Yeast-2-hybrid analysis of interactions between SLY1 and DELLA orthologues
from various species (as indicated). b, Yeast-3-hybrid analysis of interactions
between SLY, GID1 and DELLA orthologues from various species (as indicated).
c, in vitro analysis of interactions between E.coli-expressed His-tagged AtGAI and
MBP-tagged SLY1/GID2s. Anti-His serves as control and confirms that similar
amounts of AtGAI protein were used to ‘pull down’ a SLY1/GID2 protein in each
immunoprecipitation (IP) reaction, whilst anti-MBP shows how much SLY1/GID2
(quantified against anti-His, with MpSLY1 arbitrarily set at 1.00) was pulled down. Article Article d, in vitro analysis of interactions between E.coli-expressed His-tagged AtGAI and
MBP-tagged SLY1/GID2s in the presence and absence of GST-AtGID1C and GA3. Anti-His serves as control and confirms that similar amounts of AtGAI protein
were used to ‘pull down’ a SLY1/GID2 protein in each immunoprecipitation (IP)
reaction, whilst anti-MBP shows how much SLY1/GID2 (quantified against anti-
His, with SLY1/GID2 in the absence of GST-GID1C and GA3 arbitrarily set at 1.00)
was pulled down. e, Mean plant heights, red dots indicate individual heights
(n = 10), error bars indicate standard deviation, different letters (a and b) indicate
significant difference (one-way ANOVA with Tukey’s test). Extended Data Fig. 6 | Basal SLY1s exhibit broader DELLA affinities. a, Yeast-2-hybrid analysis of interactions between SLY1 and DELLA orthologues
from various species (as indicated). b, Yeast-3-hybrid analysis of interactions
between SLY, GID1 and DELLA orthologues from various species (as indicated). c, in vitro analysis of interactions between E.coli-expressed His-tagged AtGAI and
MBP-tagged SLY1/GID2s. Anti-His serves as control and confirms that similar
amounts of AtGAI protein were used to ‘pull down’ a SLY1/GID2 protein in each
immunoprecipitation (IP) reaction, whilst anti-MBP shows how much SLY1/GID2
(quantified against anti-His, with MpSLY1 arbitrarily set at 1.00) was pulled down. ended Data Fig. 6 | Basal SLY1s exhibit broader DELLA affiniti Nature Plants https://doi.org/10.1038/s41477-023-01556-0 Article Extended Data Fig. 7 | Sequence alignment of selected SLY1 orthologues. Alignment of SLY1 orthologue amino-acid sequences from various species (as
indicated) showing regions of conservation (GGF and LSL domains) and positions
affected in screens for enhanced A affinity. Black highlights identical amino-
acids, grey highlights amino acids that are similar. Red boxes highlight sites in
AtSLY1 targeted by selected amino-acid substitutions (and equivalent sites in
SLY1s from SLY1/GID2 and liverwort SLY1/GID2 clades in Extended Data Fig. 5). Extended Data Fig. 7 | Sequence alignment of selected SLY1 orthologues. acids, grey highlights amino acids that are similar. Red boxes highlight sites in Extended Data Fig. 7 | Sequence alignment of selected SLY1 orthologues. Extended Data Fig. 7 | Sequence alignment of selected SLY1 orthologues. Extended Data Fig. 7 | Sequence alignment of selected SLY1 orthologues. acids, grey highlights amino acids that are similar. Red boxes highlight sites in
AtSLY1 targeted by selected amino-acid substitutions (and equivalent sites in
SLY1s from SLY1/GID2 and liverwort SLY1/GID2 clades in Extended Data Fig. 5). Article Alignment of SLY1 orthologue amino-acid sequences from various species (as
indicated) showing regions of conservation (GGF and LSL domains) and positions
affected in screens for enhanced A affinity. Black highlights identical amino- Nature Plants https://doi.org/10.1038/s41477-023-01556-0 icle
https://doi.org/10.1038/s41477-023-0155
nded Data Fig. 8 | See next page for caption. Article Extended Data Fig. 8 | See next page for caption. Nature Plants with Tukey’s test). d, Interactions of MpSLY1 and AtSLY1 with 10 representative
Arabidopsis GRAS family proteins. e, Interactions of AtSLY1 and Atsly1 variants
with AtSCR. f, Interactions of AtSLY1 and Atsly1 variants with AtSCL4.
g, Interactions of AtSLY1 and Atsly1 variants with GAI. h, Example mutant screen
plates, mSmSLY1 (left), mAtSLY1 (centre), mOsGID2 (right). Positive candidates
(blue colonies) are indicated with arrows. https://doi.org/10.1038/s41477-023-01556-0 with Tukey’s test). d, Interactions of MpSLY1 and AtSLY1 with 10 representative
Arabidopsis GRAS family proteins. e, Interactions of AtSLY1 and Atsly1 variants
with AtSCR. f, Interactions of AtSLY1 and Atsly1 variants with AtSCL4. g, Interactions of AtSLY1 and Atsly1 variants with GAI. h, Example mutant screen
plates, mSmSLY1 (left), mAtSLY1 (centre), mOsGID2 (right). Positive candidates
(blue colonies) are indicated with arrows. Extended Data Fig. 8 | SLY1 reversion routes are evolutionarily constrained. a, Interactions of AtSLY1 and Atsly1 variants or MpSLY1 and Mpsly1 variants
with GAI. b, Interactions of MpSLY1 and Mpsly1 variants with MpDELLA. c, Mean
yeast-2-hybrid interaction strengths, Mpsly1 variants as in c, note gradient of
increasing interaction strength ranging from Mpsly1Y108H to MpSLY1. ND = not
detected; red dots indicate individual values (n = 3), error bars indicate standard
deviation, different letters (a-d) indicate significant difference (one-way ANOVA Extended Data Fig. 8 | SLY1 reversion routes are evolutionarily constrained. a, Interactions of AtSLY1 and Atsly1 variants or MpSLY1 and Mpsly1 variants
with GAI. b, Interactions of MpSLY1 and Mpsly1 variants with MpDELLA. c, Mean
yeast-2-hybrid interaction strengths, Mpsly1 variants as in c, note gradient of
increasing interaction strength ranging from Mpsly1Y108H to MpSLY1. ND = not
detected; red dots indicate individual values (n = 3), error bars indicate standard
deviation, different letters (a-d) indicate significant difference (one-way ANOVA Nature Plants
|
https://openalex.org/W3203339899
|
http://jahe.or.id/index.php/jahe/article/download/27/pdf
|
Indonesian
| null |
Penyuluhan Pembelajaran Literasi Kritis Bagi Guru SMPN di Kota Banjarmasin
|
Journal of Human and Education
| 2,021
|
cc-by-sa
| 4,203
|
Abstrak Kompetensi literasi kritis siswa SMP di Kota Banjarmasin tergolong rendah. Hasil penelitian
menunjukkan bahwa gejala itu disebabkan oleh sejumlah faktor diantaranya adalah sistem
pembelajaran literasi yang digunakan oleh guru. Ketika mengajar mereka terfokus memberikan
kompetensi pengetahuan tentang struktur dan makna teks. Akibatnya, siswa mengalami kesulitan
ketika disuruh merespon teks itu dan menggunakannya untuk menjawab persoalan kehidupannya
secara kritis. Kegiatan penyuluhan ini dilakukan untuk mengatasi masalah itu. Penyuluhan ini
dilakukan selama 30 jam yang dilakukan dengan daring melalui zoom. Materi yang diberikan kepada
para guru bahasa Indonesia, antara lain: (a) hakikat literasi kritis dan model-model pembelajaran
literasi kritis; (b) bahan ajar literasi kritis dan; dan (c) penilaian literasi kritis. Setelah dilakukan
penyuluhan, guru diminta respon dan dilakukan evaluasi untuk mengukur penguasaan mereka
terhadap materi penyuluhan. Para guru merespon kegiatan penyuluhan sangat bermanfaat dan
menyarankan untuk bisa dilakukan lebih lanjut. Sementara itu, dari sisi penguasaan, para guru sangat
menguasai materi yang diberikan narasumber. Dengan bekal materi penyuluhan itu, guru-guru akan
dapat mengarahkan pembelajaran literasi kritis secara tepat, baik dari model pembelajaran, materi
yang mereka ajarkan di kelas, maupun sistem penilaian yang digunakan dalam pembelajaran literasi
kritis sesuai dengan konteks siswa SMP di Kota Banjarmasin. Kata Kunci: Penyuluhan, Literasi, Kritis Penyuluhan Pembelajaran Literasi Kritis Bagi Guru SMPN
Di Kota Banjarmasin Jumadi 1, Faradina 2, Lita luthfiyanti3
1,2,3,4,5 Universitas Lambung Mangkurat
Email: jumadi@ulm.ac.id1, faradina@ulm.ac.id2, lita.luthfiyanti@ulm.ac.id3 JAHE Volume 1 Nomor 2 Tahun 2021 Halaman 42-51
Journal of Human and Education
Research & Learning in Primary Education PENDAHULUAN Makna dan konsep literasi dewasa ini
semakin berkembang. Dahulu literasi diartikan
sebagai kemampuan membaca dan menulis
sebagaimana
UNESCO
tahun
1957
yang
mendefinisikan literacy sebagai “person is
literate who can with understanding both read
and write a short simple statement on his (her)
everyday life” (2008). Namun, sekarang literasi
diartikan sebagai keterampilan membaca kata
dan dunia dengan kaitannya dalam memecahkan
masalah kehidupan. Definisi kontemporer pada
literasi ini menandai konsep baru perkembangan
literasi yang diungkapkan oleh Paulo Freire. Dari sinilah literasi kritis mulai digali dan
menemukan bentuknya sesuai dengan konteks
istilah ini digunakan. Teori literasi kritis
memandang literasi sebagai suatu tindakan
pemahaman yang memberdayakan seseorang
dengan membantunya menemukan suara mereka
dan tanggung jawab etik untuk memperbaiki
dunia mereka. Dengan kata lain, literasi adalah
kemampuan untuk memberdayakan kritik dan
analisis dengan cara-cara pengetahuan dan cara-
cara berpikir tentang dan menilai pengetahuan,
dikonstruksi di dalam dan melalui teks tertulis
(Hammond
dan
Macken-Horarik
dalam
Abednia, 2015). Kita sekarang berada pada era revolusi
industri 4.0. Pada era ini muncul berbagai
temuan
bidang
teknologi
seperti
robotik,
kecerdasan buatan, nanoteknologi, komputasi
kuantum, bioteknologi, Industry Internet of
Things (IioT), teknologi nirkabel generasi
kelima,
aditif
manufaktur,
dan
industri
kendaraan
otonomi
penuh
(Latif,
2020). Berbagai
temuan
itu
semakin
memicu
berkembangnya
teknologi
informasi
yang
memfasilitasi ketersediaan data melimpah (big
data) yang aksesnya mampu melintas batas
ruang dan waktu. Berbagai temuan itu bisa membawa
dampak positif yang sekaligus juga dampak
negatif. Dampak positif berkaitan dengan
ketersediaan sarana teknologi untuk kemudahan
akses informasi. Sementara itu, dampak negatif
terkait serbuan informasi yang tidak akurat
semakin hari semakin sulit dibendung sehingga
perlu kompetensi untuk memilah informasi. Peringatan tentang pentingnya memilih dan
memilah informasi secara akurat dapat disimak
dalam buku Everybody Lies (Davidowitz, 2019). Gejala di atas berdampak kepada dunia
pendidikan kita. Dunia pendidikan perlu perlu
generasi unggul agar mereka bisa kreatif,
inovatif, dan adaptif dalam menjalani kehidupan
di era industri 4.0. Banyak hal yang perlu
dilakukan,
yang
satu
diantaranya
adalah
pemberian kompetensi literasi kritis kepada
siswa. Kompetensi ini merupakan kompetensi
dasar yang akan membangun dan memperkokoh
sejumlah kompetensi yang lain. Karena begitu
pentingnya
kepemilikan
kompetensi
ini,
UNESCO dengan tegas menyatakan bahwa
literasi merupakan sarana yang penting bagi
efektivitas partisipasi ekonomi dan sosial. Bahkan,
literasi
memberi
andil
untuk
mengembangkan kemanusiaan dan mengurangi
kemiskinan. Literasi bisa mendorong dan
memelihara
kebersamaan
sosial
dan Ada
sejumlah
lembaga
internasional
melakukan
pemeringkatan
pencapaian
pendidikan suatu negara, yang di dalamnya
terkait dengan kompetensi literasi siswa. Abstract The critical literacy competence of junior high school students in Banjarmasin is low. The results
showed that the symptoms were caused by a number of factors including the literacy learning system
used by the teacher. When teaching them focus provides competency knowledge about the structure
and meaning of the text. As a result, students have difficulty when asked to respond to the text and use
it to answer critical life problems. This outreach activity was carried out to overcome this problem. This training was conducted for 30 hours online via zoom. The materials given to Indonesian
language teachers include: (a) the nature of critical literacy and critical literacy learning models; (b)
critical literacy teaching materials; and (c) critical literacy assessment. After the training, teachers
were asked to respond and answer questions to measure their mastery of the training material. The
teachers responded that the extension activities were very useful and suggested that further activities
could be done. Meanwhile, in terms of mastery, the teachers are very familiar with the material given
by the resource persons. With the provision of training materials, teachers will be able to direct
critical literacy learning appropriately, both from the learning model, the material they teach in class,
as well as the assessment system used in critical literacy learning in accordance with the context of
junior high school students in Banjarmasin City. Keywords: Training, Literacy, Critical. berkontribusi untuk implementasi hak asasi
manusia secara adil. PENDAHULUAN Satu di
antara lembaga itu yang dianut oleh banyak
negara di dunia adalah OECD (Organization for
Economic
Cooperation
and
Development). Lembaga
ini
mengadakan
pemeringkatan
pendidikan yang mereka beri nama PISA
(Program for International Student Assessment). Pemeringkatan dari PISA dilaksanakan setiap
tiga
tahun
sekali. Tujuannya
adalah
memperlihatkan tingkat literasi siswa yang
berusia 15 tahun di suatu negara. Ada tiga
literasi yang mereka ukur, yakni literasi JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 sebagai bagian dari kepemilikan kompetensi
literasi kritis. membaca, matematika, dan sains. Kompetensi
literasi siswa di suatu negara dianggap sudah
baik
jika
memiliki
skor
rata-rata
500
(Kompasiana, 2016). Permasalahan di atas tentu saja perlu
diatasi dengan memberikan pemahaman guru
tentang pembelajaran literasi secara tepat. Atas
dasar itu, penyuluhan literasi kritis kepada guru-
guru tersebut dilakukan. Dengan penyuluhan ini
diharapkan: (a) para guru SMPN di Kota
Banjarmasin dapat memperoleh pemahaman
tentang metode pembelajaran literasi kritis
secara
tepat
agar
dapat
meningkatkan
kemampuan mengajar literasi kritis kepada para
siswa; (b) memilih materi yang tepat dalam
proses pembelajaran literasi kritis; dan (c) dapat
memilih sistem evaluasi yang tepat dalam proses
pembelajaran literasi kritis. Sejak ikut pertama kali tahun 2000, skor
rata-rata literasi siswa di Indonesia berada di
bawah
peringkat
bawah. Tahun-tahun
selanjutnya skornya juga belum menunjukkan
peningkatan. Pada tahun 2018, misalnya, nilai
PISA cukup menyedihkan karena Indonesia
masih belum beranjak dari papan bawah. Secara
berturut-turut,
nilai
untuk
membaca,
matematika, dan sains dari hasil tes pada 2018
adalah 371, 379, dan 396. Nilai ini mengalami
penurunan dibanding tes pada tahun 2015, yang
secara berturut-turut membaca, matematika, dan
sains para siswa di Indonesia meraih skor 397,
386, 403. Dari semua skor itu, membaca
memiliki penurunan skor terendah, di bawah
skor di tahun 2012, yaitu 396 (Dewabrata,
2019). METODE Kegiatan penyuluhan ini dilaksanakan
melalui prosedur sebagai berikut. Pada tahap
awal, tim pelaksana melakukan orientasi awal
dengan melakukan wawancara dengan beberapa
guru
bahasa
Indonesia
SMP
di
Kota
Banjarmasin. Dari
hasil
wawancara
itu
terungkap
bahwa
mereka
belum
pernah
mendapat penyuluhan secara khusus tentang
pembelajaran literasi kritis. Mereka sangat
berharap bisa mendapatkan penyuluhan untuk
Informasi itu kami gunakan untuk melakukan
koordinasi dengan Ketua MGMP bidang Studi
Bahasa Indonesia di Kota Banjarmasin. Dari
hasil koordinasi itu Ketua MGMP sangat senang
dan menyambut baik jika dilakukan penyuluhan
pembelajaran literasi kritis. Akhirnya, kami
bersepakat
tentang
materi
dan
waktu
pelaksanaan penyuluhan. Bagaimana fakta di lapangan? Jumadi et
al (2020) menjelaskan bahwa dari tujuh sekolah
yang dijadikan sampel penelitian menunjukkan
skor rata-rata kompetensi literasi para siswa
sebesar 57,23 yang tergolong rendah. Hal ini
menunjukkan bahwa hasil PISA tidak jauh
berbeda dengan fakta di lapangan sebagaimana
tercermin dari hasil penelitian ini. Dari temuan penelitian di atas terungkap
bahwa
rendahnya
kompetensi
tersebut
disebabkan oleh sejumlah faktor, yakni: (a)
materi pembelajaran literasi kritis masih terfokus
kepada pemahaman struktur dan isi teks. Materi
yang diajarkan belum menyentuh kepada telaah
kritis terhadap teks dan implikasi dari isi teks itu
untuk
keperluan
kehidupan
bermasyarakat,
berbangsa, dan bernegara; (b) pembelajaran
literasi kritis tidak diajarkan secara khusus; (d)
tidak ada latihan khusus sebagai sarana
memupuk kompetensi literasi siswa; dan (d)
evaluasi yang ditanyakan belum mengarah
kepada penggalian berpikir tingkat tinggi Tahap berikutnya, tim mengurus surat izin
untuk
melakukan
penyuluhan. Dekan
membuatkan surat izin yang ditujukan kepada
Ketua MGMP. Akhirnya, secara resmi Ketua
MGMP memberikan izin kepada tim untuk
melakukan penyuluhan kepada guru-guru bahasa
Indonesia SMP di Kota Banjarmasin. Ketua
MGMP menyebarkan pengumuman kepada JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 HASIL DAN PEMBAHASAN sekolah-sekolah agar para guru bahasa Indonesia
mengikuti penyuluhan sesuai jadwal yang sudah
disepakati. Secara keseluruhan, ada sejumlah
sekolah baik dari sekolah negeri maupun swasta
yang
mengirimkan
perwakilannya
untuk
mengikuti penyuluhan. Secara keseluruhan ada
34 guru yang mengikuti penyuluhan. Penyajian
Setelah berbagai hal yang diperlukan
untuk kegiatan penyuluhan selesai dipersiapkan,
kegiatan penyuluhan dilaksanakan sesuai jadwal. Pada hari pertama, kegiatan diawali dengan
kegiatan pembukaan dengan mendengarkan
sambutan ketua MGMP bahasa Indonesia SMP
se-Kota Banjarmasin. Kegiatan dilanjutkan
penyajian materi hingga kegiatan penutup terkait
dengan
materi
hakikat
dan
karakteristik
pembelajaran literasi kritis. Pada, hari-hari
berikutnya, materi dan kegiatan dilaksanakan
sesuai jadwal sebagaimana disajikan dalam
Tabel 1 di atas. Berikut ini salah foto yang
menggambarkan pelaksanaan penyuluhan secara
d i
l l i
lik
i Z Penyuluhan ini dilaksanakan secara daring
(online) dengan menggunakan aplikasi Zoom,
Google Meet, dan WA yang telah disediakan
oleh tim. Kegiatan penyuluhan ini berlangsung
selama kurang lebih 30 jam. Jadwal dan materi
penyuluhan dapat dilihat pada tabel berikut ini. penyuluhan dapat dilihat pada tabel berikut ini. Tabel 1
Jadwal dan Materi Penyuluhan
No. Hari
Waktu
Materi
Penyajian
1. Pertama
9.00-9.15
9.15-9.30
9.30-11.30
11.30-12.15
12.15-12.30
• Pembukaan
• Sambutan ketua MGMP
• Hakikat dan Karakteristik Pembelajaran
Literasi Kritis
• Tanya jawab/diskusi
• Penutup
Zoom
2. Kedua
9.00-11.30
11.30-12.15
12.15-12.30
• Model-model pembelajaran literasi kritis
•
Tanya jawab/diskusi
• Penutup
Zoom
3. Ketiga
9.00-11.30
11.30-12.15
12.15-12.30
• Pemilihan bahan ajar literasi kritis
• Tanya jawab/diskusi
• Penutup
Zoom
4. Keempat
9.00-11.30
11.30-12.15
12.15-12.30
• Sistem
evaluasi
berbasis
HOT
dan
authentic assessment
• Tanya jawab/diskusi
• Penutup
Zoom
5. Kelima
9.00-10.00
• Survey kepuasan dan pemahaman
• Tes penguasaan materi penyuluhan
Google
Meet
Penyuluhan ini dilaksanakan
dengan
metode ceramah, diskusi, dan penugasan. Metode ceramah digunakan dalam penyajian
materi oleh tim. Para guru menyimak materi
yang disajikan dalam power point yang
ditayangkan penyuluh secara daring. Metode
diskusi digunakan untuk materi kasus yang
harus dipecahkan oleh peserta. Sementara itu,
metode
penugasan
digunakan
untuk
menyampaikan
suatu
tugas
yang
harus
dikerjakan oleh para peserta. sesuai jadwal sebagaimana disajikan dalam
Tabel 1 di atas. Berikut ini salah foto yang
menggambarkan pelaksanaan penyuluhan secara
daring melalui aplikasi Zoom. Gambar 1. Penyampaian Materi
Penyuluhan Melalui Zoom
Dari Gambar 1 tampak bahwa pemateri
sedang
menyampaikan
materi
penyuluhan
terkait dengan perlunya pembelajaran literasi
kritis di sekolah. HASIL DAN PEMBAHASAN Dari materi ini diharapkan guru
memiliki pemahaman yang komprehensif bahwa
pembelajaran membaca di sekolah hendaknya
bukan hanya membahas tentang struktur teks,
tetapi yang juga penting diberikan adalah
pembelajaran
literasi
kritis
sehingga
pembelajaran bahasa di sekolah bisa membekali
siswa agar bisa adaptif dengan tuntutan revolusi
4.0. Hari-hari berikutnya penyampaian materi
sebagaimana
diatur
dalam
jadwal. Untuk
mengaktifkan peserta penyuluhan, penyaji juga
menggunakan metode diskusi dan Tanya jawab. Gambar berikut menunjukkan hal itu. Tabel 1 Tabel 1
Jadwal dan Materi Penyuluhan
No. Hari
Waktu
Materi
Penyajian
1. Pertama
9.00-9.15
9.15-9.30
9.30-11.30
11.30-12.15
12.15-12.30
• Pembukaan
• Sambutan ketua MGMP
• Hakikat dan Karakteristik Pembelajaran
Literasi Kritis
• Tanya jawab/diskusi
• Penutup
Zoom
2. Kedua
9.00-11.30
11.30-12.15
12.15-12.30
• Model-model pembelajaran literasi kritis
•
Tanya jawab/diskusi
• Penutup
Zoom
3. Ketiga
9.00-11.30
11.30-12.15
12.15-12.30
• Pemilihan bahan ajar literasi kritis
• Tanya jawab/diskusi
• Penutup
Zoom
4. Keempat
9.00-11.30
11.30-12.15
12.15-12.30
• Sistem
evaluasi
berbasis
HOT
dan
authentic assessment
• Tanya jawab/diskusi
• Penutup
Zoom
5. Kelima
9.00-10.00
• Survey kepuasan dan pemahaman
• Tes penguasaan materi penyuluhan
Google
Meet
sesuai jadwal sebagaimana disajikan dalam
Tabel 1 di atas. Berikut ini salah foto yang
menggambarkan pelaksanaan penyuluhan secara
daring melalui aplikasi Zoom. Gambar 1. Penyampaian Materi
Penyuluhan Melalui Zoom
Dari Gambar 1 tampak bahwa pemateri
sedang
menyampaikan
materi
penyuluhan
terkait dengan perlunya pembelajaran literasi
kritis di sekolah. Dari materi ini diharapkan guru
iliki
h
k
h
if b h Penyuluhan ini dilaksanakan
dengan
metode ceramah, diskusi, dan penugasan. Metode ceramah digunakan dalam penyajian
materi oleh tim. Para guru menyimak materi
yang disajikan dalam power point yang
ditayangkan penyuluh secara daring. Metode
diskusi digunakan untuk materi kasus yang
harus dipecahkan oleh peserta. Sementara itu,
metode
penugasan
digunakan
untuk
menyampaikan
suatu
tugas
yang
harus
dikerjakan oleh para peserta. JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 Diagram 1. Persepsi Pentingnya
Penyuluhan Dilakukan Gambar 2. Peserta Penyuluhan
Melakukan Diskusi Secara Daring Melalui
Zoom. Diagram 1. Persepsi Pentingnya
Penyuluhan Dilakukan Diagram di atas menunjukan 74,3% guru
menganggap penyuluhan yang mereka ikuti
sangat penting; 22,9 persen penting; hanya
sekitar 7% cukup penting. Jadi, hampir semua
guru menganggap penyuluhan ini penting bagi
mereka. Sementara
itu,
dilihat
dari
sisi
menyampaikan materi, para guru menggap
bahwa
penyampaian
materi
sangat
jelas. Indikatornya mereka memahami materi yang
disampaikan narasumber. Diagram 2 berikut ini
menunjukkan fakta itu. Gambar 2. Peserta Penyuluhan
Melakukan Diskusi Secara Daring Melalui
Zoom. Untuk mengukur ketercapaian kegiatan
penyuluhan, pada akhir penyuluhan para guru
diminta mengisi angket dan mengerjakan tes
melalui google form. Angket digunakan untuk
melihat respon para guru terhadap kegiatan
penyuluhan
tersebut. Sementara
itu,
tes
digunakan untuk mengukur pemahaman guru
terhadap materi yang sudah disajikan dan
didiskusikan dalam penyuluhan. Diagram 2. Persepsi Penyampaian Materi Persepsi Guru Setelah Mengikuti Penyuluhan Ada dua pertanyaan yang dipakai untuk
menggali persepsi para peserta penyuluhan,
yaitu pertanyaan tentang pentingnya mengikuti
penyuluhan sebagai bekal pembelajaran di
sekolah; dan pertanyaan tentang jelas tidak
penyampaian
materi
penyuluhan. Respon
mereka dapat dilihat pada diagram berikut ini. Diagram 2. Persepsi Penyampaian Materi Dari Diagram 2 tampak bahwa para guru
sebanyak 60% menyatakan sangat mudah
memahami materi penyuluhan dan 40% mudah
memahami. Dengan demikian, penyampaian
materi penyuluhan mudah dipahami sehingga
akan mengubah pemahaman dan perilaku dalam
proses pembelajaran literasi kritis di sekolah. Penguasaan Guru Terhadap Materi Penyuluhan p
p
j
Penguasaan Guru Terhadap Materi Penyuluhan Ada beberapa hal yang dinilai untuk
mengukur guru, di antaranya pemahaman Ada beberapa hal yang dinilai untuk
mengukur guru, di antaranya pemahaman JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 kompetensi literasi kritis para siswa SMP di
Kota
Banjarmasin.,
yang
dalam
hal
ini
kompetensi literasi para siswa mereka. Diagram
berikut menggambarkan pemahaman itu. benar. Disamping pemahaman tentang konsep
literasi, guru juga diberi pemahaman tentang
metode pembelajaran literasi yang menekankan
kepada aktivitas belajar berkolaborasi. Data
berikut menunjukkan pemahaman guru ketika
nanti
melakukan
pembelajaran
literasi
di
sekolah. Grafik 6: Data Keterampilan Guru
Mengelola Kelas Diagram 4. Pemahaman Kompetensi Diagram 4. Pemahaman Kompetensi
Literasi Kritis Siswa SMP di Kota Banjarmasin Diagram 4. Pemahaman Kompetensi
Literasi Kritis Siswa SMP di Kota Banjarmasin Literasi Kritis Siswa SMP di Kota Banjarmasin Diagram 4 menunjukkan bahwa setelah
mengikuti penyuluhan, para guru sebanyak
66,7%
menjawab
benar,
yakni
tingkat
kemampuan literasi kritis siswa SMP di Kota
Banjarmasin tergolong rendah. Terkait dengan
konsep literasi kritis, pada awalnya, para guru
belum mengerti benar hakikat literasi kritis itu. Selama ini para guru sering menganggap bahwa
literasi kritis adalah literasi membaca seperti
biasa. Padahal, menurut konsep literasi kritis
sudah berkembang, bukan sekadar membaca
begitu saja. Setelah mengikuti penyuluhan ini
para guru memiliki pemahaman yang benar. Data berikut dalam menunjukkan hal itu. Grafik 6: Data Keterampilan Guru
Mengelola Kelas Data di atas menunjukkan bahwa guru
sudah dapat mengelola secara benar ketika
nantinya melakukan pembelajaran literasi kritis. Pembelajaran literasi kritis menghendaki siswa
aktif memaknai dan menggunakan teks untuk
kehidupan. Pembelajaran literasi kritis ini
diilhami pandangan pendidikan kritis (lihat
Thelin, 2005; Langeveld dalam Ibrahim,
2017). Pembelajaran seperti itu sudah mereka
kuasai. Diagram 5: Tingkat Pemahaman Literasi
Kritis Ketika
ditanya
terkait
pemilihan
pembelajaran,
mereka
tampaknya
belum
menguasai dengan baik. Topik pembelajaran
literasi kritis berbasis kepada masalah. Dengan
dasar itu, diharapkan siswa memiliki kompetensi
memecahkan
berbagai
persoalan
yang
ditemukan dalam kehidupan. Ketika disuruh
memilih topik yang bisa dijadikan materi
pembelajaran literasi kritis, ternyata persentase
yang menjawab benar dan salah seimbang. Grafik 7 menunjukkan bahwa guru yang
memilih topik pembelajaran secara benar Diagram 5: Tingkat Pemahaman Literasi
Kritis Grafik 5 di atas memperlihatkan tingkat
pemahaman para guru tentang konsep literasi
kritis yang benar. Sebanyak 72,7% peserta sudah
memahami konsep literasi kritis secara benar. Literasi kritis berkaitan dengan proses
mengembangkan kapasitas diri (efikasi diri)
untuk membaca situasi serta diiringi sebuah
sikap
pencarian
untuk
mempengaruhi
perubahan sosial yang positif (lihat Cooper
& White, 2008). Pemahaman ini sebagai modal
dalam melakukan pembelajaran literasi secara JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 Mclaughlin dan Allen (2000). Model ini
memiliki prosedur atau langkah-langkah
pembelajaran. Ada 5 prosedur yang harus
dilakukan
guru
ketika
melakukan
pembelajaran
literasi
kritis,
yakni:
menjelaskan,
demonstrasi,
bimbingan,
praktik,
dan
refleksi. Masing-masing
langkah itu memiliki kegiatan yang harus
dilakukan baik oleh guru maupun siswa. Intinya,
model
pembelajaran
ini
menekankan siswa aktif untuk menelaah
teks dan memanfaatkan teks tersebut untuk
memecahkan problem kehidupan. sebesar 48,5% yang persentasenya sama dengan
salah. Namun, kompetensi ini diulas pada waktu
dilakukan refleksi sehingga pemahaman mereka
lebih baik daripada ketika dilakukan tes. Diagram 4. Pemahaman Kompetensi
Literasi Kritis Siswa SMP di Kota Banjarmasin Grafik 7: Data Kemampuan Memilih
Topik Pembelajaran
l
ilih
d
d
d l Grafik 7: Data Kemampuan Memilih
Topik Pembelajaran Grafik 7: Data Kemampuan Memilih
Topik Pembelajaran Grafik 7: Data Kemampuan Memilih Dalam pemilihan metode dan model
pembelajaran,
tampaknya
persentase
pemahaman guru sudah baik, walaupun masih
ada sejumlah guru yang belum memahaminya
dengan tepat. Data Grafik 8 berikut ini
menunjukkan hal itu. Kemampuan terakhir yang dinilai adalah
kompetensi
menggunakan
teknik
evaluasi. Teknik evaluasi dalam pembelajaran literasi
kritis mempunyai fungsi sangat penting. Hanya
dengan
sistem
evaluasi
yang
tepatlah
kompetensi literasi siswa dapat ditingkatkan. Data berikut menunjukkan pemahaman guru
terhadap soal yang cocok untuk menggali
kompetensi
literasi
siswa. Grafik 8: Kemampuan Memilih Metode
Pembelajaran Grafik 9: Kemampuan Memilih Sistem
Evaluasi Grafik 9: Kemampuan Memilih Sistem
Evaluasi Grafik 8: Kemampuan Memilih Metode
Pembelajaran Dari Grafik 9 tampak bahwa hampir
semua guru sudah dapat memilih sistem evaluasi
yang tepat untuk pembelajaran literasi kritis
sebesar 93,9%. Data ini menunjukkan bahwa
dari guru yang mengikuti pembelajaran, hanya 2
orang yang masih belum bisa. Penilaian literasi
kritis berbasis penilaian autentik dan HOT. Dengan demikian, hampir semua guru sudah
memiliki kemampuan menilai secara benar
dalam pembelajaran literasi kritis. Dari grafik di atas tampak ada 69,7% guru
yang sudah tepat dalam memilih metode
pembelajaran, sisanya masih ada yang belum
tepat memilih, tetapi jumlahnya sedikit. Pada
tahap refleksi, materi itu disampaikan lagi
sehingga pemahaman mereka pada aspek ini
lebih baik. Ada beberapa metode pembelajaran
yang disampaikan dalam penyuluhan. Salah satu
model itu di antaranya yang dikembangkan JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 Dari paparan hasil di atas menunjukkan
bahwa persepsi guru dan pemahaman mereka
terkait seluk-beluk pembelajaran literasi kritis
para peserta penyuluhan sudah baik. Hal ini
menunjukkan bahwa penyuluhan ini sudah
mampu meningkatkan kompetensi guru untuk
melaksanakan pembelajaran literasi kritis ke
arah yang lebih baik. Kepemilikan kompetensi
mengajar bagi guru sangat penting untuk
pencapaian hasil belajar. Hilman dan Dewi
(dalam Soadik et al, 2015) menyatakan bahwa
kompetensi guru adalah hasil dari penggabungan
dari kemampuan-kemampuan yang banyak
jenisnya, dapat berupa seperangkat pengetahuan,
keterampilan, dan perilaku yang harus dimiliki,
dihayati,
dan
dikuasai
oleh
guru
dalam
menjalankan tugas keprofesionalannya. Ada
sejumlah
prinsip
yang
perlu
dikembangkan
dalam
pembelajaran
bahasa
berbasis literasi kritis. Menurut Alwasilah
(2021) prinsip-prinsip itu mencakup: (a) literasi
adalah kecakapan hidup yang memungkinkan
manusia berfungsi maksimal sebagai anggota
masyarakat; (b) literasi mencakup kemampuan
reseptif dan produktif dalam upaya berwacana
secara secara tertulis dan lisan; (c) literasi adalah
kemampuan memecahkan masalah; (d) literasi
adalah refleksi penguasaan dan apresiasi budaya;
(e) literasi adalah kegiatan refleksi diri; (f)
literasi adalah hasil kolaborasi; dan (g) literasi
adalah kegiatan melakukan interpretasi. SIMPULAN Panti Asuhan Puteri Aisyiyah dibangun
situasi sosial berbasis nilai keagamaan. Dengan
demikian, diharapkan muncul perilaku prososial. Pada anak.Perilaku prososial adalah istilah untuk
menggambarkan
minat
dan
kesenangan
membantu orang lain. Anak-anak dan remaja
prososial akan lebih mudah beradaptasi dan
memiliki lebih banyak teman atau sahabat. Merekapun lebih terampil bekerjasama dengan
teman-teman dan gurunya, serta mendapatkan
tanggapan
yang
lebih
positif
dari
lingkungannya. Dapat dikatakan kemampuan
mengolah diri untuk berperilaku prososial sangat
menentukan prestasi mereka di masa depan. Pola
pengasuhan berbasis keagamaan melatihkan
keterampilan prososial melalui kegiatan bersama
yang
menyenangkan. Apakah
sekedar
berkumpul bersama di setiap aktivitas pengajian
mingguan,
makan,
hingga
waktu
luang
untuk tertawa bersama. Dengan
perubahan
kompetensi
itu
diharapkan juga terjadi perubahan pembelajaran
literasi kritis di sekolah ke arah yang lebih baik,
yang pada gilirannya kompetensi literasi kritis
siswa juga akan berubah ke arah yang lebih baik. Hal ini merupakan kompetensi yang sangat
penting untuk terus ditingkatkan. Banks (dalam
Zaini, 2015) menyatakan bahwa kemampuan
literasi
kritis
diperlukan
untuk
membuat
keputusan,
memecahkan
masalah,
dan
keterlibatan warga negara yang efektif. Lebih
dari itu, UNESCO (dalam Kemendikbud, 2017)
menyatakan cakupan literasi berkembang luas
yang meliputi: (a) literasi sebagai suatu
rangkaian kecakapan membaca, menulis, dan
berbicara, kecakapan berhitung, dan kecakapan
dalam mengakses dan menggunakan informasi;
(b)
literasi
sebagai
praktik
sosial
yang
penerapannya dipengaruhi oleh konteks; (c)
literasi sebagai proses pembelajaran dengan
kegiatan membaca dan menulis sebagai medium
untuk merenungkan, menyelidik, menanyakan,
dan mengkritisi ilmu dan gagasan yang
dipelajari; dan (d) literasi sebagai teks yang
bervariasi menurut subjek, genre, dan tingkat
kompleksitas bahasa. Remaja juga diberikan tanggung jawab
tertentu; menyapu, cuci piring, memeriksa kunci
pintu-pintu sebelum tidur, memberi makan
binatang peliharaan. Serta perilaku sopan santun,
mengobrol satu sama lain. Pembimbingan yang
dapat melatih remaja untuk berpikir kreatif
terhadap solusi yang dibuatnya sendiri. Pada
tahap ini, diharuskan untuk mencari solusi JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 sebanyakbanyaknya dan siswa dibebaskan untuk
mencari solusi yang tidak masuk akal sekalipun
hanya untuk meningkatkan kreativitas berpikir
untuk
memperbanyak
solusi. Sebagaimana
tujuan
dari
tahap
ini
adalah
untuk
mengumpulkan sejumlah ide orisinil dari konseli
yang nantinya dipilih menjadi satu solusi yang
tepat untuk mengatasi masalahnya. Berdasarkan
pihak
pelatih
memberikan
lontaran
permasalahan
sosial
terkait
dinamika
pembelajaran online. keterbatasan
karena
pelaksanaannya
masih
dilakukan secara daring dan model pembelajaran
yang diajarkan belum dipraktikkan secara nyata,
baik dalam perencanaan pembelajaran maupun
mempraktikannya di dalam kelas. Oleh karena
itu, penyuluhan lanjutan layak dilakukan dengan
mempraktikkannya dalam situasi nyata dalam
pembelajaran. DAFTAR PUSTAKA Kompetensi literasi kritis siswa Indonesia,
termasuk siswa SMP di Kota Banjarmasin,
tergolong rendah. Hasil tes PISA dan penelitian
lapangan menunjukkan gejala itu. Hal ini tentu
tidak boleh dibiarkan karena mereka ini adalah
calon generasi emas yang hidup dalam era
industri 4.0 yang memerlukan kompetensi
literasi
kritis
dengan
baik. Rendahnya
kompetensi
itu
ada
kaitannya
dengan
kompetensi
guru
dalam
pelaksanaan
pembelajaran. Abdenia, A. (2015). Practicing Critical Literacy in
Second
Language
Reading. Dalam
International Journal of Critical Pedagogy. Vol. 6, (2). Alwasilah, A. Chaedar. 2012. Pokoknya Literasi. Bandung: PT Kiblat Buku Utama. Cooper, K., & White, R. E. (2008). Critical Literacy
for
School
Improvement:
An
Action
Research Project. Improving Schools, 11(2),
101-113. Davidowitz, S. (2019). Terjemahan Alex Tri
Kantjono Widodo. Everybody Lies. Jakarta:
Gramedia Pustaka Utama. Dewabrata, M. Hasil PISA 2018 Resmi Diumumkan,
Indonesia Alami Penurunan Skor di Setiap
Bidang. https://www.zenius.net/blog/23169/pisa-
20182-2019-standar-internasional. Diakses
25 Mei 2020. p
j
Penyuluhan
ini
telah
berhasil
memenunjukkan perubahan ke arah yang lebih
baik persepsi dan kompetensi para guru SMP di
Kota Banjarmasin terkait pembelajaran literasi
kritis. Setelah dilakukan penyuluhan, para guru
memiliki persepsi yang sangat positif terhadap
pelaksanaan penyuluhan ini. Sebagian besar
mereka menganggap penyuluhan ini penting dan
layak
ditindaklanjuti
dengan
kegiatan
pembelajaran literasi kritis lanjutan. Disamping
itu, mereka menyatakan bisa memahami materi
dengan mudah dan jelas. Hal itu juga dapat
dibuktikan dengan penguasaan mereka yang
baik terhadap materi yang disajikan, terutama
terkait dengan pemahaman hakikat literasi kritis,
metode pembelajaran literasi kritis, pemilihan
materi literasi kritis, dan sistem evaluasi yang
perlu digunakan menilai literasi kritis para
siswa. Hilman, I. & Dewi, S.Z. (2015). Mungkinkah
Membangun Kompetensi Literasi Sains di
SD/MI dengan Kompetensi Guru Indonesia? Dalam Soadik, et all. (Eds.). Membaca Imaji
dan Kreativitas Anak Melalui Literasi. Bandung: UPI. Ibrahim, T. (2017). Dialog Landasan Pedagogik. Bandung: Rizki Press. Bandung: Rizki Press. Jumadi; Nasrullah; Syaharuddin; Mutiani; Jumriani;
& Abbas, E.W. (2020). Competency Of
Critical Literation of Students Living in the
Bank of River Area in Banjarmasin City. International
Journal
of
Psychosocial
Rehabilitation, Vol. 24, Issue 08, 2020
ISSN: 1475-7192. Kemendikbud. (2017). Peta Jalan Gerakan Literasi
Nasional. Jakarta: Sekretariat Kemendikbud. Kompasiana. (2016). PISA
dan
Literasi
Indonesia.https://www.kompasiana.com/
frncscnvt/5c1542ec677ffb533d6105/pisa-
dan-literasi-indonesia. Sesuai dengan harapan guru, penyuluhan
ini perlu ditindaklanjuti. Walaupun penyuluhan
ini
sudah
berhasil,
tetapi
masih
punya JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021 Latif, Y. (2020). Pendidikan yang Berkebudayaan:
Histori,
Konsepsi,
dan
Aktualisasi
Pendidikan
Transformatif. Jakarta:
Gramedia Pustaka Utama. McLaughlin, M. & Allen, M. B. (2002). DAFTAR PUSTAKA Guided
Comprehension: A Teaching Model for
Grades 3-8. Newark, DE: International
Reading Association. Thelin, W. H. (2005). Understanding Problems in
Critical Classrooms. College Composition
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Melalui Literasi. Bandung: UPI. JOURNAL OF HUMAN AND EDUCATION VOLUME 1 NOMOR 2 TAHUN 2021
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Drak2 Does Not Regulate TGF-β Signaling in T Cells
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RESEARCH ARTICLE
Drak2 Does Not Regulate TGF-β Signaling in
T Cells Tarsha L. Harris1,2, Maureen A. McGargill1,2* 1 Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States
of America, 2 Integrated Biomedical Sciences Program, University of Tennessee Health Science Center,
Memphis, Tennessee, United States of America * Maureen.mcgargill@stjude.org * Maureen.mcgargill@stjude.org Academic Editor: Heinz Wiendl, University of
Münster, GERMANY Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper. Funding: This work was supported by a Juvenile
Diabetes Research Foundation Career Development
Award to MAM, 2-2007-105 and ALSAC charities. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. * Maureen.mcgargill@stjude.org RESEARCH ARTICLE Abstract Drak2 is a serine/threonine kinase expressed highest in T cells and B cells. Drak2-/- mice
are resistant to autoimmunity in mouse models of type 1 diabetes and multiple sclerosis. Resistance to these diseases occurs, in part, because Drak2 is required for the survival of
autoreactive T cells that induce disease. However, the molecular mechanisms by which
Drak2 affects T cell survival and autoimmunity are not known. A recent report demonstrated
that Drak2 negatively regulated transforming growth factor-β (TGF-β) signaling in tumor cell
lines. Thus, increased TGF-β signaling in the absence of Drak2 may contribute to the resis-
tance to autoimmunity in Drak2-/- mice. Therefore, we examined if Drak2 functioned as a
negative regulator of TGF-β signaling in T cells, and whether the enhanced susceptibility to
death of Drak2-/- T cells was due to augmented TGF-β signaling. Using several in vitro as-
says to test TGF-β signaling and T cell function, we found that activation of Smad2 and
Smad3, which are downstream of the TGF-β receptor, was similar between wildtype and
Drak2-/- T cells. Furthermore, TGF-β-mediated effects on naïve T cell proliferation, activated
CD8+ T cell survival, and regulatory T cell induction was similar between wildtype and
Drak2-/- T cells. Finally, the increased susceptibility to death in the absence of Drak2 was
not due to enhanced TGF-β signaling. Together, these data suggest that Drak2 does not
function as a negative regulator of TGF-β signaling in primary T cells stimulated in vitro. It is
important to investigate and discern potential molecular mechanisms by which Drak2 func-
tions in order to better understand the etiology of autoimmune diseases, as well as to vali-
date the use of Drak2 as a target for therapeutic treatment of these diseases. OPEN ACCESS
Citation: Harris TL, McGargill MA (2015) Drak2 Does
Not Regulate TGF-β Signaling in T Cells. PLoS ONE
10(5): e0123650. doi:10.1371/journal.pone.0123650
Academic Editor: Heinz Wiendl, University of
Münster, GERMANY
Received: November 10, 2014
Accepted: February 10, 2015
Published: May 7, 2015
Copyright: © 2015 Harris, McGargill. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. OPEN ACCESS Citation: Harris TL, McGargill MA (2015) Drak2 Does
Not Regulate TGF-β Signaling in T Cells. PLoS ONE
10(5): e0123650. doi:10.1371/journal.pone.0123650 Drak2 and TGF-β Signaling in T Cells infections and tumors. Inhibition of Drak2, a serine-threonine kinase, may be an alternative
approach to inhibit autoreactive T cells without acting as an immunosuppressant. Drak2-/- mice are resistant to autoimmune disease in mouse models of type 1 diabetes and
multiple sclerosis [1,2]. In both of these disease models, the accumulation of autoreactive T
cells in the target organ is significantly reduced in the absence of Drak2. The reduced accumu-
lation of autoreactive T cells is due to an increased susceptibility to death of the Drak2-/- T cells
[2,3]. Interestingly, despite this increased sensitivity to death in the T cells, the Drak2-/- mice ef-
fectively eliminate infectious pathogens and retain the ability to combat tumors as well as wild-
type mice [2,4–7]. Thus, Drak2 is an ideal protein to target in order to treat autoimmune
disorders without compromising immunity to pathogens and tumors. However, the substrates
and downstream effects of Drak2 signaling that contribute to autoimmunity require further
elucidation to validate its potential as a therapeutic target and to further understand how these
autoimmune diseases develop. Drak2 has been shown to interact with several proteins in in vitro recombinant assays and
in cell lines. These proteins include myosin light chain [8], calcineurin homologous protein [9],
Protein kinase D [10], p70S6 kinase [11], and TGF-β receptor I (TGF-βRI) [12]. However,
most of these interactions have not been confirmed in T cells and therefore, it is not clear
which of these interactions may affect autoimmune disease. As TGF-β is a critical suppressor of autoimmunity, the interaction of Drak2 and the TGF-
βRI is an intriguing possibility to explain how Drak2 contributes to autoimmunity. TGF-β is
a pleiotropic cytokine that elicits numerous effects on various cell types [13]. In T cells spe-
cifically, TGF-β inhibits proliferation of naïve T cells, induces development of regulatory T
cells, and enhances apoptosis of activated T cells. A recent study proposed that Drak2 func-
tions as a negative regulator of TGF-β signaling by inhibiting the phosphorylation and re-
cruitment of Smad2 and Smad3 to the TGF-βRI in cell lines [12]. Thus, the absence of Drak2
in T cells may render these cells more susceptible to TGF-β signaling, which could prevent
autoimmunity. However, it has not been tested if Drak2 functions as a negative regulator of
TGF-β in T cells, and consequently, whether Drak2-/- T cells are more sensitive to TGF-β
signaling. Therefore, we investigated whether Drak2 functions as a negative regulator of TGF-β signal-
ing in T cells, and further if the enhanced susceptibility to apoptosis in Drak2-/- T cells was due
to augmented TGF-β signaling. We found that TGF-β signaling via Smad2 and Smad3 was not
enhanced in the absence of Drak2 in T cells, and that Drak2-/- T cells did not exhibit enhanced
responses to TGF-β signaling during in vitro assays. These data suggest that Drak2 does not
function as an inhibitor of TGF-β signaling in T cells. Moreover, in the absence of TGF-β sig-
naling, Drak2-/- T cells remained more susceptible to apoptosis, suggesting that the increase in
cell death observed in vitro, was not due to enhanced TGF-β-mediated signals. These data pro-
vide insight into the role of Drak2 in autoimmune diseases by showing that Drak2 may not
suppress TGF-β signaling in T cells, and therefore may contribute to autoimmune disease via
other molecular pathways. Introduction T cells play crucial roles in tumor surveillance and protection against invading pathogens. However, if not properly regulated, T cells can attack normal healthy cells of the body. This de-
fective response may lead to tissue destruction and devastating autoimmune diseases such as
type 1 diabetes and multiple sclerosis. Treatments to modify the progression of autoimmune
diseases often include immunosuppressant medications that lead to enhanced susceptibility to Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 1 / 14 Plate-bound anti-CD3 and anti-CD28 stimulation Tissue culture-treated plates were incubated for one hour with 30μg/ml goat anti-hamster IgG
in PBS (Vector Laboratories, Burlingame, CA), then washed and incubated for one hour with
1μg/ml or 2μg/ml anti-CD3 (eBioscience). Plates were washed before addition of cells and 1μg/
ml anti-CD28 (eBioscience). Flow Cytometric Analysis Single cell suspensions from in vitro cultures were stained with antibodies specific for CD4,
CD8, CD25, CD45.1, and CD45.2 (eBioscience and BioLegend). Cells were analyzed on a
FACSCalibur or LSRFortessa (BD Biosciences,). Cell death and viability was determined utiliz-
ing Annexin V (BD Biosciences) or Fixable Viability Dye (eBioscience), according to manufac-
turer’s instructions. Analysis was performed with FlowJo software (TreeStar, Inc.). To detect
Foxp3+ cells, suspensions were stained with antibodies specific for CD4, CD8, and CD25. Cells
were then fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set ac-
cording to manufacturer’s instructions (eBioscience) and stained with anti-Foxp3 antibody
(eBioscience). For analysis of phosphorylated Smad2/3, cells were stained with antibodies spe-
cific for CD4 and CD8, then fixed with 1X BD Phosflow Lyse/Fix Buffer and permeabilized
with BD Phosflow Perm Buffer III according to manufacturer’s instructions (BD Biosciences)
and stained with anti-pSmad2/3 antibody (BD Biosciences). FACs purification of lymphoid populations T cells were purified from the spleen and lymph nodes of mice by FACS sorting with antibodies
specific for CD4, CD8, CD25, CD44, and CD62L (eBioscience). Naïve T cells were
CD25-CD44lo or CD25-CD44loCD62Lhi. Cell sorting was performed using the iCyt Reflection
or SY3200 Cell Sorters (Sony Biotechnology). Mice B6.Drak2-/- mice were previously described and backcrossed 19 generations to C57BL/6 [1]. OT-II mice were obtained from Kristin Hogquist, TGF-βDNRII mice were obtained from
Hongbo Chi, C57BL/6, CD451/1, and OT-I mice were purchased from Jackson Laboratories. Mice were held under specific pathogen-free conditions at St. Jude Children’s Research
Hospital. 2 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Ethics Statement All studies were reviewed and approved by the St. Jude Animal Ethics Committee under proto-
col number 486-100303-05/14. St. Jude is AAALAC accredited and complies with all federal,
state, and local laws. Magnetic separation of CD4+ T cells T cells were purified from the lymph nodes of mice by negative selection with biotin-conjugated
antibodies specific for B220, CD8, CD11b, DX5, and MHC class II (eBioscience), followed by sep-
aration with streptavidin-conjugated magnetic beads (Miltenyi Biotech). Stimulation of OT-II cells Naïve OT-II CD4+ T cells were sorted, CFSE labeled, and stimulated in vitro for three days
in the presence or absence of 10-fold TGF-β titrations with 10μM OVA323 peptide-pulsed,
irradiated splenocytes. After three days, the cells were harvested and analyzed by flow
cytometry. In vitro Treg induction Naïve CD4+ T cells were purified and stimulated with plate-bound anti-CD3 and soluble anti-
CD28 for 72 hours with 20ng/ml IL-2 and increasing amounts of TGF-β. Cells were analyzed
for Foxp3 expression. Fluorescent microscopy Wildtype and Drak2-/- CD4+ cells were negatively selected with Miltenyi beads and stimulated
with 1μg/ml anti-CD3 coated on poly L-lysine-coated coverglass slides and 1μg/ml soluble
anti-CD28 for 24 hours. TGF-β was added to some samples for the final 20 minutes. Cells were
fixed with 4% methanol-free formaldehyde, permeabilized in 0.1% Triton-X in PBS, washed
with PBS, blocked with 1% BSA, and incubated with anti-Smad2 antibody (Cell Signaling)
overnight. Cells were stained with Alexa Fluor 647 goat anti-Rabbit, Alexa Fluor 488 Phalloi-
din, and DAPI (Invitrogen Life Technologies). Images were collected utilizing a Nikon C1Si
laser scanning confocal microscope. Stimulation of OT-I cells Naïve OT-I CD8+ T cells were sorted and labeled with 5,6-carboxyfluorescein diacetate suc-
cinimidyl ester (CFSE) (Molecular probes) at 0.4 μM in pre-warmed PBS (0.1% FCS) for 10
minutes at 37°C, then washed twice with RP10 advanced media (RPMI advanced media,
10% FCS, Hepes, Pen-Strep, L-glutamine, BME, gentamicin). Cells were stimulated in vitro
for two days with 100pM OVA257 peptide-pulsed, CD45.1 splenocytes that were irradiated
at 3000 rads. Alternatively, 2 x 106 FACS-sorted, naïve OT-I CD8+ T cells were stimulated with 4.5 x
106 200 nM OVA257 peptide-pulsed, CD45.1 splenocytes at 37°C for two days in RP10 ad-
vanced media. The cells were harvested, washed, and replated with naïve splenocytes in the
presence of 5ng/ml TGF-β (R&D Systems) with or without 20 ng/ml recombinant mouse IL- 3 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells 2 (BD Biosciences), IL-7 (Invitrogen Life Technologies), or IL-15 (R&D Systems). Two days
later, fresh media and cytokines were added, and two days later, cells were harvested, stained
and analyzed by flow cytometry. 2 (BD Biosciences), IL-7 (Invitrogen Life Technologies), or IL-15 (R&D Systems). Two days
later, fresh media and cytokines were added, and two days later, cells were harvested, stained
and analyzed by flow cytometry. Western Blot analysis Spleen and lymph nodes were harvested from wildtype and Drak2-/- mice. Whole splenocytes
and FACS-sorted naïve CD4+ and CD8+ T cells were stimulated for two hours with plate-
bound anti-CD3 and anti-CD28 with or without 2 ng/ml TGF-β for one additional hour. Cells
were harvested and frozen at -80°C. Frozen cell pellets were lysed (50mM Tris, 150mM NaCl,
1% Triton X-100, 0.5% sodium deoxycholate, 2mM EDTA, 10% glycerol with phosphatase and
protease inhibitors (Calbiochem). Protein concentration was determined using a BCA Protein
Assay (Thermo Scientific). Equal amounts of protein were denatured in sample buffer (10%
SDS, 20% Glycerol, 0.2M Tris HCl, 0.05% Bromophenol Blue), separated by SDS-PAGE, and
transferred to PVDF membranes for immunoblot analysis. TGF-β signaling via Smad proteins is not enhanced in Drak2-/- T cells
compared to wildtype T cells Given that recent experiments in cell lines suggested that Drak2 negatively regulates TGF-β
signaling [12], and enhanced TGF-β signaling in T cells could contribute to the resistance to
autoimmune disease, we tested whether Drak2 functions as a negative regulator of TGF-β
signaling in T cells. TGF-β receptor engagement results in the phosphorylation of the
Smad2/Smad3 signaling complex, which then translocates from the cytoplasm into the nu-
cleus to facilitate TGF-β-mediated transcription. To determine if Smad2 translocation into
the nucleus was increased in the absence of Drak2, we activated CD4+ T cells with anti-CD3
and anti-CD28 antibodies for 24 hours, and then utilized confocal fluorescent microscopy to 4 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Fig 1. Smad2 translocation is not enhanced in Drak2-/- T cells compared to wildtype T cells. Wildtype and Drak2-/- CD4+ cells were negatively
selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were
treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were
collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments. PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 doi:10.1371/journal.pone.0123650.g002 Fig 1. Smad2 translocation is not enhanced in Drak2-/- T cells compared to wildtype T cells. Wildtype and Drak2-/- CD4+ cells were negatively
selected with Miltenyi magnetic beads and stimulated on anti-CD3-coated coverglass slides along with soluble anti-CD28 for 24 hours. Half of the cells were
treated with TGF-β for the final 20 minutes of culture. Cells were fixed, permeabilized, and stained with DAPI, Phalloidin, and anti-Smad2. Images were
collected via confocal microscopy. n = 2 mice per group. Data are representative of two independent experiments. doi:10.1371/journal.pone.0123650.g001 analyze Smad2 localization following addition of TGF-β. As expected, Smad2 translocation
into the nucleus was not observed in stimulated T cells without exogenous TGF-β (Fig 1). The addition of TGF-β during the final 20 minutes of culture elicited Smad2 translocation
into the nuclear region of both wildtype and Drak2-/- T cells (Fig 1). Importantly, there were
no differences in Smad2 translocation between wildtype and Drak2-/- T cells in response to
exogenous TGF-β. We also examined phosphorylation of Smad2 by western blot in lysates from purified CD4+
T cells, CD8+ T cells, or whole splenocytes. In all cell types, Smad2 was phosphorylated in re-
sponse to TGF-β treatment; however, the extent of phosphorylation was not increased in
Drak2-/- cells compared to wildtype cells (Fig 2a). Finally, to determine if Drak2-/- cells are hy-
persensitive to lower concentrations of TGF-β, we analyzed the phosphorylation of the Smad2/
Smad3 complex by flow cytometry in response to decreasing amounts of TGF-β. Again, even at
the lower doses of TGF-β, the phosphorylation of Smad2/3 was similar in wildtype and 5 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 The effects of TGF-β on T cells are comparable between wildtype and
Drak2-/- T cells Many of the downstream mechanisms utilized by TGF-β to regulate T cells remain unclear. Al-
though we did not observe enhanced TGF-β signaling in Drak2-/- T cells via Smad proteins, it
was possible that Drak2 regulated the pathway through alternative mechanisms. Therefore,
we explored the effects of TGF-β on several T cell functions in vitro. TGF-β suppresses T cell
receptor-induced proliferation of naïve T cells in vitro [14]. Thus, we examined if naïve
Drak2-/- T cells were more sensitive to TGF-β-mediated inhibition of proliferation than naive
wildtype T cells. Naïve OT-II and OT-II.Drak2-/- CD4+ T cells were stimulated with OVA323-
pulsed splenocytes in the presence or absence of TGF-β, and analyzed for proliferation. The
number of live, divided CD4+ T cells decreased in response to TGF-β (Fig 3a). However, the ef-
fect of TGF-β inhibition was comparable between OT-II and OT-II.Drak2-/- T cells. We also
tested the effect of TGF-β on proliferation of naïve CD8+ T cells, by stimulating OT-I and
OT-I.Drak2-/- T cells with OVA257-pulsed splenocytes in the presence of TGF-β. Similar to
CD4+ T cells, the number of live, divided CD8+ T cells decreased in response to TGF-β, and the
amount of suppression was similar between OT-I and OT-I.Drak2-/- T cells (Fig 3b), again sug-
gesting that TGF-β signaling was not enhanced in the absence of Drak2. TGF-β can abrogate survival signals provided by IL-15, but not those elicited by IL-2 and
IL-7 in expanding CD8+ T cells [15]. To determine if TGF-β function in response to opposing
cytokines is altered in the absence of Drak2, we explored the antagonistic effects of TGF-β on
cell recovery and survival of activated CD8+ cells. OT-I and OT-I.Drak2-/- cells were stimulated
with OVA257-pulsed splenocytes for two days. Cells were then washed and cultured with exog-
enous IL-2, IL-7, or IL-15 with or without TGF-β for an additional four days. The addition of
TGF-β decreased the number of live CD8+ T cells compared to culture in media alone (Fig 4a). Adding IL-2, IL-7, and IL-15 enhanced the recovery of live CD8+ T cells compared to culture
in media alone. The addition of TGF-β masked the increased recovery in response to IL-15, but
not IL-2 and IL-7. Decreased cell recovery in response to TGF-β compared to culture in media
alone correlated with an increase in the proportion of Annexin V+ apoptotic cells (Fig 4b). Together, these data show that Drak2-/- T cells do not exhibit enhanced
TGF-β signaling via Smad2 or Smad2/3 complex phosphorylation compared to wildtype T
cells, suggesting that Drak2 does not function as a negative regulator of TGF-β signaling in pri-
mary T cells activated in vitro. Drak2-/- cells (Fig 2b). Together, these data show that Drak2-/- T cells do not exhibit enhanced
TGF-β signaling via Smad2 or Smad2/3 complex phosphorylation compared to wildtype T
cells, suggesting that Drak2 does not function as a negative regulator of TGF-β signaling in pri-
mary T cells activated in vitro. The effects of TGF-β on T cells are comparable between wildtype and
Drak2-/- T cells The
addition of TGF-β abrogated the survival effects of IL-15, but did not alter the anti-apoptotic
effects of IL-2 and IL-7. However, the ability of TGF-β to oppose the effects of IL-15, but not
IL-2 and IL-7 was comparable between OT-I and OT-I.Drak2-/- T cells, suggesting that these
TGF-β-mediated effects are not enhanced in the absence of Drak2. These data further indicate
that TGF-β signaling and function is not increased in Drak2-/- T cells compared to wildtype T
cells following in vitro stimulation. Drak2 and TGF-β Signaling in T Cells Fig 2. Smad2 and Smad2/3 complex phosphorylation is not enhanced in Drak2-/- T cells compared to wildtype T cells. A) Wildtype and Drak2-/-
splenocytes, and FACS sorted naïve CD4+ and CD8+ T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for
additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading cont
Cells were pooled from 9 wildtype and 8 Drak2-/- mice. Data are representative of two independent experiments. B) Wildtype and Drak2-/- splenocytes w
stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested,
stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSma
expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-
Whitney U-test. Data are representative of 3 independent experiments. doi:10.1371/journal.pone.0123650.g002 Fig 2. Smad2 and Smad2/3 complex phosphorylation is not enhanced in Drak2-/- T cells compared to wildtype T cells. A) Wildtype and Drak2-/-
splenocytes, and FACS sorted naïve CD4+ and CD8+ T cells were stimulated for 2 hours with anti-CD3 and anti-CD28, with or without 2 ng/ml TGF-β for one
additional hour. Cells were lysed and analyzed by western blot with antibodies specific for Smad2, phosphorylated Smad2, and HSP90 as a loading control. Cells were pooled from 9 wildtype and 8 Drak2-/- mice. Data are representative of two independent experiments. B) Wildtype and Drak2-/- splenocytes were
stimulated for 2 hours with anti-CD3 and anti-CD28 with or without increasing concentrations of TGF-β for one additional hour. The cells were harvested,
stained with antibodies specific for CD4, CD8, and pSmad2/3, and analyzed by flow cytometry. The average mean fluorescence intensity (MFI) of pSmad2/3
expression is shown for 3 mice per group. There was no significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-
Whitney U-test. Data are representative of 3 independent experiments. doi:10.1371/journal.pone.0123650.g002 6 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Drak2-/- cells (Fig 2b). doi:10.1371/journal.pone.0123650.g003 Drak2 doi:10 1371/journal pone 0123650 g003 Fig 3. TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and
Drak2-/- T cells. A) CD4+CD25-CD44lo naïve cells were purified from OT-II and OT-II.Drak2-/- mice and
stimulated with irradiated splenocytes loaded with 10μM OVA323 peptide in the presence or absence of
10-fold TGF-β titrations for three days. The number of live, divided Foxp3-CD4+ cells are shown for each
titration. Cells were obtained from one OT-II or OT-II.Drak2-/- mouse and tested in quadruplicate. Data are
representative of five separate experiments. B) CD8+CD25-CD44loCD62Lhi naïve cells were purified from
OT-I and OT-I.Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA257 peptide in the
presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow
cytometry. The number of live, divided CD8+ cells are shown for each titration. Cells were obtained from one
OT-I or OT-I.Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate
experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according
to the Mann-Whitney U-test. Fig 3. TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and
Drak2-/- T cells. A) CD4+CD25-CD44lo naïve cells were purified from OT-II and OT-II.Drak2-/- mice and
stimulated with irradiated splenocytes loaded with 10μM OVA323 peptide in the presence or absence of
10-fold TGF-β titrations for three days. The number of live, divided Foxp3-CD4+ cells are shown for each
titration. Cells were obtained from one OT-II or OT-II.Drak2-/- mouse and tested in quadruplicate. Data are
representative of five separate experiments. B) CD8+CD25-CD44loCD62Lhi naïve cells were purified from
OT-I and OT-I.Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA257 peptide in the
presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow
cytometry. The number of live, divided CD8+ cells are shown for each titration. Cells were obtained from one
OT-I or OT-I.Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate
experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according
to the Mann-Whitney U-test. Fig 3. TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and
Drak2-/- T cells. Drak2 Another function of TGF-β is the induction of peripheral regulatory T cells [16]. As regulatory
T cells are critical to prevent autoimmune diseases, we explored if there were alterations in
TGF-β-mediated differentiation of induced regulatory T cells. Naïve wildtype and Drak2-/-
CD4+ T cells were purified and stimulated in vitro with anti-CD3, anti-CD28, and IL-2, with in-
creasing amounts of TGF-β (Fig 5). The addition of TGF-β increased Foxp3 expression, indica-
tive of regulatory T cell induction. However, we did not observe an enhanced induction in the 7 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Fig 3. TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and
Drak2-/- T cells. A) CD4+CD25-CD44lo naïve cells were purified from OT-II and OT-II.Drak2-/- mice and
stimulated with irradiated splenocytes loaded with 10μM OVA323 peptide in the presence or absence of
10-fold TGF-β titrations for three days. The number of live, divided Foxp3-CD4+ cells are shown for each
titration. Cells were obtained from one OT-II or OT-II.Drak2-/- mouse and tested in quadruplicate. Data are
representative of five separate experiments. B) CD8+CD25-CD44loCD62Lhi naïve cells were purified from
OT-I and OT-I.Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA257 peptide in the
presence or absence of 10-fold TGF-β titrations Two days later cells were harvested and analyzed by flow Fig 3. TGF-β-mediated inhibition of naïve T cell proliferation is comparable between wildtype and
Drak2-/- T cells. A) CD4+CD25-CD44lo naïve cells were purified from OT-II and OT-II.Drak2-/- mice and
stimulated with irradiated splenocytes loaded with 10μM OVA323 peptide in the presence or absence of
10-fold TGF-β titrations for three days. The number of live, divided Foxp3-CD4+ cells are shown for each
titration. Cells were obtained from one OT-II or OT-II.Drak2-/- mouse and tested in quadruplicate. Data are
representative of five separate experiments. B) CD8+CD25-CD44loCD62Lhi naïve cells were purified from
OT-I and OT-I.Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA257 peptide in the
presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow
cytometry. The number of live, divided CD8+ cells are shown for each titration. Cells were obtained from one
OT-I or OT-I.Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate
experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according
to the Mann-Whitney U-test. Drak2 A) CD4+CD25-CD44lo naïve cells were purified from OT-II and OT-II.Drak2-/- mice and
stimulated with irradiated splenocytes loaded with 10μM OVA323 peptide in the presence or absence of
10-fold TGF-β titrations for three days. The number of live, divided Foxp3-CD4+ cells are shown for each
titration. Cells were obtained from one OT-II or OT-II.Drak2-/- mouse and tested in quadruplicate. Data are
representative of five separate experiments. B) CD8+CD25-CD44loCD62Lhi naïve cells were purified from
OT-I and OT-I.Drak2-/- mice and stimulated with splenocytes loaded with 100pM OVA257 peptide in the
presence or absence of 10-fold TGF-β titrations. Two days later, cells were harvested and analyzed by flow
cytometry. The number of live, divided CD8+ cells are shown for each titration. Cells were obtained from one
OT-I or OT-I.Drak2-/- mouse and tested in quadruplicate. Data are representative of three separate
experiments. There was no significant difference in the response of the wildtype and Drak2-/- cells according
to the Mann-Whitney U-test. doi:10.1371/journal.pone.0123650.g003 8 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equ
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells w
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or O
Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments. *P < 0.05 (Mann-Whitney U-test). doi:10.1371/journal.pone.0123650.g004
Drak2 and TGF-β Signaling in T doi:10.1371/journal.pone.0123650.g004 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice an
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I.
Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments.
*P < 0.05 (Mann-Whitney U-test).
doi:10.1371/journal.pone.0123650.g004 Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice and
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I. Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments. *P < 0.05 (Mann-Whitney U-test). doi:10.1371/journal.pone.0123650.g004 Fi
4 TGF β
di t d
t
i
t ki
bl b t
ildt
d Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice and
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I. Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments. *P < 0.05 (Mann-Whitney U-test). Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice and
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I. Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments. *P < 0.05 (Mann-Whitney U-test). 9 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice an
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I.
Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments.
*P < 0.05 (Mann-Whitney U-test).
doi:10.1371/journal.pone.0123650.g004 These data
also suggest that TGF-β functions similarly in wildtype and Drak2-/- T cells that were activated
in vitro. Therefore, Drak2 may not act as a negative regulator of TGF-β signaling in T cells. percent (Fig 5a) or number (Fig 5b) of Foxp3+CD4+ cells in the absence of Drak2. These data
also suggest that TGF-β functions similarly in wildtype and Drak2-/- T cells that were activated
in vitro. Therefore, Drak2 may not act as a negative regulator of TGF-β signaling in T cells. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype
T cells is independent of TGF-β signaling in vitro Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype
T cells is independent of TGF-β signaling in vitro We previously showed that Drak2-/- T cells exhibit enhanced susceptibility to death in vivo,
which promotes resistance to type 1 diabetes and multiple sclerosis [2]. In addition, we found
that following in vitro stimulation with anti-CD3 and anti-CD28, a greater proportion of
Drak2-/- T cells were apoptotic compared to wildtype T cells (Fig 6a and 6b, left portion of Drak2
T cells were apoptotic compared to wildtype T cells (Fig 6a and 6b, left portion of
Fig 6. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro. A)
CD4+CD25-CD44lo or B) CD8+CD25-CD44lo naïve cells were purified from wildtype, Drak2-/-, DNRII, and DNRII. Drak2-/- mice and stimulated with anti-CD3
and anti-CD28 for 2–3 days. The percent of nonviable CD4+ or CD8+ T cells is shown. Cells were obtained from one mouse per group and tested in
quadruplicate. Data are representative of four separate experiments. **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). doi:10.1371/journal.pone.0123650.g006 Fig 6. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro. A)
CD4+CD25-CD44lo or B) CD8+CD25-CD44lo naïve cells were purified from wildtype, Drak2-/-, DNRII, and DNRII. Drak2-/- mice and stimulated with anti-CD3
and anti-CD28 for 2–3 days. The percent of nonviable CD4+ or CD8+ T cells is shown. Cells were obtained from one mouse per group and tested in
quadruplicate Data are representative of four separate experiments **P < 0 01 ***P < 0 001 (Mann-Whitney U-test) Fig 6. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro. A)
CD4+CD25-CD44lo or B) CD8+CD25-CD44lo naïve cells were purified from wildtype, Drak2-/-, DNRII, and DNRII. Drak2-/- mice and stimulated with anti-CD3
and anti-CD28 for 2–3 days. The percent of nonviable CD4+ or CD8+ T cells is shown. Cells were obtained from one mouse per group and tested in
quadruplicate. Data are representative of four separate experiments. **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). Fig 6. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype T cells is independent of TGF-β signaling in vitro. A)
CD4+CD25-CD44lo or B) CD8+CD25-CD44lo naïve cells were purified from wildtype, Drak2-/-, DNRII, and DNRII. Drak2-/- mice and stimulated with anti-CD3
and anti-CD28 for 2–3 days. The percent of nonviable CD4+ or CD8+ T cells is shown. Fig 4. TGF-β-mediated responses to opposing cytokines are comparable between wildtype and
Drak2-/- T cells. CD8+CD25-CD44loCD62Lhi naïve cells were purified from OT-I and OT-I.Drak2-/- mice an
stimulated with 100nM OVA257–pulsed splenocytes for 2 days. Cells were harvested and replated at equal
numbers with or without various cytokine combinations. Cytokines were replenished 2 days later. Cells were
harvested and analyzed by flow cytometry on day 6. A) The number of live, CD8+ cells and B) percent
Annexin V+ of CD8+ cells are shown for each cytokine condition. Cells were obtained from one OT-I or OT-I.
Drak2-/- mouse and tested in quadruplicate. Data are representative of two independent experiments.
*P < 0.05 (Mann-Whitney U-test).
doi:10.1371/journal.pone.0123650.g004 A) CD4+CD25-CD44lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with
1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3+ cells of electronically gated CD4+ cells is shown. There was no
significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney
U-test. doi:10.1371/journal.pone.0123650.g005 Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. A) CD4+CD25-CD44lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with
1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 da
The A) percent and B) number of Foxp3+ cells of electronically gated CD4+ cells is shown. There was n
significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney
U-test. doi:10.1371/journal.pone.0123650.g005 Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. A) CD4+CD25-CD44lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with
1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 da
The A) percent and B) number of Foxp3+ cells of electronically gated CD4+ cells is shown. There was n Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. ) C
+C
C
lo
/ Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. A) CD4+CD25 CD44lo
ï
ll
ifi d f
ildt
d D
k2 /
i
d ti
l t d Fig 5. TGF-β-mediated regulatory T cell induction is not altered in the absence of Drak2. A) CD4+CD25-CD44lo naïve cells were purified from wildtype and Drak2-/- mice and stimulated with
1μg/ml anti-CD3 and 1μg/ml anti-CD28 with 20ng/ml IL-2 alone or plus 10-fold TGF-β titrations for 3 days. The A) percent and B) number of Foxp3+ cells of electronically gated CD4+ cells is shown. There was no
significant difference in the response of the wildtype and Drak2-/- cells according to the Mann-Whitney
U-test. doi:10.1371/journal.pone.0123650.g005 10 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Drak2 and TGF-β Signaling in T Cells percent (Fig 5a) or number (Fig 5b) of Foxp3+CD4+ cells in the absence of Drak2. Discussion Drak2 contributes to organ-specific autoimmune disease and is an ideal protein to target to
treat these diseases without causing generalized immune suppression. Therefore, it is pertinent
to understand the molecular and cellular mechanisms by which Drak2 functions in order to
further comprehend the etiology of autoimmune disease. In addition, insight into the function
of Drak2 is critical to validate it as a therapeutic target. TGF-β is a multifunctional cytokine that controls many aspects of T cell behavior and elicits
protective effects in several autoimmune diseases [13]. It has been suggested that Drak2 func-
tions as a negative regulator of TGF-β signaling [12]. As TGF-β can inhibit proliferation, sur-
vival, and differentiation of T cells, enhanced TGF-β signaling in Drak2-/- T cells could
contribute to the resistance to autoimmune disease in the Drak2-/- mice via one or more of
these mechanisms. However, our data suggest that in primary T cells stimulated in vitro, Drak2
does not function as a negative regulator of this pathway. Smad2/3 signaling after TGF-β stim-
ulation was not enhanced in Drak2-/- T cells compared to wildtype T cells. Importantly, the im-
pact of TGF-β on T cell behavior was not enhanced in the absence of Drak2 as evidenced by
equal inhibition of naïve T cell proliferation, comparable effects on activated CD8+ T cell accu-
mulation and survival, and similar induction of regulatory T cells in wildtype and Drak2-/-
T cells. The previous studies that suggested Drak2 negatively regulates TGF-β signaling were per-
formed in tumor cell lines [12]. It is possible that Drak2 inhibits TGF-β in certain tumor cells,
but not in primary T cells. Mutations that lead to tumorigenesis could facilitate a role for
Drak2 regulation in TGF-β signaling. Thus, as reported in certain tumors, Drak2 may function
to negatively regulate TGF-β signaling and promote tumorigenesis [12]. However, this is con-
trary to other reports that describe Drak2 as a tumor suppressor [18–20]. Therefore, the role of
Drak2 in different types of tumors is also controversial and needs to be studied further. Inter-
estingly, we have shown that Drak2-/- mice respond similarly to wildtype mice in various in
vivo tumor models, again suggesting that the role of Drak2 in cell lines may not mimic its role
under physiological conditions [7]. Enhanced susceptibility to death of Drak2-/- T cells compared to wildtype
T cells is independent of TGF-β signaling in vitro Cells were obtained from one mouse per group and tested in
quadruplicate. Data are representative of four separate experiments. **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). doi:10.1371/journal.pone.0123650.g006 doi:10.1371/journal.pone.0123650.g006 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 11 / 14 Drak2 and TGF-β Signaling in T Cells graph). Although we did not observe differences in TGF-β signaling in the absence of Drak2,
there may be alternative TGF-β-mediated effects on T cell survival. Thus, we sought to deter-
mine if the survival defect in Drak2-/- T cells compared to wildtype T cells was due to enhanced
TGF-β signaling. To test this, we compared T cell survival between wildtype and Drak2-/- T
cells that exhibit impaired TGF-β signaling due to expression of a dominant-negative TGF-β
receptor II (DNRII) transgene. The DNRII transgene is a kinase-dead mutant that blocks sig-
naling through the endogenous TGF-β receptor by competing for TGF-β binding [17]. Naïve
CD4+ and CD8+ T cells were sorted from wildtype, Drak2-/-, DNRII, and DNRII.Drak2-/- mice. The purified T cells were stimulated in vitro with anti-CD3 and anti-CD28. We found that
even with the severe reduction in TGF-β signaling, there was an increase in the proportion of
nonviable DNRII.Drak2-/- CD4+ (Fig 6a, right portion of graph) and CD8+ (Fig 6b, right por-
tion of graph) T cells compared to DNRII CD4+ and CD8+ T cells. These data show that the en-
hanced death in the Drak2-/- T cells following in vitro stimulation is not due to increased TGF-
β signaling, and suggest that alternative signaling pathways play a role. Acknowledgments The authors would like to thank the St. Jude Cell and Tissue Imaging Core, the St. Jude Animal
Resource Center, Benjamin A. Edwards, and Ashley Castellaw for excellent
technical assistance. Drak2 and TGF-β Signaling in T Cells type 1 diabetes was also due to the absence of Drak2 in T cells (TLH and MAM manuscript
submitted). Another possible explanation for the discrepancy between our results in primary T cells and
the previous data in tumor cell lines is that during development, the Drak2-/- T cells may have
compensated for the loss of Drak2 through modifications of alternate pathways involved in
TGF-β regulation. For example, increased levels of Smad7, a negative regulator of TGF-β sig-
naling [21], could mask alterations in TGF-β signaling in the absence of Drak2. Therefore,
Drak2-/- T cells may exhibit altered signaling pathways that function differently compared to
physiological conditions in wildtype T cells, which warrants further investigation. Nevertheless, our data presented here indicate that TGF-β signaling is not enhanced in
Drak2-/- T cells following in vitro stimulation. Consequently, Drak2 may not function as a neg-
ative regulator of TGF-β signaling in T cells, which are critical for the induction of autoimmu-
nity. Therefore, further investigation of the potential molecular mechanisms by which Drak2
functions during autoimmune disease is required to gain insight into the etiology of
these diseases. Author Contributions Conceived and designed the experiments: TLH MAM. Performed the experiments: TLH
MAM Analyzed the data: TLH MAM Wrote the paper: TLH MAM Conceived and designed the experiments: TLH MAM. Performed the experiments: TLH
MAM. Analyzed the data: TLH MAM. Wrote the paper: TLH MAM. Conceived and designed the experiments: TLH MAM. Performed the experiments: TLH
MAM. Analyzed the data: TLH MAM. Wrote the paper: TLH MAM. MAM. Analyzed the data: TLH MAM. Wrote the paper: TLH MAM. PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 Discussion Furthermore, it is important to investigate the molecular mechanisms of Drak2 in primary
T cells, as these are the cells relevant to the induction or resistance to autoimmunity. The im-
portance of Drak2 specifically in T cells during autoimmunity was highlighted in our previous
studies, which demonstrated that the resistance to disease in the mouse model of multiple scle-
rosis was due to Drak2-deficiency in T cells [2]. In addition, we found that the resistance to 12 / 14 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 References 1. McGargill MA, Wen BG, Walsh CM, Hedrick SM. A deficiency in Drak2 results in a T cell hypersensitivi-
ty and an unexpected resistance to autoimmunity. Immunity. 2004; 21: 781–791. doi: 10.1016/j.immuni. 2004.10.008 PMID: 15589167 2. McGargill MA, Choy C, Wen BG, Hedrick SM. Drak2 regulates the survival of activated T cells and is re-
quired for organ-specific autoimmune disease. J Immunol Baltim Md 1950. 2008; 181: 7593–7605. PMID: 19017948 3. Ramos SJ, Hernandez JB, Gatzka M, Walsh CM. Enhanced T cell apoptosis within Drak2-deficient
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19017949 4. Wang S, Welte T, McGargill M, Town T, Thompson J, Anderson JF, et al. Drak2 contributes to West
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2091. PMID: 18641347 5. Ramos SJ, Hardison JL, Stiles LN, Lane TE, Walsh CM. Anti-viral effector T cell responses and traffick-
ing are not dependent upon DRAK2 signaling following viral infection of the central nervous system. Au-
toimmunity. 2007; 40: 54–65. doi: 10.1080/08916930600996700 PMID: 17364498 6. Schaumburg CS, Gatzka M, Walsh CM, Lane TE. DRAK2 regulates memory T cell responses following
murine coronavirus infection. Autoimmunity. 2007; 40: 483–488. doi: 10.1080/08916930701651139
PMID: 17966037 7. Edwards BA, Harris TL, Floersh H, Lukens JR, Zaki MH, Vogel P, et al. Drak2 is not required for tumor
surveillance and suppression. Int Immunol. 2015; doi: 10.1093/intimm/dxu146 8. Sanjo H, Kawai T, Akira S. DRAKs, novel serine/threonine kinases related to death-associated protein
kinase that trigger apoptosis. J Biol Chem. 1998; 273: 29066–29071. PMID: 9786912 9. Matsumoto M, Miyake Y, Nagita M, Inoue H, Shitakubo D, Takemoto K, et al. A serine/threonine kinase
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(+)/H(+) exchanger. J Biochem (Tokyo). 2001; 130: 217–225. PMID: 11481038 PLOS ONE | DOI:10.1371/journal.pone.0123650
May 7, 2015 13 / 14 Drak2 and TGF-β Signaling in T Cells 10. Newton RH, Leverrier S, Srikanth S, Gwack Y, Cahalan MD, Walsh CM. Protein kinase D orchestrates
the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen
generation. J Immunol Baltim Md 1950. 2011; 186: 940–950. doi: 10.4049/jimmunol.1000942 11. Mao J, Luo H, Han B, Bertrand R, Wu J. Drak2 is upstream of p70S6 kinase: its implication in cytokine-
induced islet apoptosis, diabetes, and islet transplantation. J Immunol Baltim Md 1950. PLOS ONE | DOI:10.1371/journal.pone.0123650
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loop to control TGF-β/Smads signaling by binding to type I TGF-βreceptor. Cell Rep. 2012; 2:
1286–1299. doi: 10.1016/j.celrep.2012.09.028 PMID: 23122956 13. Rubtsov YP, Rudensky AY. TGFbeta signalling in control of T-cell-mediated self-reactivity. Nat Rev
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tion and autoimmune disease. Immunity. 2000; 12: 171–181. PMID: 10714683 18. Doherty GA, Byrne SM, Austin SC, Scully GM, Sadlier DM, Neilan TG, et al. Regulation of the apopto-
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English
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Functional Identification of Api5 as a Suppressor of E2F-Dependent Apoptosis In Vivo
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PLOS genetics
| 2,006
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cc-by
| 13,878
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Functional Identification of Api5
as a Suppressor of E2F-Dependent
Apoptosis In Vivo Erick J. Morris1,2, William A. Michaud1,2,3,4, Jun-Yuan Ji1,2, Nam-Sung Moon1,2, James W. Rocco1,2,3,4,
Nicholas J. Dyson1,2*
1 Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Charlestown, Massachusetts, United States of America, 2 Harvard Medical School,
Boston, Massachusetts, United States of America, 3 Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 4
Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States of America Morris1,2, William A. Michaud1,2,3,4, Jun-Yuan Ji1,2, Nam-Sung Moon1,2, James W. Rocco1,2,3,4,
1 2* 1 Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Charlestown, Massachusetts, United States of America, 2 Harvard Medical School,
Boston, Massachusetts, United States of America, 3 Division of Surgical Oncology, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 4
Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, United States of America Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase
progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors
that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a
model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in
vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic
interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of
both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these
approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical
determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is
specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of
E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11
expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor
cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be
selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation. and the apoptotic machinery are less well defined. Functional Identification of Api5
as a Suppressor of E2F-Dependent
Apoptosis In Vivo E2F-
induced apoptosis is a property associated with some E2F
family members (notably mammalian E2F1 and Drosophila
dE2F1) but not with others [11,12]. After acute DNA damage
in mammalian cells, E2F1 is selectively modified and activates
transcription from a subset of E2F-regulated promoters,
resulting in activation of a large number of apoptotic
regulators (for review, see [13]). However, the relative
importance of these targets appears to be context dependent. In various studies, E2F1-induced apoptosis has been reported
to be p53 dependent, p53 independent, Apaf1 dependent,
Apaf1 independent, and p73 dependent [14–22]. Other studies PLoS Genetics | www.plosgenetics.org * To whom correspondence should be addressed. E-mail: dyson@helix.mgh.
harvard.edu Editor: Bruce Clurman, Fred Hutchinson Cancer Research Center, United States of
America Citation: Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, et al. (2006) Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo. PLoS Genet
2(11): e196. doi:10.1371/journal.pgen.0020196 Abbreviations: Aac11, antiapoptosis clone-11; Api5, apoptosis inhibitor-5; AO,
acridine orange; CycE; cyclin E; dap, dacapo; dIAP1, Drosophila inhibitor of
apoptosis-1; dp, dimerization partner; dsRNA, Double-stranded RNA; E2F, E2-
promoter binding factor; EGFP, enhanced green fluorescent protein; pRB, retino-
blastoma protein; RNAi, RNA interference; rpr, reaper; Tet, tetracycline Citation: Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, et al. (2006) Functional identification of Api5 as a suppressor of E2F-de
2(11): e196. doi:10.1371/journal.pgen.0020196 Generation of E2F-Dependent Phenotypes Generation of E2F-Dependent Phenotypes Generation of E2F Dependent Phenotypes
We sought visible phenotypes that were caused by E2F-
induced apoptosis and were suitable for genetic screening. We used the Gal4-UAS system to express the Drosophila E2f
gene (dE2f1) in a tissue-specific manner [29] and screened a
collection of 50 Gal4 drivers that provided a broad assort-
ment of developmentally regulated patterns. We compared
the effects of expressing dE2f1 with the effects of expressing
known regulators of cell cycle progression such as cyclin E
(CycE), dacapo (dap), and human p21, or apoptosis regulators
such as reaper (rpr) or baculovirus caspase inhibitor, p35
(Figure 1). Each of these transgenes caused lethality when
combined with specific subsets of drivers, and in some cases
gene expression resulted in visible phenotypes. Interestingly,
in this general survey we noted that the consequences of
expressing dE2f1 closely paralleled the effects of expressing
the Drosophila proapoptic gene, rpr, but showed far less
similarity with the effects of expressing the cell cycle
regulator CycE. Because we sought to study dE2F1-dependent
apoptosis, we selected the combinations of transgenes in
which dE2f1 expression gave a visible phenotype that was
phenocopied by the expression of rpr, but not by CycE, and we
used these to generate stable stocks bearing dE2F1-depend-
ent phenotypes. have found E2F1-induced apoptosis to be inhibited by the
expression of either pRB [23] or TopBP1 [24], by the addition
of serum [25], or by the activation of Akt signaling [26]. The
large number of E2F-inducible genes, together with incon-
sistencies between studies carried out in different cell lines,
raises the issue of whether there is one general mechanism of
E2F-induced apoptosis or whether E2F induces apoptosis in
different ways in different cell types. Studies in Drosophila
show that the overall impact of E2F regulation on the DNA-
damage response in vivo varies greatly between cell types
[27]. We found several novel dE2F1-dependent phenotypes that
gave stable stocks and appeared to be amenable to genetic
screening (Figure 1). A dE2F1-dependent wing phenotype,
generated by the Drosophila Actin 88F (Act88F) promoter, was
particularly useful and was characterized in more detail
(Figure 2). Transheterozygous crosses of Act88F-Gal4 and UAS-
dE2f1 produced gnarled and ventrally curved wings that
frequently contained blisters (Figure 2A and 2B). This
phenotype was found to be 100% penetrant and was
phenocopied by expression of other proapoptotic genes
from the same driver, such as rpr (Figure 2C). Generation of E2F-Dependent Phenotypes The expression
of the cell cycle genes CycE (Figure 2D) or string/cdc25 (stg)
(unpublished data), in contrast, gave no significant wing
curvature or gnarling. To date, the study of E2F-induced apoptosis has been
carried out primarily in tissue culture cells, and the analysis
of the factors that are important for this process has been
restricted to the testing of a few candidate genes. Because of
this, it is highly likely that many of the genes that are most
important for E2F-induced apoptosis in vivo have yet to be
identified. To identify these genes, a genetic screening
approach is required. Previous efforts have concentrated on
E2F-stimulated proliferation [28] and, thus far, the genetic
tools needed to study E2F-induced apoptosis have not been
described. To fill this void, we have exploited the Gal4/UAS
misexpression system in Drosophila to generate transgenic
lines with dosage-sensitive phenotypes that are caused by
dE2F1-induced cell death. Here we describe these stocks and
their utility to identify mutations that modify the extent of
E2F-induced apoptosis. Moreover, we link this in vivo
approach with in vitro studies in Drosophila cultured cells
designed specifically to validate the genetic interactions on
apoptosis per se. Using these reagents we show that apoptotic
inhibitor-5/antiapoptosis clone-11 (Api5/Aac11), a highly
conserved family of antiapoptotic proteins that have not
previously been linked to E2F, function as strong and specific
suppressors of E2F-dependent apoptosis. Api5/Aac11 is rate-
limiting for E2F-induced phenotypes in Drosophila in multiple
cell types and developmental contexts, and the genetic
interaction between E2F and Api5/Aac11 is conserved
between Drosophila and human cells. These results illustrate The pattern of expression generated by the Act88F driver
was visualized using a UAS-enhanced green fluorescent protein
(EGFP) transgene. Act88F is expressed during the develop-
ment of flight and thoracic muscles [30]. Consistent with this,
the earliest EGFP expression was detected in indirect flight
muscles of the pupae (unpublished data). In addition, we
observed significant EGFP expression in cells of the newly
eclosed wing blade (Figure 2E–2H). Coexpression of dE2f1
resulted in significantly fewer EGFP expressing cells in the
wing (Figure 2I–2J), a change that we quantified fluorometri-
cally in single flies (Figure 2K). To confirm that the reduction of EGFP-positive cells was
due to apoptosis, wings of newly eclosed adults were stained
with acridine orange (AO) to identify apoptotic cells. In wild-
type discs, very few cells labeled with AO shortly after
eclosion (Figure 2L and 2N). Synopsis Synopsis the value of genetic approaches for the study of E2F-
dependent apoptosis, showing that despite the extensive
molecular studies of the E2F transcriptional program, addi-
tional tiers of regulation exist that have a significant impact
on E2F-induced processes in vivo. The retinoblastoma protein (pRB) was the first human tumor
suppressor to be described, and it works by limiting the activity of
the E2F transcription factor. The pRB pathway is inactivated in most
forms of cancer, and, accordingly, most tumor cells have deregu-
lated E2F. Uncontrolled E2F drives cell proliferation, but it also
sensitizes cells to die (apoptosis). E2F-induced apoptosis is not well
understood, but it affects the development of cancer and,
potentially, could be exploited for cancer treatment. To date,
however, there have been very few studies of E2F-induced
apoptosis in animal models. The authors describe a series of genetic
tools that allow systematic studies of E2F-induced apoptosis in
Drosophila. As validation, this approach identified some known
regulators of E2F-dependent apoptosis and also identified Api5, a
little-studied gene that had not previously been linked to E2F, as a
potent suppressor of E2F-induced cell death. The effects of Api5 on
E2F occur in several different tissues and are conserved from flies to
humans. This last point is significant since Api5 is upregulated in
cancer cells. The discovery of the E2F–Api5 interaction demonstrates
that important modulators of E2F-induced apoptosis are waiting to
be discovered and that they can be found using Drosophila. November 2006 | Volume 2 | Issue 11 | e196 PLoS Genetics | www.plosgenetics.org Introduction A proper balance between cell proliferation and apoptosis
is crucial for organism development and function. Perturba-
tions in this balance underlie a variety of pathological
conditions, including cancer (for review, see [1]). E2-pro-
moter binding factor (E2F) family proteins are important
regulators of cell cycle progression and a major target for
regulation by the retinoblastoma protein (pRB) tumor
suppressor protein family (for review, see [2,3]). The pRB
pathway is functionally inactivated in most tumor cells, and
the resulting change in E2F activity is thought to allow
unchecked cell proliferation. In addition to their ability to drive cell proliferation, E2F
proteins sensitize cells to apoptosis (for review, see [4,5]). E2F-
induced apoptosis limits the consequences of E2F dereg-
ulation to such an extent that tumorigenesis selects not only
for lesions in the pRB pathway but also for mutations that
suppress the apoptotic potential of E2F (for review, see [6,7]). Studies in mice show that apoptosis significantly limits
tumorigenic growth following pRB inactivation, and the cell
types that are most prone to tumorigenesis following the
inactivation of pRB-family members are those that are
intrinsically resistant to apoptosis [8–10]. Received July 10, 2006; Accepted October 3, 2006; Published November 17, 2006 Received July 10, 2006; Accepted October 3, 2006; Published November 17, 2006 Copyright: 2006 Morris et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited. Abbreviations: Aac11, antiapoptosis clone-11; Api5, apoptosis inhibitor-5; AO,
acridine orange; CycE; cyclin E; dap, dacapo; dIAP1, Drosophila inhibitor of
apoptosis-1; dp, dimerization partner; dsRNA, Double-stranded RNA; E2F, E2-
promoter binding factor; EGFP, enhanced green fluorescent protein; pRB, retino-
blastoma protein; RNAi, RNA interference; rpr, reaper; Tet, tetracycline While the effects of E2F on the control of cell cycle
progression are well known, the connections between E2F November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 1834 Api5 Abrogates E2F-Dependent Apoptosis Generation of E2F-Dependent Phenotypes However, dE2f1 expression 1835 November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 Api5 Abrogates E2F-Dependent Apoptosis Figure 1. Summary of the Gal4-Driven Phenotypic Comparison Screen
(A) Gal4 driver lines were crossed to various UAS alleles at 25 8C in order to identify novel dominant phenotypes. Phenotype modification was compared
relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable ‘‘escaper’’ flies
from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P). (B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested. doi:10.1371/journal.pgen.0020196.g001 Figure 1. Summary of the Gal4-Driven Phenotypic Comparison Screen Figure 1. Summary of the Gal4-Driven Phenotypic Comparison Screen
(A) Gal4 driver lines were crossed to various UAS alleles at 25 8C in order to identify novel dominant phenotypes. Phenotype modification was compared
relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable ‘‘escaper’’ flies
from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P). (B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested. doi:10.1371/journal.pgen.0020196.g001 g
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yp
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(A) Gal4 driver lines were crossed to various UAS alleles at 25 8C in order to identify novel dominant phenotypes. Phenotype modification was compared
relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable ‘‘escaper’’ flies
from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P). (B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested. d i 10 1371/j
l
0020196 001 November 2006 | Volume 2 | Issue 11 | e196 PLoS Genetics | www.plosgenetics.org 1836 Api5 Abrogates E2F-Dependent Apoptosis Figure 2. Generation and Characterization of Tissue-Specific dE2F1-Dependent Apoptotic Phenotypes in Drosophila
The Act88F-Gal4 driver was crossed to either a wild-type chromosome (w1118; þ) (A), UAS-dE2f1 (B), UAS-rpr (C), or UAS-CycE (D). Expression of dE2f1 and
rpr, but not CycE, was sufficient to induce a ventrally curved, blistered, and gnarled wing phenotype. In order to visualize expression patterns, Act88F-
Gal4 was crossed to either a wild-type chromosome (Canton-S) (E and F) or UAS-EGFP transgene (G and H). Act88F drives expression in the newly eclosed
adult fly wing blade cells, and coexpression of dE2f1 removes many EGFP-positive cells (I and J). EGFP expression was quantitatively determined in
single transgenic flies by fluorescent spectrophotometry (K). Expression of dE2f1 significantly reduced the levels of EGFP and was rescued by
coexpression of Rbf, dDpDN, or p35, but not p21. Newly eclosed wild-type wings (L and N) or dE2f1-expressing wings (M and O) were stained with AO to
visualize apoptotic cells. Cells expressing dE2f1 had strong, punctuate AO staining in the distal wing region where EGFP is mainly expressed early after
eclosion. DIC, differential interference contract. doi:10 1371/journal pgen 0020196 g002
Api5 Abrogates E2F Dependent Apoptosis racterization of Tissue-Specific dE2F1-Dependent Apoptotic Phenotypes in Drosophila ure 2. Generation and Characterization of Tissue-Specific dE2F1-Dependent Apoptotic Phenotypes in Drosophila The Act88F-Gal4 driver was crossed to either a wild-type chromosome (w1118; þ) (A), UAS-dE2f1 (B), UAS-rpr (C), or UAS-CycE (D). Expression of dE2f1 and
rpr, but not CycE, was sufficient to induce a ventrally curved, blistered, and gnarled wing phenotype. In order to visualize expression patterns, Act88F-
Gal4 was crossed to either a wild-type chromosome (Canton-S) (E and F) or UAS-EGFP transgene (G and H). Act88F drives expression in the newly eclosed
adult fly wing blade cells, and coexpression of dE2f1 removes many EGFP-positive cells (I and J). EGFP expression was quantitatively determined in
single transgenic flies by fluorescent spectrophotometry (K). Expression of dE2f1 significantly reduced the levels of EGFP and was rescued by
coexpression of Rbf, dDpDN, or p35, but not p21. Newly eclosed wild-type wings (L and N) or dE2f1-expressing wings (M and O) were stained with AO to
visualize apoptotic cells. Cells expressing dE2f1 had strong, punctuate AO staining in the distal wing region where EGFP is mainly expressed early after
eclosion. DIC, differential interference contract. induced phenotype, we crossed Act88F-Gal4,UAS-dE2f1 re-
combinant stocks with various UAS and mutant alleles
(Figure 3) and examined their genetic interactions. As
expected, the wing phenotype was completely suppressed by
the coexpression of the Drosophila pRB ortholog, Rbf (Figure
3D), or by the coexpression of a dominant-negative form of
the dE2F1 heterodimerization partner, dDp (Figure 3E). These
proteins also blocked dE2F1-induced apoptosis, as measured
by the loss of EGFP-expressing cells (Figure 2K). Conversely,
expression of additional dE2f1 or the coexpression of func-
tional dDp strongly enhanced the wing defects (Figure 3I). PLoS Genetics | www.plosgenetics.org PLoS Genetics | www.plosgenetics.org Modification of the dE2F1 phenotype was compared
relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study. doi:10.1371/journal.pgen.0020196.g003 Figure 3. Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic Stock Various alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-
Gal4,UAS-EGFP/þ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/þ (w1118) recombinant stock phenotype (B) is strongly
phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent
phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a
heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog. (I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 8C to
various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Modification of the dE2F1 phenotype was compared
relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study. doi:10.1371/journal.pgen.0020196.g003 Drosophila Apaf-1-related killer (dArk) were strong, dominant
suppressors of the Act88F-Gal4,UAS-dE2f1 phenotype (Figure
3H). In addition, loss-of-functional alleles of the endogenous
caspase inhibitor dIAP1 were strong enhancers of the
phenotype (Figure 3I). These interactions indicate that
apoptosome-caspase signaling is required for dE2F1-induced
apoptosis and that this in vivo E2F-dependent phenotype can
be dominantly modified by mutations in downstream
apoptotic signaling pathways. Interestingly, Act88F-Ga-
l4,UAS-dE2f1 was unaltered by the expression of various
dominant-negative alleles of Drosophila p53 (dp53) or by the
introduction of mutant alleles of dp53 (Figure 3I). Similar
results were found with other dE2F1-dependent phenotypes
(unpublished data). In mammalian cells, E2F-induced apop-
tosis can be either p53 dependent or p53 independent (for
review, see [33]); in Drosophila, dE2F1-induced apoptosis
appears to be primarily independent of dp53. Drosophila Apaf-1-related killer (dArk) were strong, dominant
suppressors of the Act88F-Gal4,UAS-dE2f1 phenotype (Figure
3H). In addition, loss-of-functional alleles of the endogenous
caspase inhibitor dIAP1 were strong enhancers of the
phenotype (Figure 3I). These interactions indicate that
apoptosome-caspase signaling is required for dE2F1-induced
apoptosis and that this in vivo E2F-dependent phenotype can
be dominantly modified by mutations in downstream
apoptotic signaling pathways. Interestingly, Act88F-Ga-
l4,UAS-dE2f1 was unaltered by the expression of various
dominant-negative alleles of Drosophila p53 (dp53) or by the
introduction of mutant alleles of dp53 (Figure 3I). Similar
results were found with other dE2F1-dependent phenotypes
(unpublished data). November 2006 | Volume 2 | Issue 11 | e196 Taken together, with the suppression observed by dominant-
negative dDp, these data strongly suggest that the wing
phenotype is dependent on dE2F1-induced transcription. Control UAS transgenes, such as UAS-EGFP and UAS-beta- caused a punctate pattern of AO staining within the blistered
portion of the distal wing blade (Figure 2M and 2O). By 30
min after eclosion, both wild-type and dE2f1-expressing wings
demonstrate significant levels of AO staining (unpublished
data), consistent with previous reports of programmed cell
death in the wing [31]. The punctate pattern of AO staining
induced by dE2F1 was significantly different, and easily
distinguishable, from the diffuse pattern of staining seen
during the later wave of programmed cell death that occurs
in the newly eclosed wing. These observations show that dE2f1
expression induces premature cellular death, and we infer
that these dE2F1-induced changes perturb the multicellular
architecture of the wing epithelia, causing defects that
become set in place during wing maturation. As a further test of the processes involved in this dE2F1- 1837 November 2006 | Volume 2 | Issue 11 | e196 Api5 Abrogates E2F-Dependent Apoptosis Figure 3. Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transg
Various alleles were analyzed for modification of the dE2f1-dependent phenotyp
Gal4,UAS-EGFP/þ (w1118) recombinant stock as control (A). The Act88F-Gal4,UA
phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) co
phenotype was also suppressed by various apoptotic regulators including coexpre
heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog. (I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-G
various transgenic and mutant alleles and phenotypes analyzed in transheterozyg
relative to control chromosome w1118. See Materials and Methods for mutant allel
doi:10.1371/journal.pgen.0020196.g003 Figure 3. Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic Stock
Various alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-
Gal4,UAS-EGFP/þ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/þ (w1118) recombinant stock phenotype (B) is strongly
phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent
phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a
heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog. (I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 8C to
various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity In addition to apoptosis, wing gnarling and blistering can
be induced by a variety of different mechanisms that include
changes in cell fate, adhesion, and proliferation. To confirm
that Aac11 affects dE2F1-dependent apoptosis, rather than
simply causing a synergistic disruption in tissue development,
we moved away from the context in which we had discovered
the connection between Aac11 and dE2F1 and reconstructed
this genetic interaction in cultured Drosophila cells (Figure 6). A dE2F1-expression construct, or lacZ as control protein, was
introduced into SL2 cells together with a GFP-expression
construct that allowed us to visualize the transfected cells. As
expected from the proapoptotic activity of dE2F1, very few
GFP-positive cells were found in dE2F1-transfected cultures
compared to the lacZ control after 48 h (Figure 6A–6D). The
level of GFP expression was measured by fluorimetry, and this
enabled the extent of dE2F1-induced cell killing to be
quantified (Figure 6E). The effects of dE2F1 were both time
and dosage dependent and quantitatively similar to the
changes seen when the proapoptotic Drosophila gene, hid, was
expressed as a positive control (unpublished data). As
expected, the effects of dE2F1 in this assay were inhibited
by the coexpression of RBF1 (Figure 6E). The P-element insertion l(2)k06710 was a strong and
specific enhancer of dE2F1-dependent phenotypes in the
wing, eye, and bristles (Figure 4). The recessive-lethal
l(2)k06710 insertion failed to complement the genomic
deficiency Df(2L)H20, and a similar set of interactions were
observed using this deletion (unpublished data). Df(2L)H20
spans 36A8–9 to 36F1 of Chromosome 2L and uncovers the
l(2)k06710 insertion site within the first exon of the Drosophila
Aac11 gene (Figure 5). Aac11 is a member of the API5 gene family (Figure 5B). Alignment of the human, mouse, frog, fly, mosquito, and
plant API5 homolog products shows a high level of
conservation throughout the majority of the protein, as well
as a number of conserved protein domains (Figure 5C and 5D
and Discussion). Interestingly, the human Aac11/API5 gene,
also known as AAC-11/API5L1/FIF/MIG8 and hereafter re-
ferred to as API5 (NCBI Homologene), is located at
Chromosome 11p12–13 in a region frequently associated
with chromosomal aberrations including amplification in
glioma and breast tumor cells [34,35]. Api5 is an antiapop-
totic protein first described in a mammalian cDNA screen for
prosurvival genes; its expression promoted long-term cell
survival in the absence of serum [36]. In mammalian cells, E2F-induced apop-
tosis can be either p53 dependent or p53 independent (for
review, see [33]); in Drosophila, dE2F1-induced apoptosis
appears to be primarily independent of dp53. galactosidase (lacZ), had no effect on the Act88F-Gal4,UAS-dE2f1
phenotype (Figure 3I). We also tested for dominant interactions with various
alleles of known cell cycle regulators (Figures 2K and 3I). Unlike Rbf, the expression of the human p21 cyclin-depend-
ent kinase inhibitor protein or the Drosophila p21 homolog
dap failed to suppress the Act88F-Gal4,UAS-dE2f1 phenotype. Loss-of-function mutations in CycE, stg, Cdk1, Cdk2, or Cdk4
also failed to suppress the phenotype. Indeed, mutant alleles
of CycE and the overexpression of p21 caused a slight
enhancement of the wing defects. Taken together, with the
inability of CycE or stg to generate a similar phenotype, these
results suggest that the Act88F-driven dE2F1-dependent wing
phenotype is unlikely to be caused by cell cycle activation. A clear pattern of strong genetic interactions emerged
when alleles of apoptotic regulators were tested. The dE2F1-
dependent phenotype was strongly suppressed by coexpres-
sion of the caspase inhibitors baculovirus p35 or Drosophila
thread/inhibitor of apoptosis protein-1 (dIAP1) (Figure 3F and 3G). Accordingly, caspase expression alone phenocopied the
dE2F1 wing phenotype (Figure 3C). Caspase activity is
triggered by activation of apoptosome complexes which
regulate initiator caspase activation (for review, see [32]). Accordingly, heterozygous loss-of-function alleles in the Taken together, these results show that expression of
dE2F1 under the control of the Act88F-Gal4 driver triggers
cell death. Act88F-Gal4,UAS-dE2f1 wings have a visible
phenotype that can be readily modified by transgenes that
affect E2F1 activity and by transgenes that induce or block
cell death. Moreover, this phenotype can be dominantly
enhanced or suppressed by heterozygous mutations in genes November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 1838 Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis Aac11R alleles were combined with various wing Gal4 drivers
(engrailed, apterous, and Act88F), we observed dominant gnarled
and blistered wing phenotypes that resembled the effects of
dE2F1 expression (Figure 4L). These effects were dramatically
enhanced by the presence of a single l(2)k06710 allele (Figure
4M). When tested in the eye, expression of UAS-Aac11R gave
no phenotype alone but strongly enhanced the blackened and
rough eye phenotypes caused by GMR-regulated expression
of dE2f1 and dDp (Figure 4I–4K). Blacked areas of the eye have
been previously described (burned and scorched phenotypes)
and are characterized by pupal disc neurodegeneration [41]. Taken together, these results indicate that RNAi-mediated
depletion of Aac11 is sufficient to enhance dE2F1-induced
phenotypes as well as to induce dominant phenotypes in the
fly wing. encoding known apoptotic regulators. Hence, it represents a
sensitized background that can be used to screen for
mutations that have a significant impact on dE2F1-induced
apoptosis. Identification of Aac11 as a Modifier of dE2F1-Dependent
Phenotypes Identification of Aac11 as a Modifier of dE2F1-Dependent
Phenotypes PLoS Genetics | www.plosgenetics.org Identification of Aac11 as a Modifier of dE2F1-Dependent
Phenotypes We screened a collection of recessive-lethal P-element
transposon insertions for mutations that modified the Act88F-
Gal4,UAS-dE2f1 wing phenotype (see Materials and Methods). Dominant modifiers were retested against four other dE2F1-
dependent phenotypes that were generated in the eye and in
bristle cells and were retested against other apoptotic
phenotypes to assess the specificity of the interactions (see
below). The primary question arising from this type of screen
was whether the modifiers isolated in this way do genuinely,
and specifically, affect E2F-dependent apoptosis. Therefore,
as described below, we have taken one such modifier and have
characterized the interaction in detail. November 2006 | Volume 2 | Issue 11 | e196 Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity Api5 expression has
been linked to tumorigenesis in a number of studies, although
its function is unknown [36–39]. Three lines of evidence confirmed that the loss of GFP in
this assay was due to E2F1-induced apoptosis. First, a
significant increase in the number of Hoechst 33258–positive
apoptotic chromatin-condensed nuclei was observed follow-
ing dE2F1 expression, compared to lacZ-transfected controls
(Figure 6F). Second, transfection of dE2F1 induced the
activation of both initiator and effector caspases (Figure 6G
and 6H). Third, the effects of dE2F1 were blocked by the
coexpression of either RBF1 or the baculovirus caspase
inhibitor p35 (Figure 6E). The interaction between dE2f1 and Aac11 is highly specific;
l(2)k06710 did not modify rough eye phenotypes generated by
the expression of rpr, hid, dp53, CycE, or p21 (Table 1). In
addition, the effects of the Aac11 insertion on dE2F1 are most
likely downstream of RBF1 since the l(2)06710 insertion did
not modify phenotypes from Rbf transgenes (Table 1). l(2)k06710 had no effect on an Act88F-Gal4,UAS-ced3 wing
phenotype that phenocopies Act88F-Gal4,UAS-dE2f1 (Table 1),
demonstrating that the mutation does not indirectly affect
the Act88F promoter or some aspect of Gal4 function. As
control, the Act88F-Gal4,UAS-ced3 phenotype was totally
suppressed by coexpression of p35 but unaffected by Rbf or
dominant-negative dDp (Figure S1). Using this assay, we asked whether Aac11 activity was a
limiting factor for dE2F1-induced apoptosis by measuring the
level of dE2F1-induced death in cells depleted of Aac11 by
RNA interference (RNAi). Cells were treated with luciferase
double-stranded RNA (dsRNA) as nonspecific control, RBF1
dsRNA as positive control, or Aac11 dsRNA for 3 d and then
cotransfected with GFP and either lacZ or dE2F1 (along with
each dsRNA, respectively). As expected, depletion of RBF1
significantly enhanced dE2F1-dependent death compared to
lacZ-transfected controls (Figure 6I). Depletion of Aac11 also
enhanced dE2F1-induced apoptosis to a level that was
comparable to that caused by the depletion of RBF1 (Figure Tissue-specific RNAi was used to confirm that these
interactions were attributed to reduced levels of Aac11. An
inverted repeat specific to Aac11 (exons 1–3) was cloned
downstream of UAS binding sites [40], and transgenic UAS-
Aac11 RNAi (UAS-Aac11R) alleles were established. When UAS- 1839 November 2006 | Volume 2 | Issue 11 | e196 Api5 Abrogates E2F-Dependent Apoptosis Figure 4. Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity Aac11 Loss-of-Function Specifically Enhances Multiple dE2f1-Dependent Phenotypes In Vivo
Through genetic screening, the P-element insertion l(2)k06710 was identified as a strong enhancer of the Act88F-Gal4;UAS-dE2f1 apoptotic wing
phenotype (A). The arrow in (A) indicates enhanced ventral wing curvature and blistering in the l(2)k06710-enhanced dE2f1-dependent wing phenotype. In secondary screening, l(2)k06710 was found to enhance multiple dE2f1-dependent phenotypes in different tissues including a nos-Gal4;UAS-dE2f1
notum bristle phenotype (B–D) and a GMR-Gal4,UAS-dE2f1,UAS-dDp rough eye phenotype (E–H). The l(2)k06710 insertion enhanced bristle degeneration
(compare arrows in [B] and [C]) and bristle stubble (arrowheads in [C]) induced by dE2f1 without inducing bristle phenotypes under heterozygous
conditions alone (D). Expression of a UAS-Aac11 RNAi allele also enhanced the dE2f1-induced rough eye resulting in a blackened phenotype (I–K). Expression of the UAS-Aac11 RNAi allele under engrailed results in dominant posterior wing blistering which is enhanced by the l(2)k06710 P-insertion (L
and M) doi:10.1371/journal.pgen.0020196.g004 PLoS Genetics | www.plosgenetics.org November 2006 | Volume 2 | Issue 11 | e196 1840 Api5 Abrogates E2F-Dependent Apoptosis Figure 5. Aac11 Is a Member of the Api5 Protein Family
(A) P-element l(2)k06710 insertion in the Drosophila Aac11 gene. The genomic locus of Aac11 on Chromosome 2L depicting P-insertion l(2)k06710 (þ187
nucleotides) in Aac11 exon1. This transposon failed to complement Df(2L)H20 covering region 36C9 but not the nearby deficiency Df(2L)VA18. (B) Phylogenetic tree of Api5 family proteins. Nine Homologene-annotated homolog sequences (NCBI) were aligned using MegAlign PhyloTree
(DNAStar software, http://dnastar.com), using a Clustal method with PAM250 residue weight table. Additional species expressed-sequence tags are
present but not included here. (C) Schematic of conserved Api5 protein domains. (D) ClustalW multiple alignment of human, mouse, frog, fly, mosquito, and plant Api5 homologs with gray and black depicting amino acid similarity and
identity, respectively. doi:10.1371/journal.pgen.0020196.g005 il Figure 5. Aac11 Is a Member of the Api5 Protein Family g
p
y
(A) P-element l(2)k06710 insertion in the Drosophila Aac11 gene. The genomic locus of Aac11 on Chromosome 2L depicting P-insertion l(2)k06710 (þ187
nucleotides) in Aac11 exon1. This transposon failed to complement Df(2L)H20 covering region 36C9 but not the nearby deficiency Df(2L)VA18. (B) Phylogenetic tree of Api5 family proteins. Nine Homologene-annotated homolog sequences (NCBI) were aligned using MegAlign PhyloTree
(DNAStar software, http://dnastar.com), using a Clustal method with PAM250 residue weight table. Additional species expressed-sequence tags are
present but not included here. (C) Schematic of conserved Api5 protein domains. November 2006 | Volume 2 | Issue 11 | e196 Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity (D) ClustalW multiple alignment of human, mouse, frog, fly, mosquito, and plant Api5 homologs with gray and black depicting amino acid similarity and
identity, respectively. doi:10.1371/journal.pgen.0020196.g005 6I). Aac11 depletion alone did not result in growth or cell
cycle phenotypes under these conditions (Figure 6K),
indicating that the changes were unlikely to result from a
nonspecific effect on cell number. These data indicate that
endogenous Aac11, like RBF1, suppresses the apoptotic
activity of dE2F1. submaximal levels to ensure that either an increase or a
decrease in transcription could be measured. As expected,
dE2F1 transfection activated transcription from the wild-type
PCNA promoter but not a promoter with mutant E2F binding
sites (Figure 6J). The wild-type PCNA promoter, but not the
E2F-binding mutant, is activated by RBF1 RNAi [27]. However, no change in dE2F1-mediated activation was
observed in Aac11-depleted cells (Figure 6J). This indicates
that Aac11 depletion alters dE2F1-dependent apoptosis
without generally elevating dE2F1-dependent transcription;
hence, Aac11 most likely acts downstream of dE2F1-mediated
transcriptional activation. Although these data suggest that
Aac11 does not generally affect E2F-dependent transcrip- To ask whether Aac11 might repress the transcriptional
activity of dE2F1, we tested whether depletion of Aac11 by
RNAi altered dE2F19s ability to activate transcription. Drosophila SL2 cells were treated with Aac11 dsRNA, or
nonspecific dsRNA to the white gene as control for 5 d, and
then transiently transfected with a wild-type or mutant
PCNA-lucif reporter construct. The reporter was titrated to Table 1. Genetic Interaction of l(2)k06710 in Secondary Screen Phenotypes
Phenotype
Genotype
Phenotype
Interaction
Other dE2F1-dependent phenotypes
nos-Gal4,UAS-dE2f1
Deformed bristles
Enhanced
GMR-Gal4,UAS-dE2f1
Rough eye
Enhanced
GMR-Gal4,UAS-dE2f1,UAS-dDp
Rough eye
Enhanced
Other apoptotic phenotypes
Act88F-Gal4,UAS-ced3
Gnarled wing
No effect
GMR-rpr
Rough eye
No effect
GMR-hid
Rough eye
No effect
GMR-Gal4,UAS-dp53
Rough eye
No effect
Other cell-cycle phenotypes
en-Gal4,UAS-Rbf
Notched wing
No effect
GMR-p21
Rough eye
No effect
GMR-Gal4,UAS-p21
Rough eye
No effect
GMR-Gal4,UAS-cycE
Rough eye
No effect
doi:10.1371/journal.pgen.0020196.t001
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November 2006 | Volume 2 | Issue 11 | e196
1841 Table 1. Genetic Interaction of l(2)k06710 in Secondary Screen Phenotypes November 2006 | Volume 2 | Issue 11 | e196 Figure 6. RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi
(A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss. PLoS Genetics | www.plosgenetics.org Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity (E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35
cotransfection (*p , 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G)
caspase-3 activation, and (H) caspase-9 activation 48 h after transfection. (I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p , 0.01 by t-test). Cell survival was determined by GFP assay 48 h after
transfection. (J) Aac11 RNAi does not affect dE2F1 transcriptional activation of the Drosophila PCNA promoter. (K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div ¼ days in vitro after RNAi). (L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into
media with and without serum, and cell survival was determined 3 d after RNAi. doi:10.1371/journal.pgen.0020196.g006
PLoS Genetics | www.plosgenetics.org
November 2006 | Volume 2 | Issue 11 | e196
1842
Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis Figure 6. RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi Figure 6. RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi sfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss. ti
t
f GFP i dE2F1 t
f
ti
d
t t d i
ifi
t GFP l
f
dE2F1 th t (A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss. (E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35
cotransfection (*p , 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G)
caspase-3 activation, and (H) caspase-9 activation 48 h after transfection. (I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p , 0.01 by t-test). Cell survival was determined by GFP assay 48 h after
transfection. (J) A
11 RNAi d
t
ff
t dE2F1 t
i ti
l
ti
ti
f th
D
hil
PCNA
t (K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div ¼ days in vitro after RNAi). Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity (L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into
media with and without serum, and cell survival was determined 3 d after RNAi. d i 10 1371/j
l
0020196 006 PLoS Genetics | www.plosgenetics.org November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 1842 Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis tional activity, we cannot rule out potential effects of Aac11
on a subset of E2F promoters. of Api5 was also insufficient to block death induced by
vinblastine, staurosporine, rotenone, or tumor necrosis
factor-alpha and had no effect on cell death induced by the
retroviral expression of p53 or p73 (unpublished data). Hence, in human cells, as in Drosophila, Api5 proteins provide
a very specific protection against E2F-induced apoptosis, and
its protective activity distinguishes between these paradigms
of cell death. To test the idea that Aac11 affects dE2F1 in a manner that
is distinct from RBF1, we examined the effects of depleting
both Aac11 and RBF1. No significant changes in cell survival
or proliferation were observed following the depletion of
either RBF1 or Aac11, or both, in the presence of serum
(Figure 6L). However, SL2 cells are more sensitive to
apoptosis following serum deprivation. In these conditions,
cells that would normally survive if Aac11 and RBF1 were
depleted individually died when both proteins were depleted
(Figure 6L). This synthetic lethality confirms that Aac11 and
RBF1 have independent functions and raises the possibility
that Aac11 may act generally to protect RBF1-deficient cells
from E2F-induced apoptosis in contexts where survival
signals are limiting. Tumor studies have shown that Api5 is preferentially
expressed in squamous cell carcinoma versus adenocarcino-
ma in non–small cell lung cancer [38]. We hypothesized that
endogenous Api5 might be an important regulator of survival
in squamous cell carcinoma and tested this by reducing Api5
expression (Figure 8). shRNA constructs to Api5 were
designed, tested for their ability to deplete transfected
FLAG-tagged Api5 (Figure 8A), and then expressed from
lentiviral vectors (LLP) to target the endogenous Api5
protein (51-kDa doublet) in human squamous cell carcinoma
029 cells (JHU-029) (Figure 8B). JHU-029 cells are deficient
for p16INK4a [42] and endogenously express nuclear-localized
Api5 (Figure 8C and 8D). Api5 Suppresses E2F1-Induced Apoptosis in Human
Tumor Cells, and Api5 Depletion Is Tumor Cell Lethal Accordingly, the ability
of E2F1 to induce levels of apoptotic caspase-3 and PARP
cleavage was abrogated by exogenous Api5 (Figure 7F and
7G). However, the expression of Api5 did not prevent E2F1-
mediated induction of the E2F target genes p14ARF and CycE
(Figure 7H). Thus, in human cells, as in Drosophila, Api5
suppresses E2F-induced apoptosis and most likely acts
downstream of E2F-induced transcription, although we
cannot rule out effects on other promoters at the moment. To explore the specificity of Api5 action, control or Api5-
expressing Saos-2 cells were treated with or without
tetracycline, to induce E2F1, in the presence or absence of
the DNA-damaging agent camptothecin. Whereas Api5
protected cells from E2F1-induced death, it failed to protect
against apoptosis induced by camptothecin (Figure 7I). Moreover, the ability of Api5 to protect against E2F1-induced
death was overridden by camptothecin treatment. Expression PLoS Genetics | www.plosgenetics.org Aac11 Inhibits dE2F1-Dependent Apoptosis without
Generally Affecting dE2F1 Transcriptional Activity Compared to control WI38 human
diploid fibroblasts, endogenous Api5 is highly expressed and
RNAi depletion of Api5 resulted in reduced survival with
higher sensitivity in the tumor cells (Figure 8E). In keeping
with the synthetic lethality between RBF1 and Aac11
depletion in SL2 cells, apoptosis was even more evident
when Api5-depleted cells were maintained in low-serum
(unpublished data). Taken together, the extensive pattern of
genetic interactions between E2F1 and Api5, and the
conservation of these interactions from flies to humans,
underscores the significance of Api5 for E2F1-dependent
apoptosis. Depletion of Api5 in E2F-deregulated tumor cells
results in reduced survival, and this raises the possibility that
Api5 may be a useful target for antineoplastic therapy. November 2006 | Volume 2 | Issue 11 | e196 Api5 Suppresses E2F1-Induced Apoptosis in Human
Tumor Cells, and Api5 Depletion Is Tumor Cell Lethal An underlying premise to this work is the idea that our
understanding of E2F-dependent apoptosis is incomplete. Since much of the current information about E2F is derived
from studies in mammalian cells, it is important to know
whether novel functional interactions discovered in a genetic
screen in Drosophila are also relevant to studies of the
mammalian factor. To test the evolutionary conservation of
the genetic interaction between dE2F1 and Aac11, we
examined the effects of their homologs in human cells. Lines
of Saos-2 cells, a human osteosarcoma cell line that is both Rb
and p53 deficient, were generated containing a tetracycline
(Tet)-responsive transgene controlling human E2F1 expres-
sion. A cDNA for the human API5 gene was cloned and used
to generate paired cell lines, with or without exogenous Api5. Using these lines, we examined the effects of elevated E2F1
(Figure 7). E2F1 expression in Saos-2 cells causes extensive apoptosis
(Figure 7A–7D) beginning at approximately 30 h post-
induction and preceded and accompanied by elevated
expression of various E2F target genes such as p14ARF and
CycE (Figure 7H). Tet-induction in this system results in
significant elevation of E2F1 over endogenous as observed by
Western analysis (Figure 7F–7H). Remarkably, the over-
expression of Api5 strongly inhibited E2F1-induced apoptosis
(Figure 7A–7D), giving significantly enhanced cell survival
even when very high levels of E2F1 expression were sustained
for over 1 wk in culture (Figure 7E). Accordingly, the ability
of E2F1 to induce levels of apoptotic caspase-3 and PARP
cleavage was abrogated by exogenous Api5 (Figure 7F and
7G). However, the expression of Api5 did not prevent E2F1-
mediated induction of the E2F target genes p14ARF and CycE
(Figure 7H). Thus, in human cells, as in Drosophila, Api5
suppresses E2F-induced apoptosis and most likely acts
downstream of E2F-induced transcription, although we
cannot rule out effects on other promoters at the moment. E2F1 expression in Saos-2 cells causes extensive apoptosis
(Figure 7A–7D) beginning at approximately 30 h post-
induction and preceded and accompanied by elevated
expression of various E2F target genes such as p14ARF and
CycE (Figure 7H). Tet-induction in this system results in
significant elevation of E2F1 over endogenous as observed by
Western analysis (Figure 7F–7H). Remarkably, the over-
expression of Api5 strongly inhibited E2F1-induced apoptosis
(Figure 7A–7D), giving significantly enhanced cell survival
even when very high levels of E2F1 expression were sustained
for over 1 wk in culture (Figure 7E). Discussion (F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells. (H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells. (I) Api5 expression blocks death induced by E2F1 (þT) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO
vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT. doi:10.1371/journal.pgen.0020196.g007
PLoS Genetics | www.plosgenetics.org
November 2006 | Volume 2 | Issue 11 | e196
1844 Figure 7. Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription
(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background Following E2F1 ind Figure 7. Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription Figure 7. Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription
(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by
treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell de
(E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-do
every other day. (F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells. (H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells. (I) Api5 expression blocks death induced by E2F1 (þT) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DM
vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT. doi:10 1371/journal pgen 0020196 g007 Figure 7. Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription
(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet
treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death. (E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing
every other day. (F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells. Discussion E2F-dependent apoptosis has been implicated in a wide
variety of pathophysiological settings, including DNA damage
signaling, neurodegeneration, and in the consequences of
pRB inactivation in cancer cells (for review, see [5,43,44]). Very little is known about the regulation of E2F-dependent
apoptosis in vivo; most of our current knowledge comes from
studies of cultured cell lines. Because of this paucity of
information, we predict that many of the genes that have the
greatest impact on E2F-dependent apoptosis in vivo have yet
to be identified. Here we describe a series of tools for the study of E2F-
induced apoptosis in Drosophila. Placing dE2F1 expression
under the control of the Act88F-Gal4 driver induces prema-
ture apoptosis in the developing wing, giving a gnarled and
blistered wing phenotype. These effects are dosage sensitive
and can be modified not only by changing the levels or
activity of dE2F1 but also by coexpressing regulators of cell
death and by heterozygous mutations in genes known to
function in cell death pathways. Indeed, similar phenotypes
can be generated by the misexpression of proapoptotic genes
from the same driver. To explore the specificity of Api5 action, control or Api5-
expressing Saos-2 cells were treated with or without
tetracycline, to induce E2F1, in the presence or absence of
the DNA-damaging agent camptothecin. Whereas Api5
protected cells from E2F1-induced death, it failed to protect
against apoptosis induced by camptothecin (Figure 7I). Moreover, the ability of Api5 to protect against E2F1-induced
death was overridden by camptothecin treatment. Expression We note that Drosophila may be particularly advantageous
for the study of E2F-induced apoptosis. Since flies have only
one activator E2F gene, and one DP gene, the way that
signaling pathways converge on E2F may be easier to dissect 1843 November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 Api5 Abrogates E2F-Dependent Apoptosis Figure 7. Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription
(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet
treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death. (E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing
every other day. Discussion (H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells. (I) Api5 expression blocks death induced by E2F1 (þT) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO
vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT. doi:10.1371/journal.pgen.0020196.g007 expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 ind
ental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-indu
cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d afte y
y
(F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells. (H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells. (I) Api5 expression blocks death induced by E2F1 (þT) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO
vehicle control (Veh) Saos-2 cell survival was assayed at 48 h by MTT (H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells. (I) Api5 expression blocks death induced by E2F1 (þT) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO
vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT. doi:10.1371/journal.pgen.0020196.g007 November 2006 | Volume 2 | Issue 11 | e196 PLoS Genetics | www.plosgenetics.org 1844 Api5 Abrogates E2F-Dependent Apoptosis Figure 8. Depletion of Human Api5 in p16INK4a-Deficient Squamous Ce
Carcinoma Cells Results in Reduced Survival versus Normal Huma
Fibroblasts
(A) RNAi-mediated depletion of transfected Api5. Various shRN
constructs were tested for their ability to deplete FLAG-Api5 transfecte
U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfecte
constructs strongly depleted Api5 expression after 3 d as determined b effector caspase activity as reported in mammalian settings in
vivo [20,45,46]. Moreover, we also found that this activity is
dominantly modified by dIAP1 in vivo. However, some aspects
of the mammalian interactions do not appear to be present. For example, we failed to find any evidence for genetic
interactions between dE2f1 and dp53. November 2006 | Volume 2 | Issue 11 | e196 PLoS Genetics | www.plosgenetics.org Discussion Several different path-
ways for E2F-induced apoptosis have been proposed for
mammalian cells, and the Drosophila model may most closely
resemble p53-independent forms of apoptosis induced by the
mammalian E2Fs. Understanding mechanisms of p53-inde-
pendent apoptosis by E2F1 is particularly important given the
high incidence of p53 mutations in human tumors. While the Act88F-Gal4,UAS-dE2f1 wing phenotype provides
a sensitized background to identify modifiers of E2F-depend-
ent apoptosis, modification of this phenotype does not
necessarily implicate a gene in dE2F-induced death. Muta-
tions that have synergistic or antagonistic effects on the
processes of wing development that are disrupted by dE2F1
will also change the severity of the phenotype. In this case, the
genetic interaction would inform us about the context in
which dE2F1 is being expressed but would not give us insight
into specific activities of E2F. A related, but different, issue is
the possibility that a mutation that affects cellular sensitivity
to apoptosis acts in general, rather than being specific to the
process of E2F-induced apoptosis. We therefore designed a
series of secondary assays to distinguish between classes of
modifiers. One way to eliminate developmental context as the reason
for a genetic interaction is to look for interactions in different
tissues and different stages of development. We found that
UAS-dE2f1 expression from three additional drivers (sca-Gal4,
nos-Gal4, GMR-Gal4) gave visible phenotypes that could also be
suppressed by coexpression of RBF1, dominant-negative dDP,
or baculovirus p35 (GMR-Gal4, nos-Gal4; the sca-Gal4 pheno-
type is too severe). These phenotypes can also be dominantly
modified by heterozygous mutations, and we infer that
mutants that genetically interact with dE2F1 in multiple
different contexts are more likely to be informative. Of
course, a potential weakness of this rationale is that if E2F-
dependent apoptosis is controlled by tissue specific mecha-
nisms, then the genetic interactions might only occur in one
specific context. As an alternative strategy that completely
removes any potential contribution from developmental
context, we have also established assays for dE2F1-induced
apoptosis in tissue-culture cells. This assay system allows
candidate genes to be tested by both overexpression and loss-
of-function approaches (RNAi). In addition, the tissue culture
assays allow effects on the level and rate of dE2F1-dependent
apoptosis to be quantified precisely and open the way to more
mechanistic studies of genetic interactors. Figure 8. Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell
Carcinoma Cells Results in Reduced Survival versus Normal Human
Fibroblasts Figure 8. Materials and Methods Fly transgenes, stocks, and crosses. Unless otherwise noted, all fly
crosses were conducted at 25 8C and phenotypes are depicted from
female progeny. The initial Gal4 screen was conducted by crossing
approximately 50 unique Gal4 lines to four different UAS-dE2f1 lines
(3rd, 2BX, 5AII, and 3CII) at 18 8C, 25 8C, and, in some cases, 30 8C. The
recessive-lethal P-element transposon collection (approximately
2,200 lines) was a generous gift from Dr. Spyros Artavanis-Tsakonas
[51] and was F1 screened through the Act88F-Gal4,UAS-dE2f1 wing
phenotype and rescreened against the battery of secondary screens
described in the Results section. We isolated 30 strong suppressors
and two strong enhancers from the primary screen and the secondary
screens narrowed these to ten insertions, in four different loci, that
interacted with at least two additional dE2f1-dependent phenotypes
but failed to modify other apoptosis phenotypes. From these,
mutations in three loci acted as suppressors, and mutation in one
locus was an enhancer (Aac11). The following stocks were used for these
studies (stock identification numbers): w1118, CycA03946, CycEAR95,
stg01235, Cdk12, Cdk405428, Cdk406503, thl(3)j5C8, Aac11k06710 (10645),
Df(2L)H20 (3180), Df(2L)VA18 (6105), GUS-dp53DN259H, GUS-dp53DNCt,
UAS-EGFP (5431), UAS-lacZ (8529), UAS-dE2f1,UAS-dDp (4774), nanos-
Gal4 (4442), GMR-Gal4 (1104), and apterous-Gal4 (G2–1) (Bloomington
stock center); UAS-ced3 (6–6), UAS-caspase-1 (7–1) (Teiichi Tanimura);
UAS-dp53, dp534, dp533þ (Michael Brodsky); dArkCD4 (John Abrams);
engrailed-Gal4, sca-Gal4, UAS-p21, GMR-p21, UAS-dacapo (Iswar Har-
iharan); Act88F-Gal4 (2nd) (Eric Fyrberg); Cdk1E10, UAS-CycE (Christian
Lehner), UAS-rpr, GMR-rpr, UAS-hid, UAS-grim, GMR-hid, UAS-p35,
UAS-dIAP1 (Kristin White). The UAS-Rbf (4) stock was described
previously [52]. The following double-balanced or recombinant stocks
were created for these studies: (a) Act88F-Gal4,UAS-dE2f1 (B2)/CyO ftz
lacZ; (b) Act88F-Gal4,UAS-EGFP (A)/CyO ftz lacZ; (c) Act88F-Gal4,UAS-
EGFP,UAS-dE2f1 (3A)/CyO ftz lacZ; (d) Act88F-Gal4,UAS-ced3 (4)/CyO ftz
lacZ; (e) GMR-Gal4,UAS-dp53/CyO ftz lacZ; (f) GMR-Gal4/CyO; UAS-
dE2f1/TM6b; (g) GMR-Gal4,UAS-dE2f1,UAS-dDp/CyO ftz lacZ; (h) Sca-
Gal4/CyO, UAS-dE2f1/TM6b; (i) nos-Gal4,UAS-dE2f1/CyO ftz lacZ; (j) en-
Gal4,UAS-Rbf/CyO, ftz, lacZ; (k) GMR-Gal4,UAS-p21/CyO ftz lacZ; (l) GMR-
Gal4,UAS-CycE/CyO ftz lacZ; (m) en-Gal4,UAS-Aac11 RNAi (9A)/CyO ftz The discovery that Api5 is a potent suppressor of E2F1-
induced apoptosis adds new significance to these observa-
tions. The genetic interactions described here suggest that
Api5 may contribute to human malignancy by limiting the
extent of E2F1-dependent cell death, and we suggest that this
activity is particularly important when cells need to survive
under suboptimal conditions. Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis Api5 proteins are important and specific determinants of
E2F-induced death. As described here, the evidence for a
functional connection between E2F and Api5 is compelling:
(1) mutation of Aac11 enhances the phenotypes caused by
elevated levels of dE2F1 in the Drosophila wing, eye, and
bristles, (2) RNAi-mediated inhibition of Aac11 enhances
dE2F1-induced phenotypes in vivo and in tissue culture cells,
(3) RNAi depletion of Aac11 not only enhances dE2F1-
induced apoptosis but also is synthetic-lethal with depletion
of RBF1, (4) raising the level of Api5 strongly suppresses
E2F1-induced apoptosis, and (5) depletion of Api5 is
specifically lethal to tumor cells with deregulated E2F. These
genetic interactions are relatively specific in both Drosophila
and human cells; manipulating the levels of Api5/Aac11 did
not affect apoptosis induced by caspases, hid, rpr, p53, or
DNA-damaging agents. Future experiments are now necessary to identify the
molecular functions of Api5/Aac11. Orthologs of the API5
gene family are highly conserved in species as diverse as
plants and humans, but there are no obvious family members
in worms or yeast. Api5 proteins share a number of conserved
domains including a putative transactivation-domain flanked
by two acidic domains, an LxxLL motif, a putative leucine
zipper domain, and a nuclear localization sequence. The
presence of these motifs suggests that Api5 family proteins
might be transcriptional regulators. Various deletion mutants
of Api5 possess strong transactivation activity when fused to
the DNA binding domain of Gal4 [47]. However, to date, no
target genes for Api5 have been described. Perhaps one of the greatest advantages of the Drosophila
system is the opportunity for broad-based genetic screens,
and the tools described here allow novel interactors to be
quickly characterized and categorized. Further screening with
Act88F-Gal4,UAS-dE2f1 could reveal additional mutations
that, like Api5/Aac11, have a major impact on E2F-induced
apoptosis in vivo. One of the most interesting aspects of the
E2F–Api5 genetic interaction is the finding that it has been
conserved between flies and humans during evolution. The
discovery of the connection between Api5 and E2F under-
scores the point that although molecular studies have
provided a great deal of information about the E2F-tran-
scriptional program, not all of the genes that have a major
impact on E2F-induced apoptosis in vivo have been identi-
fied. Api5 Abrogates E2F-Dependent Apoptosis These results illustrate the need for genetic screens for
mutations that have a significant impact on E2F-induced
apoptosis and highlight the potential that components
isolated in this way may be highly relevant in other species. g
g
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API5 was initially isolated as a gene whose expression
promoted cell survival following serum deprivation [36]. Multiple studies have shown that the API5 mRNA transcript is
strongly expressed in transformed cell lines [36,37,47,48]. Consistent with this, we also found Api5 protein levels
significantly elevated in tumor cell lines with known lesions in
the pRB pathway (JHU-029 cells versus WI38 fibroblasts in
Figure 7E and unpublished data). Recently, Api5 expression
was reported to be repressed by myb [49] and activated by
mutant p53 [39]. In this latter study, API5 was one of a cluster
of genes that were upregulated by three different dominant
gain-of-function tumor-derived p53 missense mutants. In-
triguingly, in these cells, whereas wild-type p53 repressed
Api5, the p53 mutant alleles significantly activated Api5
expression. Therefore, the API5 promoter may be specifically
deregulated in tumors cells harboring dominant p53 muta-
tions. In addition to its survival-promoting activity, Api5
overexpression has been reported to induce cervical tumor
cell invasiveness, and its expression has been found to be
upregulated in some metastatic lymph node tissues [37],
raising the possibility that it may be a metastatic oncogene. Api5 expression has been linked to poor prognosis in non–
small cell lung cancer, and particularly in squamous cell
carcinoma [38]. PLoS Genetics | www.plosgenetics.org Discussion Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell
Carcinoma Cells Results in Reduced Survival versus Normal Human
Fibroblasts (A) RNAi-mediated depletion of transfected Api5. Various shRNA
constructs were tested for their ability to deplete FLAG-Api5 transfected
U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected
constructs strongly depleted Api5 expression after 3 d as determined by
anti-FLAG immunoblot. (B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells
were infected with lentiviral encoding scramble or API5-AB shRNAs and
selected with puromycin for 3 d. Expression of endogenous Api5 was
determined by immunoblotting with affinity-purified anti-Api5 poly-
clonal antibody (G3162). y
(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and
excluded from the nucleolus. JHU-029 cells were stained with the G3162
polyclonal antibody after 4% paraformaldehyde fixation. p y
y
p
y
(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to
normal human fibroblasts. JHU-029 cells, as well as normal human WI38
diploid fibroblasts, were infected with lentiviral empty vector or vectors
encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10%
fetal calf serum–containing media. Cell survival was determined by MTT
assay at the indicated days post–lentiviral infection. Api5 and control
actin expression was determined from equally loaded protein from day 3
lysates. Mutations that specifically affect dE2F1-induced apoptosis
could be distinguished from mutations that modify apoptosis
in general using Act88F-Gal4,UAS-ced3. Heterologous misex-
pression of this C. elegans caspase from the same Act88F-Gal4
driver gave a phenotype that was very similar to Act88F-
Gal4,UAS-dE2f1 but was insensitive to changes in E2F activity. As an alternative, Act88F-Gal4,UAS-human caspase-1 could also
be used for this purpose (unpublished data). Other transgenes
such as GMR-hid and GMR-rpr provided additional tests for
specificity, using specific proapoptotic molecules and the eye
rather than the wing as the context to score interactions. y
doi:10.1371/journal.pgen.0020196.g008 in Drosophila than in mammalian cells (eight E2F and three DP
genes have been described to date). Several of the known
features of E2F1-induced apoptosis in mammalian cells are
conserved in flies. For example, in the Drosophila wing, we find
that dE2F1-dependent death is regulated by both dArk/Apaf1-
dependent apoptosome function and requires downstream Using these tools, we made the unexpected finding that November 2006 | Volume 2 | Issue 11 | e196 1845 Api5 Abrogates E2F-Dependent Apoptosis Materials and Methods Api5 fusion protein was prepared and used to inject two rabbits for
polyclonal production (Genemed Synthesis, http://www.genemedsyn. com). Two bleeds were screened against transfected tagged and
untagged full-length Api5 to verify antigenicity. Positive bleeds were
affinity-purified against PVDF membrane–bound GST-Api5, eluted
with 100 mM glycine (pH 2.5), and Centricon-purified. Specificity of
the pAB3162 affinity-purified antibody was confirmed using both
transfected and endogenous Api5 with and without shRNA depletion
of the specific bands. Creation of inducible E2F1 and stable Api5–expressing Saos-2 cells. The Tet-inducible Saos-2 cell line was created by transfecting
pCDNA6-TR (Invitrogen) into Saos-2 and selecting blasticidin (2.5
lg/ml)-resistant clones to create Saos-2-TR. The E2F1 cDNA was
cloned into pCDNA4-TO (Invitrogen) and transfected into Saos-2-TR,
and blasticidin (2.5 lg/ml)- plus zeocin (100 lg/ml)-resistant clones
were isolated and tested for induciblity with 0.1 lg/ml tetracycline. Saos2-TR-E2F1 was transformed with retrovirus containing either
pLPC (a gift from Scott Lowe) or pLPC containing HA-tagged API5
cDNA (cloned from human cDNA library) and selected with 1 lg/ml
puromycin to create cells stably expressing Api5. Creation of inducible E2F1 and stable Api5–expressing Saos-2 cells. The Tet-inducible Saos-2 cell line was created by transfecting
pCDNA6-TR (Invitrogen) into Saos-2 and selecting blasticidin (2.5
lg/ml)-resistant clones to create Saos-2-TR. The E2F1 cDNA was
cloned into pCDNA4-TO (Invitrogen) and transfected into Saos-2-TR,
and blasticidin (2.5 lg/ml)- plus zeocin (100 lg/ml)-resistant clones
were isolated and tested for induciblity with 0.1 lg/ml tetracycline. Saos2-TR-E2F1 was transformed with retrovirus containing either
pLPC (a gift from Scott Lowe) or pLPC containing HA-tagged API5
cDNA (cloned from human cDNA library) and selected with 1 lg/ml
puromycin to create cells stably expressing Api5. p
y
g
y
AO staining and EGFP quantification in single flies. AO staining of
apoptotic cells in the wing was performed as described [54]. AO
positive cells were visualized with fluorescent microscopy under FITC
filters (excitation k 490 nm, emission k 520 nm) and pseudocolor-
depicted. As positive control, we detected AO positive cells in the
posterior compartment of newly eclosed wings from en-Gal4/UAS-dp53
progeny (unpublished data). EGFP fluorescence in individual newly
eclosed female flies was quantified in single flies as described [55]. EGFP fluorescence was determined at excitation k 488 nm, emission k
511 nm (cutoff k 495 nm) with buffer background subtraction. Fluorescent measurements were determined within a linear range
from a single UAS-EGFP transgene; two UAS-EGFP transgenes
produced 23 relative fluorescent units (RFU) (unpublished data). Materials and Methods We did not detect squelching since expression of control protein
from a single UAS-lacZ transgene had no effect on EGFP fluorescence
(unpublished data). shRNA construction and lentiviral infection. A series of API5
targeting shRNAs were created in pBS-U6 as described [61]. Targeting
sequences were as follows: API5-AB 59-GGCCAGCATAAAGATGCC-
TAT-39; API5-CD 59-GGGTTGTTCAGCCAAATACTT-39; API5-EF 59-
GGCCGACCTAGAACAGACCTT-39. Sequences were subcloned from
pBS-U6 into Lentiviral vector LLP, and high-titer lentivirus was
produced as previously described [62]. Cell culture and transient transfections. Drosophila Schneider line 2
(SL2 cells; ATCC, http://www.atcc.com) and mammalian cells were
grown as previously described [52,56]. Drosophila cell (CellFectin
reagent; Invitrogen, http://www.invitrogen.com) and human cell
(Fugene-6 reagent; Roche, http://www.roche.com) transfections were
performed according to the manufacturer’s recommended instruc-
tions. Luciferase reporter assays were performed as described [52]. The wild-type and E2F-binding mutant PCNA-luciferase reporter was
kindly provided [57]. Accession Numbers The National Center for Biotechnology Information (NCBI) (http://
www.ncbi.nlm.nih.gov) GeneID numbers for genes (and products)
discussed herein are H. sapiens API5 (8539), CASP1 (834), CASP3 (836),
E2F1 (1869), TP53 (7157), TP73 (7161), CCNE1 (898), CycE/CDKN1A
(1026), ARF/CDKN2A (1029), RB1 (5925); D. melanogaster E2f (42550),
Dp (36461), Aac11 (35053), Ark (36914), rpr (40015), cdk1/cdc2 (34411),
cdk2/cdc2c (42453), cdk4 (36854), hid/W (40009), PCNA/mus209 (37290),
dIAP1/thread (39753), dap (36001), CycA (39340), CycE (34924), grim
(40014), p53 (2768677), Rbf (31027), stg (43466), Act88F (41885); C. elegans ced3 (178272); baculovirus p35 (1403968). p
f
p
f p
p
p
Flow cytometric analysis. Cell cycle analysis using FACS CellQuest
(Becton Dickinson, http://www.bd.com) of ethanol-fixed, propidium
iodide–stained SL2 cells was performed as described [60]. Supporting Information Figure S1. Genetic Characterization of a Recombinant Act88F-
Gal4,UAS-ced3 Transgenic Stock Various alleles were analyzed for modification of the ced3-dependent
phenotype in trans. Coexpression of p35 (C), but not Rbf (D) or
dominant-negative dDp (E), completely suppressed the ced3 caspase
wing phenotype. (F) The l(2)06710 Aac11 mutant does not modify the
ced3-dependent phenotype. y p
Apoptosis and viability assays. Caspase-3 (DEVD-AFC peptide
substrate) and caspase-9 (LEHD-AFC peptide substrate) enzymatic
assays (R&D Systems, http://www.rndsystems.com) were performed
according to the manufacturer’s recommendations. Cleaved, acti-
vated fluorescent substrate was measured in RFU and normalized to
total protein content (Bio-Rad, http://www.bio-rad.com). Cell viability
was determined by 0.4% trypan blue exclusion hemocytometry or
MTT assay [58]. Apoptotic chromatin condensation was assayed in
GFP-cotransfected live cells by incubating cultures with membrane-
permeant 50 lg/ml Hoechst 33258 dye (20 min; room temperature). Nuclei with apoptotic condensed chromatin was visualized under
epifluorescence and scored in five nonoverlapping fields per
condition expressed relative to total transfected. Transfected SL2
cell survival in six-well trays was determined by GFP cotransfection
viability assay [59] with 0.3 lg/well pAct-GFPUS9 (Act5c promoter)
expression construct. Cells were harvested by trituration, pelleted by
centrifugation, resuspended in PBS, and transferred to 96-well trays
for GFP fluorescence quantification. Plasmids used for these studies
were pIE4-dE2f1, pIE4-Rbf, pIE4-lacZ, and pIE4-p35. Found at doi:10.1371/journal.pgen.0020196.sg001 (1.2 MB PDF). Found at doi:10.1371/journal.pgen.0020196.sg001 (1.2 MB PDF). Acknowledgments p
[
]
Drosophila SL2 cell RNAi. All RNAi for Drosophila SL2 cells was
performed as described [52]. Double-stranded RNA was synthesized
with T7 RiboMax (Promega, http://www.promega.com). Cells were
RNAi depleted using 50 lg of dsRNA for each gene and normalized
with luciferase dsRNA for co-RNAi treatments. For RNAi-trans-
fection experiments, 15 lg of dsRNA was included in each trans-
fection after the initial RNAi depletion. Depletion of RBF1 was
confirmed by Western analysis (unpublished data). Depletion of
Aac11 RNA was confirmed on microarray analysis (unpublished data). Western analysis, antibodies, and immunocytochemistry. Western
blot and immunohistochemical analysis was performed using stand-
ard techniques. Antibodies used in this study include those against
cleaved caspase-3 (9661; Cell Signaling, http://www.cellsignal.com),
PARP (Ab2; EMD Biosciences, http://www.emdbiosciences.com), E2F1
(SC193; Santa Cruz Biotechnology, http://www.scbt.com), HA (Clone-
11; Covance, http://www.covance.com), FLAG (M2; Sigma, http://
www.signaaldrich.com), cyclin E (SC247; Santa Cruz Biotechnology),
and p14ARF (Ab2; NeoMarkers, Lab Vision Corporation, http://www. labvision.com). The Api5 polyclonal antibody was created by
subcloning full-length human API5 cDNA into pGEX, and GST- Drosophila SL2 cell RNAi. All RNAi for Drosophila SL2 cells was
performed as described [52]. Double-stranded RNA was synthesized
with T7 RiboMax (Promega, http://www.promega.com). Cells were
RNAi depleted using 50 lg of dsRNA for each gene and normalized
with luciferase dsRNA for co-RNAi treatments. For RNAi-trans-
fection experiments, 15 lg of dsRNA was included in each trans-
fection after the initial RNAi depletion. Depletion of RBF1 was
confirmed by Western analysis (unpublished data). Depletion of
Aac11 RNA was confirmed on microarray analysis (unpublished data). We thank the CBRC Transgenic Fly Core for embryo injections and
the Bloomington Drosophila Stock Center for the provision of
numerous stocks for this study. We also thank various investigators
for their generous gifts of fly stocks (see Materials and Methods). JHU-
029 cells were a generous gift from Dr. David Sidransky. Retrovirus
(pLPC) was a generous gift from Dr. Scott Lowe. We thank Drs. Max
Frolov, Olivier Stevaux, Takafumi Katayama, and Zhigang Xie for
technical assistance and our colleagues for valuable discussions. Author contributions. EJM, WAM, JWR, and NJD conceived and
designed the experiments. EJM, WAM, JYJ, JWR, and NJD performed
the experiments. EJM, WAM, JYJ, NSM, JWR, and NJD analyzed the
data. EJM, WAM, JYJ, NSM, JWR, and NJD contributed reagents/
materials/analysis tools. EJM and NJD wrote the paper. Western analysis, antibodies, and immunocytochemistry. Western
blot and immunohistochemical analysis was performed using stand-
ard techniques. Materials and Methods The synthetic-lethality ob-
served with Aac11 and RBF1 depletion in low serum further
suggests that Api5 might also promote the survival of tumor
cells harboring pRb-inactivating mutations. Identification of
new synthetic-lethal interactions is an important goal in
developing cancer-specific therapies that should, in theory,
reduce toxicity to normal cells [50]. Accordingly, RNAi-
mediated depletion of Api5 resulted in enhanced cell death
of p16-deficient squamous cell carcinoma cells, as compared
to normal human fibroblast controls. Although future studies
will be necessary to fully characterize the crosstalk between
E2F and Api5 signaling pathways, these findings indicate that
the levels of Api5 are likely to be very important for the
survival of human tumor cells with deregulated E2F. Hence,
Api5 may be an exploitable target for antineoplastic treat-
ment, particularly in tumors with pRb inactivation. 1846 November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis lacZ; and (n) en-Gal4,UAS-Aac11 RNAi (9A)/T(2:3) CyO TM6b. The UAS-
dDp dominant-negative allele (Chromosome 3) was created by subclon-
ing the dDp113–337 amino-acid fragment into the pUASt vector [29]. The UAS-Aac11 RNAi allele was created by subcloning an inverted
repeat of an XbaI fragment containing Aac11 59 UTR-exon3
sequences (primer set,
59-
GCGCTCTAGAGCTGTCTCGA-
GATCTGGTCACTC and 59-GCGCTCTAGAGCGTTTCCCTGGCA-
CAGTTTC) into the pWIZ vector [40]. P-element transformation
was performed as described [53]. All transgenic fly embryo injections
were performed by the CBRC Transgenic Fly Core. Api5 fusion protein was prepared and used to inject two rabbits for
polyclonal production (Genemed Synthesis, http://www.genemedsyn. com). Two bleeds were screened against transfected tagged and
untagged full-length Api5 to verify antigenicity. Positive bleeds were
affinity-purified against PVDF membrane–bound GST-Api5, eluted
with 100 mM glycine (pH 2.5), and Centricon-purified. Specificity of
the pAB3162 affinity-purified antibody was confirmed using both
transfected and endogenous Api5 with and without shRNA depletion
of the specific bands. Api5 fusion protein was prepared and used to inject two rabbits for
polyclonal production (Genemed Synthesis, http://www.genemedsyn. com). Two bleeds were screened against transfected tagged and
untagged full-length Api5 to verify antigenicity. Positive bleeds were
affinity-purified against PVDF membrane–bound GST-Api5, eluted
with 100 mM glycine (pH 2.5), and Centricon-purified. Specificity of
the pAB3162 affinity-purified antibody was confirmed using both
transfected and endogenous Api5 with and without shRNA depletion
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32. Acknowledgments Antibodies used in this study include those against
cleaved caspase-3 (9661; Cell Signaling, http://www.cellsignal.com),
PARP (Ab2; EMD Biosciences, http://www.emdbiosciences.com), E2F1
(SC193; Santa Cruz Biotechnology, http://www.scbt.com), HA (Clone-
11; Covance, http://www.covance.com), FLAG (M2; Sigma, http://
www.signaaldrich.com), cyclin E (SC247; Santa Cruz Biotechnology),
and p14ARF (Ab2; NeoMarkers, Lab Vision Corporation, http://www. labvision.com). The Api5 polyclonal antibody was created by
subcloning full-length human API5 cDNA into pGEX, and GST- Funding. This work was supported by National Institutes of Health
grants PO1CA095281 and GM53203 (to NJD) and F32CA88474 NRSA
(to EJM) and by fellowships from the Canadian Institute of Health
Research and the MGH Fund for Medical Discovery (to NSM). Competing interests. The authors have declared that no competing
interests exist. PLoS Genetics | www.plosgenetics.org November 2006 | Volume 2 | Issue 11 | e196 1847 Api5 Abrogates E2F-Dependent Apoptosis Api5 Abrogates E2F-Dependent Apoptosis amplification analysis of human glioma tissue and glioma derived fragment
spheroids using reverse chromosome painting (RCP). Anticancer Res 16:
2601–2606. amplification analysis of human glioma tissue and glioma derived fragment
spheroids using reverse chromosome painting (RCP). Anticancer Res 16:
2601–2606. References Adams JM (2003) Ways of dying: Multiple pathways to apoptosis. Genes Dev
17: 2481–2495. 62. Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, et al. (2003) A
lentivirus-based system to functionally silence genes in primary mamma-
lian cells, stem cells and transgenic mice by RNA interference. Nat Genet
33: 401–406. 33. Bell LA, Ryan KM (2004) Life and death decisions by E2F-1. Cell Death
Differ 11: 137–142. 34. Janka M, Fischer U, Tonn JC, Kerkau S, Roosen K, et al. (1996) Comparative M, Fischer U, Tonn JC, Kerkau S, Roosen K, et al. (1996) Compara PLoS Genetics | www.plosgenetics.org November 2006 | Volume 2 | Issue 11 | e196 November 2006 | Volume 2 | Issue 11 | e196 PLoS Genetics | www.plosgenetics.org 1848
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Study of the Surface and Dimensional Quality of the AlSi10Mg Thin-Wall Components Manufactured by Selective Laser Melting
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Citation: Waqas, M.; He, D.; Elahi,
H.; Riaz, S.; Eugeni, M.; Gaudenzi, P. Study of the Surface and Dimensional
Quality of the AlSi10Mg Thin-Wall
Components Manufactured by
Selective Laser Melting. J. Compos. Sci. 2021, 5, 126. https://doi.org/
10.3390/jcs5050126 Citation: Waqas, M.; He, D.; Elahi,
H.; Riaz, S.; Eugeni, M.; Gaudenzi, P. Study of the Surface and Dimensional
Quality of the AlSi10Mg Thin-Wall
Components Manufactured by
Selective Laser Melting. J. Compos. Sci. 2021, 5, 126. https://doi.org/
10.3390/jcs5050126 Academic Editor: Nikolaos
G. Semaltianos Keywords: selective laser melting; AlSi10Mg; surface roughness; dimensional accuracy Received: 11 April 2021
Accepted: 7 May 2021
Published: 9 May 2021 Received: 11 April 2021
Accepted: 7 May 2021
Published: 9 May 2021 Study of the Surface and Dimensional Quality of the
AlSi10Mg Thin-Wall Components Manufactured by
Selective Laser Melting Muhammad Waqas 1, Dingyong He 1
, Hassan Elahi 2,*
, Saleem Riaz 3
, Marco Eugeni 2
d P
l
G
d
i 2 Muhammad Waqas 1, Dingyong He 1
, Hassan Elahi 2,*
, Saleem Riaz 3
, Marco Eugeni
and Paolo Gaudenzi 2 1
Faculty of Materials and Manufacturing, Beijing University of Technology, No.100, Ping Le Yuan,
Beijing 100124, China; waqasm082@yahoo.com (M.W.); dyhe@bjut.edu.cn (D.H.) j
g
q
y
(
)
y
j
(
)
2
Department of Mechanical and Aerospace Engineering, Sapienza University of Rome, 00185 Ro
marco.eugeni@uniroma1.it (M.E.); paolo.gaudenzi@uniroma1.it (P.G.) g
(
); p
g
(
)
3
School of Automation, Northwestern Polytechnical University, Xi’an 170072, China;
saleemriaznwpu@mail.nwpu.edu.cn *
Correspondence: hassan.elahi@uniroma1.it Abstract: Additive manufacturing (AM), a 3D printing technique that manufactures components by
sequential addition of powder, has massively reshaped the manufacturing and engineering sectors
from batch production to manufacturing customized, innovative, state-of-the-art, and sustainable
products. Additive manufacturing of aluminum alloys by selective laser melting (SLM) is one of
the latest research trends in this field due to the fact of its advantages and vast applications in
manufacturing industries such as automobiles and aerospace. This paper investigated the surface
and dimensional quality of SLM-built AlSi10Mg parts using a response surface method (RSM) and
found the influence of the wall thickness and process parameters (i.e., laser power, scanning speed,
hatch distance) on the pieces. Thin-walled test specimens of AlSi10Mg alloy were manufactured
with different combinations of process parameters at three wall thicknesses: 1.0 mm, 2.0 mm,
and 3.0 mm. The Minitab DOE module was used to create 27 different configurations of wall
thickness and process parameters. The samples’ surface roughness and dimensional accuracy were
investigated, and the findings were evaluated using the ANOVA technique. The regression model
and the ANOVA technique showed high precision and had a particular reference value for practical
engineering applications. Citation: Waqas, M.; He, D.; Elahi,
H.; Riaz, S.; Eugeni, M.; Gaudenzi, P.
Study of the Surface and Dimensional
Quality of the AlSi10Mg Thin-Wall
Components Manufactured by
Selective Laser Melting. J. Compos. Sci.
2021, 5, 126. https://doi.org/
10.3390/jcs5050126 1. Introduction So far, the focus of research in 3D printing has been the production of parts
using polymer materials, but the invention of new AM techniques, including selective
laser sintering/melting, laser engineered net shaping, and electron beam melting, made it
possible to manufacture metallic, ceramic, and composite parts. The automobile, aerospace,
military production, and biomedical industries are leading sectors utilizing additively
manufactured parts [4,9–11]. The employment of AM techniques has granted designers
high freedom of design and enabled manufacturing industries to produce topologically
optimized components with better strength-to-weight ratios, an essential characteristic in
both automobile and aerospace designs in order to produce light-weight products. AM
has enormously revolutionized the biomedical industry with customized production of
prosthetic parts, dental implants, hearing aids, and orthopedic parts to suit the unique
needs of an individual’s physiology. AM has rapidly revamped the manufacturing process
within the last ten years and produced practically functional components, making these
techniques commercially popular [6,9,12]. Surface roughness is one of the most important factors when evaluating the surface
quality of components, and roughness has a major impact on the mechanical properties
that affect the service life of components. Therefore, the role of surface roughness in the
SLM parts has been investigated by many researchers. To discover the related factors
influencing surface roughness, various scientific studies have been carried out. g
g
The effects of laser power and scan speed on the surface roughness of the printed
aluminum alloy samples were investigated by Dad bakhsh et al. [13]. Calignano et al. [14]
concluded that with direct metal laser sintering (DMLS), the scanning speed has a signifi-
cant impact on the surface roughness. Leary [15] conducted a series of surface roughness
optimizations for Ti6Al4V specimens. The effect of input LEDs on the surface roughness of
aluminum alloy specimens with DMLS was studied by Mohammadi and Asgari [16], but
it was confined to the construction plane. Wang et al. investigated the surface roughness
of SLM-built AlSi10Mg parts and established that the surface roughness first decreases
noticeably and then rises gradually with the increase in laser energy density [17]. The
SLM process parameters’ effect on surface roughness was studied by Tian et al., and it
was observed that surface roughness increased with small hatch distances that led to the
adhesion of particles on the surface. They also found that good surface quality can be
achieved by better scan strategies [18]. Rakesh et al. 1. Introduction Additive manufacturing (AM) techniques, along with newly developed alloys and
conventional manufacturing processes, possess the ability to transform the method of
production of industrial products. AM methods are often mentioned as layered manufac-
turing, owing to the nature of the procedure which involves layer-by-layer fabrication of
a part while using CAD data from a computer [1–3]. AM is a generally used term in the
manufacturing industry, but autofab (auto fabrication), freeform manufacturing, powder
metallurgy, stereo lithography, layer-based manufacturing, and 3D printing are also com-
monly used terminologies while referring to additive manufacturing [2]. In the late 1980s,
manufacturing industries started using additive manufacturing techniques to produce pro-
totypes of their products for the purpose of assessing the difficulties associated with their
geometry, design, and form along with their usefulness. At the end of 1990s and in the early
2000s, due to the great improvements in the reproducibility and reliability of the method,
and while referring to metallic materials, owing to the employment of high-energy density
sources able to consolidate these metallic materials, application of AM techniques and 3D Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
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terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/jcs J. Compos. Sci. 2021, 5, 126. https://doi.org/10.3390/jcs5050126 J. Compos. Sci. 2021, 5, 126 2 of 16 printing began for the production of final products [2,4–6]. Nowadays, additive manufac-
turing has replaced some traditional production processes, for instance, casting and forging,
especially in some specific manufacturing industries, e.g., the automotive and aerospace
industries. The main factors for the adoption of AM in these industries are its advantages
over conventional manufacturing process such as the high degree of freedom in design
and complexity of geometry, the ability to manufacture freeform surfaces, amalgamation
of many components into one-part, minimal requirement of work tools, and the ability to
customize production [4,7–9]. Over the last two decades, AM has been transformed from
rapid prototyping-oriented 3D printers to advanced production systems having the ability
to produce fully functional components of different materials with minimal involvement
of tooling. 2.1. Equipment and Material To manufacture the sample under study, a gas-atomized AlSi10Mg powder material
was used, which is commonly used for aluminum moldings. The powder was supplied by
the PAC Corporation (USA). An SLM 280HL machine of SLM solutions was employed to
produce the sample. The aluminum substrate was heated to 150 ◦C to avoid the thermal
stresses. Prior to the start of fabrication, the AlSi10Mg powder was dried out by exposing
it to a temperature of 70 ◦C in a drying oven for 4 h. The sieving of the powder was
performed in order to filter out the particles between the size of 18–58 mm for onward
production. The machine used, an SLM 280HL, was supplied with the maximum scan
speed of 10 m/s and two fiber lasers each with laser power of 400 watts. Build platform
used to produce samples consisted of a 280 mm × 280 mm square plate. The selection of
suitable process parameters is very significant for accomplishing the best results from the
SLM technique. The scan speed, hatch distance, and laser power are vital parameters in the
SLM process. The almost eutectic structure of AlSi10Mg in the Al–Si phase diagram and
a melting temperature of 570 ◦C make it one of the most suitable and widely used alloys
in casting. This alloy, due to the fact of its hardness and good strength, is used for thin
wall and intricate geometry components prone to high loads such as in automotive and
aerospace productions [27,28]. The chemical composition of AlSi10Mg is given below in
Table 1. Table 1. Composition of AlSi10Mg alloy powder as per DIN EN 1706. ELEMENT
Si
Fe
Cu
Mn
Mg
Zn
Ti
Al
Weight %
9–11
≤0.55
≤0.05
≤0.45
0.2–0.45
≤0.1
≤0.15
Balance
2.2. Powder Characterization
The morphology of the AlSi10Mg powder particles was examined on a scanning
electron microscopy (SEM) and images were taken by a Tescan VEGA 3 LMU Scanning
Electron Microscope system as shown in Figure 1. A laser particle analyzer (Beckman
Coulter LS 13,320) was used to measure the size of powder particles. The powder particles’
size varied from an average diameter of 25–65 µm; however, some particles also had
diameters smaller and larger than the abovementioned range, having different spherical
and elliptical forms. The density of the loose powder was 2.68 gm/cm3 as per the data
sheet provided by PAC Corporation. Table 1. 1. Introduction compared the effects of argon- and
nitrogen-built environments for SLM of AlSi10Mg and concluded that samples built in a
nitrogen atmosphere reported smoother surfaces and better mechanical properties [19] nitrogen atmosphere reported smoother surfaces and better mechanical properties [19]. Han et al. [20] focused on the various process parameters that influenced dimensional
accuracy and recognized that a high scan speed, resulting in high densification, can help
to improve dimensional accuracy. Majeed et al. [21] studied the surface and dimensional
quality of SLM-built AlSi10Mg parts and defined a set of optimal process parameters. Maamoun et al. investigated the effects of the SLM process parameters on the dimensional
accuracy of the AlSi10Mg parts. The results reported a dimensional tolerance variation
from an oversize of 0.15–0.195 mm [22]. Sing et al. stated that the geometrical precision
of the lattice structures can be enhanced by cautious variations of the process parameters. The research concluded that laser power, as compared to layer thickness and scan speed,
greatly influences the dimensional accuracy of lattice structures built by SLM [23]. The J. Compos. Sci. 2021, 5, 126 3 of 16 3 of 16 dimensional quality of SLM- and DMLS-built internally cooled cutting tools was studied by
Ghani et al. They concluded that, with respect to dimensional accuracy, the DMLS method
performed better than the SLM method [24]. Fateri et al. investigated the SLM of glass,
finding that both high- and low-energy intensities led to poor dimensional quality causing
bulging and poor bonding, respectively. They showed that 100%-dimensional accuracy
is attainable by selection of the optimal process parameters [25]. Wang et al. studied the
SLS process parameters’ effect on dimensional accuracy and concluded that the accuracy
of SLS-built parts can be improved with a higher laser power, lower scanning speed, and
maximum permissible temperature of working atmosphere [26]. It is evident from the literature that there are a number of SLM process parameters that
can be controlled and varied so as to obtain the desirable surface and dimensional quality
of the parts. This study aimed to investigate the effect on the surface and dimensional
quality of SLM-built test specimens of various combinations of laser power, scan speed,
hatch distance, and wall thicknesses. 2.1. Equipment and Material Composition of AlSi10Mg alloy powder as per DIN EN 1706. Table 1. Composition of AlSi10Mg alloy powder as per DIN EN 1706. ELEMENT
Si
Fe
Cu
Mn
Mg
Zn
Ti
Al
Weight %
9–11
≤0.55
≤0.05
≤0.45
0.2–0.45
≤0.1
≤0.15
Balance Table 1. Composition of AlSi10Mg alloy powder as per DIN EN 1706. ELEMENT
Si
Fe
Cu
Mn
Mg
Zn
Ti
Al
Weight %
9–11
≤0.55
≤0.05
≤0.45
0.2–0.45
≤0.1
≤0.15
Balance 2.2. Powder Characterization 2.2. Powder Characterization The morphology of the AlSi10Mg powder particles was examined on a scanning
electron microscopy (SEM) and images were taken by a Tescan VEGA 3 LMU Scanning
Electron Microscope system as shown in Figure 1. A laser particle analyzer (Beckman
Coulter LS 13,320) was used to measure the size of powder particles. The powder particles’
size varied from an average diameter of 25–65 µm; however, some particles also had
diameters smaller and larger than the abovementioned range, having different spherical
and elliptical forms. The density of the loose powder was 2.68 gm/cm3 as per the data
sheet provided by PAC Corporation. 4 of 16 J. Compos. Sci. 2021, 5, 126
021 5 126 Figure 1. Morphology of AlSi10Mg; (a) powder particles, and (b) the powder size distribution
Figure 1. Morphology of AlSi10Mg; (a) powder particles, and (b) the powder size distribution. Figure 1. Morphology of AlSi10Mg; (a) powder particles, and (b) the powder size distribution
Figure 1. Morphology of AlSi10Mg; (a) powder particles, and (b) the powder size distribution. Surface Methodology in Combination with Design of Experiment (DOE)
ponse surface method (RSM) is a group of procedures that includes (1) out-
2.3. Response Surface Methodology in Combination with Design of Experiment (DOE) po se su ace
et od ( SM) is a g oup o p ocedu es t at i c udes ( ) out
p of experimentations in order to obtain trustworthy and satisfactory meas-
he response under study, (2) defining a set of the optimum process parame-
d to the attainment of the desired value of response, and (3) explaining the
selected process parameters upon the response with the help of 2D and 3D
ponse surfaces. The response surface methods are categorized into three dif-
of design of experiments (DOE) techniques: (1) central composite design
eveloped by Box and Wilson in 1951 and is capable of designing experimental
ive levels; (2) Box–Behnken design (BBD) proposed by Box and Behnken in
has the capacity to outline experimental plans up to three levels; (3) EIMSE-
gns presented by Allen et al. in 2003, founded on supposed “expected inte-
squared error optimal” [29,30]. 2.2. Powder Characterization imization of parameters in the SLM process can be beneficial to enhance the
The response surface method (RSM) is a group of procedures that includes (1) outlining
a group of experimentations in order to obtain trustworthy and satisfactory measurement
of the response under study, (2) defining a set of the optimum process parameters that
lead to the attainment of the desired value of response, and (3) explaining the influence
of selected process parameters upon the response with the help of 2D and 3D plots and
response surfaces. The response surface methods are categorized into three different
kinds of design of experiments (DOE) techniques: (1) central composite design (CCD) was
developed by Box and Wilson in 1951 and is capable of designing experimental plans up to
five levels; (2) Box–Behnken design (BBD) proposed by Box and Behnken in 1960, which
has the capacity to outline experimental plans up to three levels; (3) EIMSE-optimal designs
presented by Allen et al. in 2003, founded on supposed “expected integrated mean squared
error optimal” [29,30]. imization of parameters in the SLM process can be beneficial to enhance the
dimensional quality of fabricated parts [14,31–33]. This research aimed to un-
mprehensive understanding of the relationship between different sets of pro-
ters and the surface and dimensional quality of SLM-built AlSi10Mg parts. y use of optimization techniques, also tried to outline the optimized process
to attain better surface and dimensional quality. The main objectives of this
given below:
i
f SLM
’ i fl
h
f
d di
i
l
p
The optimization of parameters in the SLM process can be beneficial to enhance the
surface and dimensional quality of fabricated parts [14,31–33]. This research aimed to
undertake a comprehensive understanding of the relationship between different sets of
process parameters and the surface and dimensional quality of SLM-built AlSi10Mg parts. The study, by use of optimization techniques, also tried to outline the optimized process
parameters to attain better surface and dimensional quality. 2.2. Powder Characterization The main objectives of this
study are as given below: ation of SLM process parameters’ influence upon the surface and dimensional
of AlSi10Mg specimens;
y the impact of variations of wall thickness and process parameters on surface
•
Investigation of SLM process parameters’ influence upon the surface and dimensional
quality of AlSi10Mg specimens; y the impact of variations of wall thickness and process parameters on surface
ess and the dimensional accuracy of the specimens;
i
th
ff
t
f
t
i
t
h i
(i
d bl
ti
d
li h
•
To study the impact of variations of wall thickness and process parameters on surface
roughness and the dimensional accuracy of the specimens; mine the effect of post-processing techniques (i.e., sand blasting and polish-
he surface and dimensional quality of the specimens;
ti
i
ti
t
h i
t
d fi
t
f
ti
l
t
•
To determine the effect of post-processing techniques (i.e., sand blasting and polishing)
on the surface and dimensional quality of the specimens; g optimization techniques, to define a set of optimal process parameters
urface roughness and dimensional accuracy. q
y
p
•
By using optimization techniques, to define a set of optimal process parameters against
surface roughness and dimensional accuracy. ng Minitab 17
DOE by Using Minitab 17 re many statistical software tools available for DOE and optimization pur-
s Design Expert, Statistica, Minitab, Design-Ease. Minitab 17 was used in this
periment planning, regression analysis, and optimization of the SLM process
ce roughness and dimensional accuracy. There are many statistical software tools available for DOE and optimization purposes
such as Design Expert, Statistica, Minitab, Design-Ease. Minitab 17 was used in this study
for experiment planning, regression analysis, and optimization of the SLM process against
surface roughness and dimensional accuracy. dy proposes a BBD-based optimization approach. This approach, by outlin-
possible runs of experiment, suggests a set of optimum process parameters
ower, scan speed, hatch distance) of the selective laser melting process. Re-
ce methods are normally applied in scenarios when the importance of some
ess parameters) have already been established as a result of previous experi-
This study proposes a BBD-based optimization approach. This approach, by outlining
the least possible runs of experiment, suggests a set of optimum process parameters (i.e.,
laser power, scan speed, hatch distance) of the selective laser melting process. Response
surface methods are normally applied in scenarios when the importance of some factors
(process parameters) have already been established as a result of previous experimenta-
tion [29]. The DOE starts by defining the factors (in this case. the SLM process parameters) J. Compos. Sci. 2021, 5, 126 5 of 16 and their levels (highs and lows). In continuation of previous research work done [34,35],
three levels of four parameters (i.e., wall thickness, laser power, scan speed, and hatch
spacing) were selected. The three levels of parameters employed in this study are given
below in Table 2. Table 2. Process parameters and their levels for DOE. Parameters
Unit
Level
−1
0
1
Wall Thickness
mm
1.0
2.0
3.0
Laser Power
Watt
320
350
380
Scan Speed
mm/s
730
900
1070
Hatch Distance
µm
80
105
130 Table 2. Process parameters and their levels for DOE. Table 2. Process parameters and their levels for DOE. The powder layer thickness was kept constant at 30 µm. The experimental plan was
further tested, and surface plots were produced to describe the tendency of attainable
surface-roughness and dimensional accuracy against different sets of process parameters. Eventually, this is beneficial in investigating the process parameters’ effect upon the subse-
quent responses. 3. Experimental Results and Analysis
3.1. Experimental Approach
ntal Results and Analysis
ental Approach
t d
t
t
t
f
t t l
f 54 (27
0 In this study, two sets out of a total of 54 (27 × 02) test specimens of AlSi10Mg were
manufactured using the selective laser melting technique. As mentioned earlier, Box–
Behnken design, which is an RSM design, was applied to outline the experimental plan. First, a three-dimensional CAD model of the samples was prepared. The CAD model was
fed into the SLM machine and the manufacturing was performed. The length (L) and
height (H) of the samples were kept constant at 55 mm and 12 mm, respectively, and there
were three variations in wall thickness, i.e., 1.0 mm, 2.0 mm, and 3.0 mm, against different
combinations of process parameters (i.e., laser power, scan speed, hatch distance) to make
27 combinations as designed by the DOE technique. The wall thicknesses and process
parameters were varied systematically to study their effects on the surface and dimensional
quality of the built specimens. The specimens’ dimensions and built orientation were
given in Figure 2. The wall thickness (T) and length (L) of the samples were built in the
horizontal direction (i.e., x,y-plane), while the height was built in the vertical direction,
i.e., on the z-axis. The samples were allowed to cool down at room temperature to relieve
residual stresses. Then, samples were subjected to sand blasting at a pressure of 0.3 MPa for
4 min. The wire cutting process was applied to separate specimens from the built platform
and to obtain the final shape required for further tensile and hardness testing. The length
and height of the samples after wire cutting were 53 mm and 10 mm, respectively. The
dimensions of the final specimen after the wire cutting process are given in Figure 3. The
overall flow of the manufacturing process is given in Figure 4. study, two sets out of a total of 54 (27 × 02) test specimens of AlSi10Mg were
ed using the selective laser melting technique. As mentioned earlier, Box–
sign, which is an RSM design, was applied to outline the experimental plan. -dimensional CAD model of the samples was prepared. The CAD model was
SLM machine and the manufacturing was performed. ng Minitab 17
DOE by Using Minitab 17 Moreover, it also makes us capable of defining a set of optimal process
parameters with respect to dimensional accuracy and surface roughness of SLM-built
AlSi10Mg specimens. The Box–Behnken design-based experimental plan generated by
Minitab 17 is given below in Table 3. Table 3. Four-factor three-level Box–Behnken DOE. Table 3. Four-factor three-level Box–Behnken DOE. Run Order
Wall Thickness
(mm)
Laser Power
(Watt)
Scan Speed
(mm/s)
Hatch Distance
(µm)
1
1
320
900
105
2
3
320
900
105
3
1
380
900
105
4
3
380
900
105
5
2
350
730
80
6
2
350
1070
80
7
2
350
730
130
8
2
350
1070
130
9
1
350
900
80
10
3
350
900
80
11
1
350
900
130
12
3
350
900
130
13
2
320
730
105
14
2
380
730
105
15
2
320
1070
105
16
2
380
1070
105
17
1
350
730
105
18
3
350
730
105
19
1
350
1070
105
20
3
350
1070
105
21
2
320
900
80
22
2
380
900
80
23
2
320
900
130
24
2
380
900
130
25
2
350
900
105
26
2
350
900
105
27
2
350
900
105 The DOE plan consisted of 27 experimental runs with different combinations of
process parameters so as to minimize the experimental cost and achieve optimization. The
DOE plan does not combine all the lows or highs of all the parameters in a single run J. Compos. Sci. 2021, 5, 126 6 of 16 so as to avoid extreme conditions during experimentation. Moreover, the experimental
plan contains three center points, i.e., points where the middle values (0, 0, 0, 0) of all the
parameters are chosen in a single run (Run 25, 26, 27). In this case, the middle values of
wall thickness, laser power, scan speed, and hatch distance are 2 mm, 350 watts, 900 mm/s,
and 105 µm, respectively. A center point is repeated twice to ensure the repeatability and
dispersion of the experimental design. 2
320
900
130
2
380
900
130
2
350
900
105
2
350
900
105
2
350
900
105 3. Experimental Results and Analysis
3.1. Experimental Approach
ntal Results and Analysis
ental Approach
t d
t
t
t
f
t t l
f 54 (27
0 The length (L) and
f the samples were kept constant at 55 mm and 12 mm, respectively, and there
variations in wall thickness, i.e., 1.0 mm, 2.0 mm, and 3.0 mm, against different
ns of process parameters (i.e., laser power, scan speed, hatch distance) to make
ions as designed by the DOE technique. The wall thicknesses and process pa-
re varied systematically to study their effects on the surface and dimensional
he built specimens. The specimens’ dimensions and built orientation were
ure 2. The wall thickness (T) and length (L) of the samples were built in the
direction (i.e., x,y-plane), while the height was built in the vertical direction,
-axis. The samples were allowed to cool down at room temperature to relieve
esses. Then, samples were subjected to sand blasting at a pressure of 0.3 MPa
he wire cutting process was applied to separate specimens from the built plat-
obtain the final shape required for further tensile and hardness testing. The
height of the samples after wire cutting were 53 mm and 10 mm, respectively. ions of the final specimen after the wire cutting process are given in Figure 3. flow of the manufacturing process is given in Figure 4. Figure 2. Samples dimensions and built-up scheme. Figure 2. Samples dimensions and built-up scheme. mensions and built-up scheme. Figure 2. Samples dimensions and built-up scheme. J. Compos. Sci. 2021, 5, 126
J. Compos. Sci. 2021, 5, 126 7 of 16
7 of 17 Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after w Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after wire cutting. Figure 3. Dimensions of the sample after w Figure 4. Overall flow of the manufacturing process. Figure 4. Overall flow of the manufacturing process. Figure 4. Overall flow of the manufacturing process. i Figure 4. Overall flow of the manufacturing p
Figure 4. Overall flow of the manu
Figure 4. Overall flow of the manufacturing process. J. Compos. Sci. 3.2. Surface Roughness of as-Built Samples 3.2. Surface Roughness of as-Built Samples The purpose of experimentation, as stated earlier, was to optimize the process param-
eters of the SLM process to improve the surface quality of AlSi10Mg parts. The samples’
surface roughness was recorded using the contact method. A profilometer was used to
take a reading of the sample’s SR. The profilometer records an average SR Ra in µm by
moving its stylus over the surface of a sample in a straight line. The roughness reading
was noted on eight different points along the longitudinal axis of each sample and the
average of the values was taken. The roughness of samples in as-built condition against
each experimental run is given below in Table 4. Table 4. Average SR values in As-built condition. Run Order
WT (mm)
LP (W)
SS (mm/s)
HD (µm)
SR (µm)
1
1
320
900
105
11.575
2
3
320
900
105
8.985
3
1
380
900
105
8.873
4
3
380
900
105
12.210
5
2
350
730
80
12.156
6
2
350
1070
80
10.391
7
2
350
730
130
13.111
8
2
350
1070
130
9.563
9
1
350
900
80
11.012
10
3
350
900
80
11.610
11
1
350
900
130
13.816
12
3
350
900
130
10.146
13
2
320
730
105
9.538
14
2
380
730
105
9.993
15
2
320
1070
105
10.159
16
2
380
1070
105
10.477
17
1
350
730
105
10.240
18
3
350
730
105
12.682
19
1
350
1070
105
10.108
20
3
350
1070
105
9.587
21
2
320
900
80
11.086
22
2
380
900
80
10.945
23
2
320
900
130
11.818
24
2
380
900
130
9.182
25
2
350
900
105
9.727
26
2
350
900
105
10.869
27
2
350
900
105
10.253 Table 4. Average SR values in As-built condition. The results indicate that a minimum value of surface roughness, i.e., Ra = 8.873 µm was
recorded in the specimen of Run Order #3 having a wall thickness of 1.0 mm, laser power of
380 watts, scan speed of 900 mm/s, and hatch distance of 105 µm. The maximum value of
SR (i.e., 13.816 µm) was seen in the specimen with run order #11. The corresponding values
of wall thickness, laser power, scan speed, and hatch distance were 1.0 mm, 350 watts,
900 mm/s, and 130 µm, respectively. 3. Experimental Results and Analysis
3.1. Experimental Approach
ntal Results and Analysis
ental Approach
t d
t
t
t
f
t t l
f 54 (27
0 2021, 5, 126 8 of 16 3.2.1. Surface Roughness of Sand-Blasted Samples 3.2.1. Surface Roughness of Sand-Blasted Samples 3.2.1. Surface Roughness of Sand-Blasted Samples One of the objectives of the experiment was to study the effects of one of the post-
processing techniques (i.e., sand blasting) on the surface quality of the test specimens. As
stated previously, the specimens were exposed to sand blasting at a pressure of 0.3 MPa for
4 min. A profilometer was used to measure the SR of specimens. The profilometer records
average SR Ra in µm by moving its stylus over the surface of a sample in a straight line. The roughness reading was noted on eight different points along the longitudinal axis of
each sample and the average of the values was taken. The average SR of samples after
sand blasting against each experimental run is given below in Table 5. J. Compos. Sci. 2021, 5, 126 9 of 16 Table 5. Average SR values after sand blasting. Run Order
WT (mm)
LP (W)
SS (mm/s)
HD (µm)
SR (µm)
1
1
320
900
105
6.355
2
3
320
900
105
6.590
3
1
380
900
105
6.748
4
3
380
900
105
8.040
5
2
350
730
80
5.557
6
2
350
1070
80
5.337
7
2
350
730
130
5.442
8
2
350
1070
130
5.620
9
1
350
900
80
7.622
10
3
350
900
80
6.290
11
1
350
900
130
13.270
12
3
350
900
130
6.450
13
2
320
730
105
4.750
14
2
380
730
105
3.770
15
2
320
1070
105
4.200
16
2
380
1070
105
4.870
17
1
350
730
105
6.049
18
3
350
730
105
7.380
19
1
350
1070
105
5.947
20
3
350
1070
105
5.500
21
2
320
900
80
5.890
22
2
380
900
80
4.920
23
2
320
900
130
6.080
24
2
380
900
130
4.650
25
2
350
900
105
5.500
26
2
350
900
105
5.250
27
2
350
900
105
5.600 Table 5. Average SR values after sand blasting. It can be observed from the Table 5 that the lowest reading of surface roughness (i.e.,
Ra = 3.77 µm) was obtained in the specimen of Run Order #14, having a wall thickness of
2.0 mm, laser power of 380 watts, scan speed of 730 mm/s, and hatch distance of 105 µm. 3.2.2. Dimensional Accuracy of the Samples The highest accuracy in the height was observed in run #26
with a recorded value of the average height being 54.983 mm. The corresponding values
of wall thickness, laser power, scan speed and hatch distance were 2.0 mm, 350 watts,
900 mm/s, and 105 µm, respectively. For height of the specimens, the least precise value
(i.e., 12.078 mm) was recorded in run #23, having process parameters of wall thickness =
2.0 mm, laser power = 320 watts, scan speed = 900 mm/s, and hatch distance = 130 µm. The highest accurate value of 11.998 mm was noted in run #17 with relevant values of
wall thickness, laser power, scan speed, and hatch distance being 1.0 mm, 350 watts,
730 mm/s, and 105 µm, respectively. The summary of the graphical representation based
on experimental results is given below. The Figure 6 describes the relationship between
length and height and perception parameter values. Table 6. Average values of the length and height of the samples. Table 6. Average values of the length and height of the samples. Run Ord. 3.2.2. Dimensional Accuracy of the Samples WT (mm)
LP (W)
SS (mm/s)
HD (µm)
Length (mm)
Height
(mm)
1
1
320
900
105
54.818
12.025
2
3
320
900
105
54.920
12.030
3
1
380
900
105
54.805
12.068
4
3
380
900
105
54.933
12.050
5
2
350
730
80
54.958
12.020
6
2
350
1070
80
54.943
12.045
7
2
350
730
130
54.953
12.018
8
2
350
1070
130
54.920
12.055
9
1
350
900
80
54.795
12.073
10
3
350
900
80
54.940
12.060
11
1
350
900
130
54.895
12.033
12
3
350
900
130
54.965
12.070
13
2
320
730
105
54.883
12.025
14
2
380
730
105
54.905
12.055
15
2
320
1070
105
54.900
12.025
16
2
380
1070
105
54.920
12.035
17
1
350
730
105
54.848
11.998
18
3
350
730
105
54.950
12.010
19
1
350
1070
105
54.883
12.008
20
3
350
1070
105
54.888
12.020
21
2
320
900
80
54.920
12.028
22
2
380
900
80
54.935
12.060
23
2
320
900
130
54.888
12.078
24
2
380
900
130
54.918
12.073
25
2
350
900
105
54.913
12.065
26
2
350
900
105
54.983
12.043
27
2
350
900
105
54.963
12.038 It can be inferred from Table 6 that for the length of the specimens, the highest
deviation from the design value occurred in run #9, yielding an average height of 54.795 mm
at a wall thickness of 1.0 mm, laser power of 350 watts, scan speed of 900 mm/s, and
hatch distance of 80 µm. The highest accuracy in the height was observed in run #26
with a recorded value of the average height being 54.983 mm. The corresponding values
of wall thickness, laser power, scan speed and hatch distance were 2.0 mm, 350 watts,
900 mm/s, and 105 µm, respectively. For height of the specimens, the least precise value
(i.e., 12.078 mm) was recorded in run #23, having process parameters of wall thickness =
2.0 mm, laser power = 320 watts, scan speed = 900 mm/s, and hatch distance = 130 µm. The highest accurate value of 11.998 mm was noted in run #17 with relevant values of
wall thickness, laser power, scan speed, and hatch distance being 1.0 mm, 350 watts,
730 mm/s, and 105 µm, respectively. The summary of the graphical representation based
on experimental results is given below. 3.2.2. Dimensional Accuracy of the Samples 3.2.2. Dimensional Accuracy of the Samples 3.2.2. Dimensional Accuracy of the Samples To study the effect of process parameters on the dimensional quality of the samples
was also one of the objectives of the experimentation. The dimensional measurements
(i.e., length and height) were measured using a digital vernier caliper. The design values
for length and height were 55 mm and 12 mm, respectively, as given in Figure 2. The
measurements of height were taken at five different points along the longitudinal axis,
while length was recorded four times for every specimen. The average of these values was
taken and are given in Table 6. taken and are given in Table 6. Table 6. Average values of the length and height of the samples. Run Ord. WT (mm)
LP (W)
SS (mm/s)
HD (µm)
Length (mm)
Height
(mm)
1
1
320
900
105
54.818
12.025
2
3
320
900
105
54.920
12.030
3
1
380
900
105
54.805
12.068
4
3
380
900
105
54.933
12.050
5
2
350
730
80
54.958
12.020
6
2
350
1070
80
54.943
12.045
7
2
350
730
130
54.953
12.018
8
2
350
1070
130
54.920
12.055
9
1
350
900
80
54.795
12.073
10
3
350
900
80
54.940
12.060
11
1
350
900
130
54.895
12.033
12
3
350
900
130
54.965
12.070
13
2
320
730
105
54.883
12.025
14
2
380
730
105
54.905
12.055
15
2
320
1070
105
54.900
12.025
16
2
380
1070
105
54.920
12.035
17
1
350
730
105
54.848
11.998
18
3
350
730
105
54.950
12.010
19
1
350
1070
105
54.883
12.008
20
3
350
1070
105
54.888
12.020
21
2
320
900
80
54.920
12.028
22
2
380
900
80
54.935
12.060
23
2
320
900
130
54.888
12.078
24
2
380
900
130
54.918
12.073
25
2
350
900
105
54.913
12.065
26
2
350
900
105
54.983
12.043
27
2
350
900
105
54.963
12.038
It can be inferred from Table 6 that for the length of the specimens, the highest
deviation from the design value occurred in run #9, yielding an average height of 54.795 mm
at a wall thickness of 1.0 mm, laser power of 350 watts, scan speed of 900 mm/s, and
hatch distance of 80 µm. 3.2.1. Surface Roughness of Sand-Blasted Samples 2021, 5, 126 10 of 16 3.2.1. Surface Roughness of Sand-Blasted Samples The highest value of SR (i.e., 13.27 µm) was recorded in the specimen with run order #11. The corresponding values of wall thickness, laser power, scan speed, and hatch distance
were 1.0 mm, 350 watts, 900 mm/s, and 130 µm, respectively. The graphical representation
based on experimental data is also described below in Figure 5. This graph depicts the
relationship between the surface roughness and the values of the perspective parameter. 10 of 17 Figure 5. SR in respect to each of the parameters. 3.2.2. Dimensional Accuracy of the Samples
To study the effect of process parameters on the dimensional quality of the sample
was also one of the objectives of the experimentation. The dimensional measurements (i.e
length and height) were measured using a digital vernier caliper. The design values fo
length and height were 55 mm and 12 mm, respectively, as given in Figure 2. The meas
urements of height were taken at five different points along the longitudinal axis whil
0
8
100
0
2
3
340
360
800
700
0
8
3
0
10
0
0
9
800
0
0
0
2
1
)
m
µ
(
D
H
)s
/
m
m
(
S
S
)
W
(
P
L
5.500
5.557
5.600
5.620
5.890
5.947
6.049
6.080
6.290
6.355
3.770
6.450
6.590
6.748
7.380
7.622
4.200
4.650
4.750
4.870
4.920
5.250
5.337
5.442
SR (µm)
D Scatterplot of HD (
3
m
µ
vs SS (mm/s) vs LP (W)
)
Figure 5. SR in respect to each of the parameters. Figure 5. SR in respect to each of the parameters. 0
8
100
0
2
3
340
360
800
700
0
8
3
0
10
0
0
9
800
0
0
0
2
1
)
m
µ
(
D
H
)s
/
m
m
(
S
S
)
W
(
P
L
5.500
5.557
5.600
5.620
5.890
5.947
6.049
6.080
6.290
6.355
3.770
6.450
6.590
6.748
7.380
7.622
4.200
4.650
4.750
4.870
4.920
5.250
5.337
5.442
SR (µm)
D Scatterplot of HD (
3
m
µ
vs SS (mm/s) vs LP (W)
)
Figure 5. SR in respect to each of the parameters. D Scatterplot of HD (
3
m
µ
vs SS (mm/s) vs LP (W)
) Figure 5. SR in respect to each of the parameters
Figure 5. SR in respect to each of the parameters. J. Compos. Sci. 3.2.2. Dimensional Accuracy of the Samples The Figure 6 describes the relationship between
length and height and perception parameter values. 11 of 16
imental
d height
erimental
nd height J. Compos. Sci. 2021, 5, 126 Figure 6. All plot parameter effects for (a) length and (b) height. Figure 6. All plot parameter effects for (a) length and (b) height. Figure 6. All plot parameter effects for (a) length and (b) height. Figure 6. All plot parameter effects for (a) length and (b) height. Figure 6. All plot parameter effects for (a) length and (b) height. Figure 6. All plot parameter effects for (a) length and (b) height 3.3. Surface Roughness
Th
i
ff
t
3.3. Surface Roughness
3.3. Surface Roughness
The main effects p The main effects plot and interaction plot for SR are given in Figures 7 and 8, resp
ively
The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectiv
The main effects plot and interaction plot for SR are given in Figures 7 and 8, res
tively.
12 The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
tively. The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectively. The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
tively. o tively. Figure 7. Main effects plot for SR. 3
2
1
11.5
11.0
10.5
10.0
380
350
320
1070
900
730
130
105
80
WT
Mean
LP
SS
HD
Main Effects Plot for SR
Data Means
tively. Figure 7. Main effects plot for SR. 3
2
1
11.5
11.0
10.5
10.0
380
350
320
1070
900
730
130
105
80
WT
Mean
LP
SS
HD
Main Effects Plot for SR
Data Means
Figure 7. Main effects plot for SR. The main effect plot shows the mean of the response at every level of process param
eter. Grand mean is displayed by drawing a horizontal line. The influence of process p
rameters is observed by the slope of the line which joins different levels of the proce
parameters. A steeper line indicates greater influence on response, while a line parallel
the grand mean displays a lesser effect on the response. It is clear from Figure 7 that sca
speed had the greatest effect on surface roughness followed by laser power, while wa
thickness had the least effect on the SR. The optimum value for surface roughness w
attained at a scan speed of 900 mm/s, laser power of 380 W, and hatch distance of 105 µm
The interaction among process parameters can be visualized by an interaction pl
(Figure 8). In the plot, parallel lines point to no interaction. The interaction was very si
nificant when lines moved away from being parallel. The plot indicates that a low valu
of SR was achieved at a high value of laser power and medium value of hatch distanc
Significant interaction existed between hatch distance and wall thickness. There was
greater influence for hatch distance values of 80 µm and 130 µm at all values of wall thic
ness. There was also a significant interaction between the laser power of 380 W and wa
thickness greater than 2.0 mm. Figure 8. Interaction plot for SR. Regression Equation
The regression equation based on the chosen quadratic model is given below in Equ
tion (1). Figure 8. Interaction plot for SR. 3.3. Surface Roughness
Th
i
ff
t
3.3. Surface Roughness
3.3. Surface Roughness
The main effects The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
tively. The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectively. The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
tively. 12 of 17 The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
vely
The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectively. The main effects plot and interaction plot for SR are given in Figures 7 and 8, respec-
tively. 12 of 17 The main effects plot and interaction plot for SR are given in Figures 7 and 8, resp
ively
The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectiv
The main effects plot and interaction plot for SR are given in Figures 7 and 8, res
tively.
12 Figure 7. Main effects plot for SR. 3
2
1
11.5
11.0
10.5
10.0
380
350
320
1070
900
730
130
105
80
WT
Mean
LP
SS
HD
Main Effects Plot for SR
Data Means
Figure 7. Main effects plot for SR. 3
2
1
11.5
11.0
10.5
10.0
380
350
320
1070
900
730
130
105
80
WT
Mean
LP
SS
HD
Main Effects Plot for SR
Data Means
Figure 7. Main effects plot for SR. The main effect plot shows the mean of the response at
eter. Grand mean is displayed by drawing a horizontal line
rameters is observed by the slope of the line which joins d
parameters. A steeper line indicates greater influence on res
the grand mean displays a lesser effect on the response. It i
speed had the greatest effect on surface roughness followe
thickness had the least effect on the SR. The optimum val
attained at a scan speed of 900 mm/s, laser power of 380 W,
The interaction among process parameters can be visu
(Figure 8). In the plot, parallel lines point to no interaction. nificant when lines moved away from being parallel. The p
of SR was achieved at a high value of laser power and me
Significant interaction existed between hatch distance and
greater influence for hatch distance values of 80 µm and 130
ness. There was also a significant interaction between the l
thickness greater than 2.0 mm. Figure 7. Main effects plot for SR. Figure 7. Main effects plot for SR
Figure 7. Main effects plot for SR. ness. There was also a signific
thickness greater than 2.0 mm Figure 8. Interaction plot for SR. Figure 8. Interaction plot for SR. Figure 8. Interaction plot for SR
Figure 8. Interaction plot for SR. J. Compos. Sci. 2021, 5, 126 12 of 16 The main effect plot shows the mean of the response at every level of process parameter. Grand mean is displayed by drawing a horizontal line. The influence of process parameters
is observed by the slope of the line which joins different levels of the process parameters. A steeper line indicates greater influence on response, while a line parallel to the grand
mean displays a lesser effect on the response. 3.4. Dimensional Accuracy
3.4.1. Length of the Samples The main effects plot for length was drawn and is given below in Figure 9. Figure 9. Main effects plot for length. It is clear from the figure above that wall thickness had the greatest effect on the di-
mensional accuracy of the samples in terms of length, while scan speed had the least effect
on length. The most precise value for length was attained at a wall thickness of 3.0 mm,
laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. Regression Equation
Figure 9. Main effects plot for length. It is clear from the figure above that wall thickness had the greatest effect on the
dimensional accuracy of the samples in terms of length, while scan speed had the least
effect on length. The most precise value for length was attained at a wall thickness of 3.0
mm, laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. Figure 9. Main effects plot for length. Fi
9 M i
ff
t
l t f
l
th Figure 9. Main effects plot for length
Figure 9. Main effects plot for length. It is clear from the figure above that wall thickness had the greatest effect on the di-
mensional accuracy of the samples in terms of length, while scan speed had the least effect
on length. The most precise value for length was attained at a wall thickness of 3.0 mm,
laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. It is clear from the figure above that wall thickness had the greatest effect on the
dimensional accuracy of the samples in terms of length, while scan speed had the least
effect on length. The most precise value for length was attained at a wall thickness of 3.0
mm, laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. It is clear from the figure above that wall thickness had the greatest effect on the di-
mensional accuracy of the samples in terms of length, while scan speed had the least effect
on length. The most precise value for length was attained at a wall thickness of 3.0 mm,
laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. The main effects plot and interaction plot for SR are given in Figures 7 and 8, resp
ively
The main effects plot and interaction plot for SR are given in Figures 7 and 8, respectiv
The main effects plot and interaction plot for SR are given in Figures 7 and 8, res
tively.
12 It is clear from Figure 7 that scan speed had
the greatest effect on surface roughness followed by laser power, while wall thickness had
the least effect on the SR. The optimum value for surface roughness was attained at a scan
speed of 900 mm/s, laser power of 380 W, and hatch distance of 105 µm. The interaction among process parameters can be visualized by an interaction plot
(Figure 8). In the plot, parallel lines point to no interaction. The interaction was very
significant when lines moved away from being parallel. The plot indicates that a low value
of SR was achieved at a high value of laser power and medium value of hatch distance. Significant interaction existed between hatch distance and wall thickness. There was a
greater influence for hatch distance values of 80 µm and 130 µm at all values of wall
thickness. There was also a significant interaction between the laser power of 380 W and
wall thickness greater than 2.0 mm. Regression Equation The regression equation based on the chosen quadratic model is given below in
Equation (1). SR = −92.5 −4.40WT + 0.518LP + 0.0316SS + 0.124HD + 0.286WT ∗WT
−0.000654LP ∗LP + 0.000002SS ∗SS + 0.001657HD ∗HD + 0.0327WT ∗LP−
0.00436WT ∗SS −0.0427WT ∗HD −0.000056LP ∗SS −0.000832LP ∗HD
−0.000105SS ∗HD
(1 (1) 3.4. Dimensional Accuracy
3.4.1. Length of the Samples 3.4. Dimensional Accuracy
3.4.1. Length of the Samples Regression Equation Regression Equation
The regression equation based on the chosen quadratic model is given below in
Equation (3). The regression equation based on the chosen quadratic model is given below in
Equation (3). Height = 11.27 + 0.051WT −0.00071LP + 0.001854SS −0.00125HD −0.00875WT ∗WT
+0.000005LP ∗LP −0.000001SS ∗SS + 0.000020HD ∗HD
−0.000187WT ∗LP + 0.000500WT ∗HD −0.000001LP ∗SS
−0.000013LP ∗HD + 0.000001SS ∗HD
(3) (3) −0.000013LP ∗HD + 0.000001SS ∗HD −0.000013LP ∗HD + 0.000001SS ∗HD 3.4.2. Height of the Samples
3.4.2. Height of the Samples The main effects plot for the height was drawn and is given below in Figure 10. It is
clear from the figure above that laser power and scan speed had the greatest effect on the
dimensional accuracy of the samples in terms of height. The most precise value for height
was attained at a wall thickness of 1.0 mm, laser power of 320 W, scan speed of 730 mm/s,
and hatch distance of 105 µm. g
p
The main effects plot for the height was drawn and is given below in Figure 10. It is
clear from the figure above that laser power and scan speed had the greatest effect on the
dimensional accuracy of the samples in terms of height. The most precise value for height
was attained at a wall thickness of 1.0 mm, laser power of 320 W, scan speed of 730 mm/s,
and hatch distance of 105 µm. Figure 10. Main effects plot for height. 3
2
1
12.06
12.05
12.04
12.03
12.02
380
350
320
1070
900
730
130
105
80
WT
Mean
LP
SS
HD
Main Effects Plot for Height
Data Means
Figure 10. Main effects plot for height. Main Effects Plot for Height
Data Means Figure 10. Main effects plot for height
Figure 10. Main effects plot for height. Regression Equation Regression Equation
Regression Equation Regression Equation
Regression Equation The regression equation based on the chosen quadratic model is given below in
Equation (2). The regression equation based on the chosen quadratic model is given below in Equa
tion (2). Length = 49.04 + 0.380WT + 0.0279LP + 0.00110SS + 0.00118HD −0.0498WT ∗WT
49.04
0.380
0.0279
0.00110
0.00118
0.0498
0 000041
*
0 000002
*
0 000208
*
Length
WT
LP
SS
HD
WT WT
LP LP
HD HD
WT
LP
=
+
+
+
+
−
−
+ −0.000041LP ∗LP −0.000002HD ∗HD + 0.000208WT ∗LP
0.000041
0.000002
0.000208
0 000143
*
0 000750
*
0 000005
*
LP LP
HD HD
WT
LP
WT
SS
WT
HD
LP HD
−
−
+
+ (2)(2) +
−0.000143WT ∗SS −0.000750WT ∗HD + 0.000005LP ∗HD
(2)
0.000143
*
0.000750
*
0.000005
*
WT
SS
WT
HD
LP HD
−
−
+
(2 −0.000143WT ∗SS −0.000750WT ∗HD + 0.000005LP ∗HD
0.000143
*
0.000750
*
0.000005
*
WT
SS
WT
HD
LP H
−
−
+ −0.000001SS ∗HD
0.000001
*
SS
HD
− −0.000001SS ∗HD
0.000001
*
SS
HD
− 3.4. Dimensional Accuracy
3.4.1. Length of the Samples It is clear from the figure above that wall thickness had the greatest effect on the
dimensional accuracy of the samples in terms of length, while scan speed had the least
effect on length. The most precise value for length was attained at a wall thickness of 3.0
mm, laser power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm. J. Compos. Sci. 2021, 5, 126 13 of 16
ast effec
3.0 mm 3.5. Selection of Optimum Process Parameters Optimizing the process parameters for the responses was also one of the aims of
the study. Minitab 17 was used to define the optimal process parameters to obtain better
surface and dimensional qualities. The optimization plot and set of optimum SLM process
parameters are given below in Figure 11. The composite desirability is a single measure that combines the individual desirability
of all the responses into a single measure. The figure below illustrates those responses were
optimized and a composite desirability of 0.7472 was achieved. These optimized values of
the response were obtained at a wall thickness of 2.7172 mm, laser power of 320 W, scan
speed of 730 mm/s, and hatch distance of 94.1414 µm. Thus, these values combined make
up a set of optimum process parameters against SR, length, and height as responses. J. Compos. Sci. 2021, 5, 126 14 of 16
p
se values
h i ht Figure 11. Optimization plot for SR, height, and length. Figure 11. Optimization plot for SR, height, and length. Figure 11. Optimization plot for SR, height, and length
Figure 11. Optimization plot for SR, height, and length. 4. Conclusions
4. Conclusions In this research work, the influence of SLM process parameters (i.e., wall thickness,
laser power, scan speed, and hatch distance) on the surface and dimensional quality of
AlSi10Mg specimens was studied. Response surface methodology was applied for DOE,
analysis, prediction, and optimization purposes. ANOVA was applied to observe the ef-
fect of input factors on the responses. The conclusions made on the basis of the current
study are listed below:
In this research work, the influence of SLM process parameters (i.e., wall thickness,
laser power, scan speed, and hatch distance) on the surface and dimensional quality of
AlSi10Mg specimens was studied. Response surface methodology was applied for DOE,
analysis, prediction, and optimization purposes. ANOVA was applied to observe the effect
of input factors on the responses. The conclusions made on the basis of the current study
are listed below: (1)
With p-value and F-test value, wall thickness proved to have the most substantial
influence on the length of the specimens in terms of dimensional accuracy. Length
accuracy improved greatly when wall thickness varied from 1.0 to 2.0 mm. A further
improvement in accuracy was observed when wall thickness varied from 2.0 to
3.0 mm; (2)
The response estimation model developed for length of the specimens was significant
and yielded an R2 of 82.14%. This means that the developed model fits more than
80% of the response data; (3)
The values for SR, length, and height were predicted on the basis of respective
regression equations developed with regression analysis of the responses. The SR,
length, and height were predicted accurately with minimum error of 0.005%, 0.0%,
and 0.002%, respectively, as compared to experimental values; (4)
The most precise value for height was achieved at a wall thickness of 1.0 mm, laser
power of 320 W, scan speed of 730 mm/s, and hatch distance of 105 µm; (5)
The most accurate value for length was attained at a wall thickness of 3.0 mm, laser
power of 350 W, scan speed of 730 mm/s, and hatch distance of 130 µm; (6)
Multi-objective optimization methods can be used to optimize different process
parameters of the SLM process simultaneously; p
p
y
(7)
More statistical methods, such as the Taguchi method, artificial neural networks,
fuzzy logic, genetic algorithms, and grey relational analysis, can be applied to ana-
lyze results. J. Compos. Sci. 2021, 5, 126 15 of 16 15 of 16 Author Contributions: Conceptualization, D.H. References 1. Kruth, J.-P.; Levy, G.; Klocke, F.; Childs, T. Consolidation phenomena in laser and powder-bed based layered manufacturing. CIRP Ann. 2007, 56, 730–759. [CrossRef] onner, B.P.; Manogharan, G.P.; Martof, A.N.; Rodomsky, L.M.; Rodomsky, C.M.; Jordan, D.C.; Limperos, J.W
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g
g
p
g p
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, ,
[
]
3. Maamoun, A.H.; Elbestawi, M.; Dosbaeva, G.K.; Veldhuis, S.C. Thermal post-processing of AlSi10Mg parts produced by Selective
Laser Melting using recycled powder Addit Manuf 2018 21 234–247 [CrossRef] g
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f
4. Martin, J.H.; Yahata, B.D.; Hundley, J.M.; Mayer, J.A.; Schaedler, T.A.; Pollock, T.M. 3D printing of high-strength aluminum alloys. Nature 2017, 549, 365–369. [CrossRef] 4. Martin, J.H.; Yahata, B.D.; Hundley, J.M.; Mayer, J.A.; Schaedler, T.A.; Pollock, T.M. 3D printing of high-strength aluminum alloys. Nature 2017, 549, 365–369. [CrossRef] 5. Majeed, A.; Zhang, Y.F.; Lv, J.X.; Peng, T.; Waqar, S.; Atta, Z. Study the effect of heat treatment on the relative density of SLM built
parts of AlSi10Mg alloy. In Proceedings of the 48th International Conference on Computers and Industrial Engineering (CIE
2018), Auckland, New Zealand, 2–5 December 2018. 5. Majeed, A.; Zhang, Y.F.; Lv, J.X.; Peng, T.; Waqar, S.; Atta, Z. Study the effect of heat treatment on the relative density of SLM built
parts of AlSi10Mg alloy. In Proceedings of the 48th International Conference on Computers and Industrial Engineering (CIE
2018), Auckland, New Zealand, 2–5 December 2018. )
6. Sing, S.L.; An, J.; Yeong, W.Y.; Wiria, F.E. Laser and electron-beam powder-bed additive manufacturing of metallic implants: A
review on processes, materials and designs. J. Orthop. Res. 2016, 34, 369–385. [CrossRef] )
6. Sing, S.L.; An, J.; Yeong, W.Y.; Wiria, F.E. Laser and electron-beam powder-bed additive manufacturing of metallic implants: A
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,
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,
[
]
7. Elahi, H.; Eugeni, M.; Gaudenzi, P. A review on mechanisms for piezoelectric-based energy harvesters. Energies 2018, 11, 1850. [CrossRef] 7. Elahi, H.; Eugeni, M.; Gaudenzi, P. A review on mechanisms for piezoelectric-based energy harvesters. Energies 2018, 11, 1850. [CrossRef] 7. Elahi, H.; Eugeni, M.; Gaudenzi, P. A review on mechanisms for piezoelectric-based energy harvester
[CrossRef] 8. Elahi, H. The investigation on structural health monitoring of aerospace structures via piezoelectric aeroelastic energy harvesting. Microsyst. Technol. 2020, 1–9. [CrossRef] 8. Abbreviations Abbreviations
Nomenclature
ASTM
American Society for Testing and Materials
AM
Additive Manufacturing
SLM
Selective Laser Melting
EBM
Electron Beam Melting
DED
Directed Energy Deposition
BBD
Box–Behnken Design
RSM
Response Surface Methodology
SEM
Scanning Electron Microscope
3D
Three Dimensional
CAD
Computer-Aided Design
DOE
Design of Experiment
CCD
Central Composite Design
BBD
Box–Behnken Design
EIMSE
Expected Integrated Mean Squared Error
SEM
Scanning Electron Microscope
SR
Surface Roughness
WT
Wall Thickness
Auto Fab
Auto Fabrication
LP
Laser Power
SS
Scan Speed
HD
Hatch Distance 4. Conclusions
4. Conclusions and M.W.; methodology, H.E., S.R.; software, M.W.;
validation, D.H., M.W. and P.G.; formal analysis, H.E.; investigation, M.W.; resources, H.E.; data
curation, D.H.; writing—original draft preparation, M.W.; writing—review and editing, S.R.; visual-
ization, M.E.; supervision, D.H.; project administration, P.G.; funding acquisition H.E. All authors
have read and agreed to the published version of the manuscript. Funding: This work was supported by the China National Key R&D Program (2018YFD0400800 and
2018YFB1106303), the National Natural Science Foundation of China (51975006), and the PostGradu-
ate Education Project of Beijing (11000101010). Conflicts of Interest: The authors declare no conflict of interest. Abbreviations
Nomenclature
ASTM
American Society for Testing and Materials
AM
Additive Manufacturing
SLM
Selective Laser Melting
EBM
Electron Beam Melting
DED
Directed Energy Deposition
BBD
Box–Behnken Design
RSM
Response Surface Methodology
SEM
Scanning Electron Microscope
3D
Three Dimensional
CAD
Computer-Aided Design
DOE
Design of Experiment
CCD
Central Composite Design
BBD
Box–Behnken Design
EIMSE
Expected Integrated Mean Squared Error
SEM
Scanning Electron Microscope
SR
Surface Roughness
WT
Wall Thickness
Auto Fab
Auto Fabrication
LP
Laser Power
SS
Scan Speed
HD
Hatch Distance References 2017, 13, 103–112. [CrossRef] 19. Rakesh, C.S.; Priyanka, N.; Jayaganthan, R.; Vasa, N.J. Effect of build atmosphere on the mechanical properties of AlSi10Mg
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20. Han, X.; Zhu, H.; Nie, X.; Wang, G.; Zeng, X. Investigation on Selective Laser Melting AlSi10Mg Cellular Lattice Strut: Molten
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piezoelectric energy harvester based on flag-flutter. Micromachines 2020, 11, 933. [CrossRef] 10. Gibson, I.; Rosen, D.W.; Stucker, B. Design for Additive Manufacturing. In Additive Manufacturing Technologies; Springer Science
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Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements
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Variable levels of drift in tunicate cardiopharyngeal gene
Variable levels of drift in tunicate cardiopharyngeal gene
regulatory elements. regulatory elements. William Colgan
Broad Institute
Alexis Leanza
Thomas Jefferson University
Ariel Hwang
University of North Carolina
Melissa B. DeBiasse
Whitney Laboratory for Marine Bioscience
Isabel Llosa
Swarthmore College
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Colgan, William; Leanza, Alexis; Hwang, Ariel; DeBiasse, Melissa B.; Llosa, Isabel; Rodrigues, Daniel;
Adhikari, Hriju; Barreto Corona, Guillermo; Bock, Saskia; Carillo-Perez, Amanda; Currie, Meagan; Darkoa-
Larbi, Simone; Dellal, Daniel; Gutow, Hanna; Hokama, Pascha; Kibby, Emily; Linhart, Noah; Moody, Sophi
Naganuma, Allison; Nguyen, Diep; Stanton, Ryan; Stark, Sierra; Tumey, Cameron; Velleca, Anthony; Ryan
Joseph F.; and Davidson, Brad, "Variable levels of drift in tunicate cardiopharyngeal gene regulatory
elements." (2019). Student Papers, Posters & Projects. Paper 42. https://jdc.jefferson.edu/student_papers/42
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accepted for inclusion in Student Papers, Posters & Projects by an authorized administrator of the Jefferson Digita
Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu. Authors
Authors
William Colgan, Alexis Leanza, Ariel Hwang, Melissa B. DeBiasse, Isabel Llosa, Daniel Rodrigues, Hriju
Adhikari, Guillermo Barreto Corona, Saskia Bock, Amanda Carillo-Perez, Meagan Currie, Simone Darkoa-
Larbi, Daniel Dellal, Hanna Gutow, Pascha Hokama, Emily Kibby, Noah Linhart, Sophia Moody, Allison
Naganuma, Diep Nguyen, Ryan Stanton, Sierra Stark, Cameron Tumey, Anthony Velleca, Joseph F. Ryan,
and Brad Davidson Variable levels of drift in tunicate cardiopharyngeal gene
Variable levels of drift in tunicate cardiopharyngeal gene
regulatory elements.
regulatory elements. Colgan, William; Leanza, Alexis; Hwang, Ariel; DeBiasse, Melissa B.; Llosa, Isabel; Rodrigues, Daniel;
Adhikari, Hriju; Barreto Corona, Guillermo; Bock, Saskia; Carillo-Perez, Amanda; Currie, Meagan; Darkoa-
Larbi, Simone; Dellal, Daniel; Gutow, Hanna; Hokama, Pascha; Kibby, Emily; Linhart, Noah; Moody, Sophia;
Naganuma, Allison; Nguyen, Diep; Stanton, Ryan; Stark, Sierra; Tumey, Cameron; Velleca, Anthony; Ryan,
Joseph F.; and Davidson, Brad, "Variable levels of drift in tunicate cardiopharyngeal gene regulatory
elements." (2019). Student Papers, Posters & Projects. Paper 42. https://jdc.jefferson.edu/student_papers/42 This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital
Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is
a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections
from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested
readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been
accepted for inclusion in Student Papers, Posters & Projects by an authorized administrator of the Jefferson Digital
Commons. For more information, please contact: JeffersonDigitalCommons@jefferson.edu. Authors
Authors
William Colgan, Alexis Leanza, Ariel Hwang, Melissa B. DeBiasse, Isabel Llosa, Daniel Rodrigues, Hriju
Adhikari, Guillermo Barreto Corona, Saskia Bock, Amanda Carillo-Perez, Meagan Currie, Simone Darkoa-
Larbi, Daniel Dellal, Hanna Gutow, Pascha Hokama, Emily Kibby, Noah Linhart, Sophia Moody, Allison
Naganuma, Diep Nguyen, Ryan Stanton, Sierra Stark, Cameron Tumey, Anthony Velleca, Joseph F. Ryan,
and Brad Davidson This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/student_papers/42 Abstract Background: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene
expression or developmental patterning. The rules governing this process of developmental systems drift, includ‑
ing the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly
delineated. Results: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of
two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that
trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory
elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory ele‑
ments we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyn‑
geal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites
lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. Conclusions: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory ele‑
ments. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene
function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be
governed by distinct structural constraints that will be difficult to predict based on network architecture. Conclusions: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory ele‑
ments. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene
function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be
governed by distinct structural constraints that will be difficult to predict based on network architecture. Keywords: Gene regulatory networks, Developmental systems drift, Tunicates, Heart development, Selective
constraints ene regulatory networks, Developmental systems drift, Tunicates, Heart development, Selective Keywords: Gene regulatory networks, Developmental systems drift, Tunicates, Heart development, Selective
constraints © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Variable levels of drift in tunicate
cardiopharyngeal gene regulatory elements William Colgan1, Alexis Leanza3, Ariel Hwang4, Melissa B. DeBiasse5, Isabel Llosa2, Daniel Rodrigues2,
Hriju Adhikari2, Guillermo Barreto Corona2, Saskia Bock2, Amanda Carillo‑Perez2, Meagan Currie2,
Simone Darkoa‑Larbi6, Daniel Dellal7, Hanna Gutow2, Pascha Hokama8, Emily Kibby9, Noah Linhart10,
Sophia Moody11, Allison Naganuma2, Diep Nguyen12, Ryan Stanton2, Sierra Stark13, Cameron Tumey2,
Anthony Velleca14, Joseph F. Ryan5 and Brad Davidson2* Authors
Authors Colgan et al. EvoDevo (2019) 10:24
https://doi.org/10.1186/s13227-019-0137-2 EvoDevo RESEARCH
Variable levels of drift in tunicate
cardiopharyngeal gene regulatory elements
William Colgan1, Alexis Leanza3, Ariel Hwang4, Melissa B. DeBiasse5, Isabel Llosa2, Daniel Rodrigues2,
Hriju Adhikari2, Guillermo Barreto Corona2, Saskia Bock2, Amanda Carillo‑Perez2, Meagan Currie2,
Simone Darkoa‑Larbi6, Daniel Dellal7, Hanna Gutow2, Pascha Hokama8, Emily Kibby9, Noah Linhart10,
Sophia Moody11, Allison Naganuma2, Diep Nguyen12, Ryan Stanton2, Sierra Stark13, Cameron Tumey2,
Anthony Velleca14, Joseph F. Ryan5 and Brad Davidson2*
Open Access Backgroundh The prevalence and nature of
such cooperative binding interactions and the resulting
impact on drift are outstanding questions in evolutionary
developmental biology [3]. Previous studies of tunicate developmental systems
drift have focused on comparisons to the relatively well-
characterized regulatory networks underlying embryonic
development in C. robusta [44]. For some genes, includ-
ing the key developmental transcription factor Otx, con-
servation of the trans-regulatory environment promotes
conserved expression patterns and mutual intelligibility
in cross-species analysis despite extensive binding site
rearrangements within cis-regulatory elements [24, 45]. In other cases, expression is conserved despite diver-
gence of the trans-regulatory factors and associated
cis-regulatory elements, leading to loss of cross-species
intelligibility [26]. Drift in trans-factors is also indicated
by species-specific deployment of distinct signaling path-
ways in otherwise conserved developmental programs,
including the program driving muscle progenitor lineage
induction [46, 47]. These findings align with the hypoth-
esis that the extreme genomic divergence between tuni-
cate species has resulted in profound levels of drift within
developmental GRNs [37]. Although developmental systems drift in GRNs
appears to be a common phenomenon in metazoan evo-
lution, it can be difficult to study due to the requirement
for rigorous cross-species analysis within well-character-
ized networks [11, 12, 20, 21]. Cross-species assays are
used to determine the intelligibility of characterized cis-
regulatory elements between two species and thus evalu-
ate hypotheses regarding the amount of drift. Mutual
intelligibility of a cis-regulatory element suggests that
only cis drift has occurred [22–24]. In contrast, partial
or complete loss of intelligibility indicates that trans drift
has occurred [10, 25, 26]. It should be noted that results
from cross-species analysis are not definitive. Altera-
tions in GRN structure may be associated with shifts in
temporal or spatial expression that are difficult to detect
either because they are subtle or because available tech-
niques (such as reporter assays) do not accurately reflect
endogenous expression. Thus, in general, experimental
evidence for developmental systems drift does not rule
out a role for selection in driving observed shifts in GRN
architecture. Extensive characterization of the C. robusta cardi-
opharyngeal GRN makes it an attractive model for
comparative studies examining developmental systems
drift (Fig. 1a–c) [42, 48, 49]. The heart in C. robusta can
be traced back to two blastomeres (the B7.5 cells, also
termed cardiopharyngeal founder cells) which express
the bHLH transcription factor Mesp (Fig. 1a) [50–52]. Backgroundh shifts in phenotype [4–9]. However, mutations can accu-
mulate in cis-regulatory elements without altering gene
network function, contributing to developmental systems
drift [10–12]. Drift can also occur in trans due to muta-
tions that impact the expression or coding sequence of
upstream transcription factors (as defined in relation to
a specific target gene) [5]. In general, the organization of
binding motifs within cis-regulatory elements is loosely
constrained. This structural flexibility presumably reflects
independent, non-cooperative binding of upstream tran-
scription factors [3, 13, 14]. However, within a limited
subset of regulatory elements, the binding site organiza-
tion is more tightly constrained. This structural rigidity The gene regulatory networks (GRNs) that orchestrate
development are largely composed of trans-regulatory
factors (i.e., transcription factors) and cis-regulatory ele-
ments (i.e., enhancers and silencers) [1]. Connections
within these networks are dictated by transcription fac-
tor binding sites within each regulatory element [1–3]. Mutations that alter binding site composition are a major
driver of developmental changes underlying evolutionary © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. scription factors [3, 13, 14]. However, within a limited
subset of regulatory elements, the binding site organiza-
tion is more tightly constrained. This structural rigidity
*Correspondence: bdavids1@swarthmore.edu
2 Swarthmore College, Swarthmore, USA
Full list of author information is available at the end of the article *Correspondence: bdavids1@swarthmore.edu
2 Swarthmore College, Swarthmore, USA
Full list of author information is available at the end of the article *Correspondence: bdavids1@swarthmore.edu
2 Swarthmore College, Swarthmore, USA
Full list of author information is available at the end of the article Colgan et al. EvoDevo (2019) 10:24 Colgan et al. EvoDevo (2019) 10:24 Page 2 of 17 Page 2 of 17 combination of stringent developmental conservation
and extreme genomic divergence makes the tunicates a
powerful model for revealing the constraints that shape
adaptation and developmental systems drift [37]. presumably reflects cooperative, position-specific inter-
actions between bound transcription factors and asso-
ciated co-factors [14–19]. Backgroundh a Initial specification of the cardiopharyngeal founder cells (pink) through exclusive up-regulation of Mesp and subsequent expression of Ets1/2. b Signal-dependent regulation
of early trunk ventral cell genes by Ets1/2 and an unknown ATTA-binding co-factor. Ets1/2 activation in the TVCs is dependent on FGF9/16/20 signaling transduced by the MapK pathway. c
Presumptive modules differentially regulated by FoxF, Hand-like, or GATAa. FoxF is portrayed as the primary regulator of TVC migration, while GATAa regulates a highly conserved heart “kernel”
i
j
i
i h BMP2/4 i
li
F1 H1 H2
d K1
h
h i
l
Fi
i b
d
W
i
l
d C
l [41 42] d Si
lifi d
i
h l
b
d Fig. 1 Ciona robusta cardiopharyngeal gene regulatory network and tunicate phylogeny. a–c Regulatory network diagrams for cardiopharyngeal founder lineage cells during three embryonic
stages. Schematics on the left indicate stage and cell lineage. Background colors delineate discrete regulatory modules. Solid lines indicate regulatory connections supported by functional
enhancer analysis, while dashed lines indicate regulatory connections supported by expression data. Circles represent signal dependent activation and double slanted lines represent signal
transduction. a Initial specification of the cardiopharyngeal founder cells (pink) through exclusive up-regulation of Mesp and subsequent expression of Ets1/2. b Signal-dependent regulation
of early trunk ventral cell genes by Ets1/2 and an unknown ATTA-binding co-factor. Ets1/2 activation in the TVCs is dependent on FGF9/16/20 signaling transduced by the MapK pathway. c
Presumptive modules differentially regulated by FoxF, Hand-like, or GATAa. FoxF is portrayed as the primary regulator of TVC migration, while GATAa regulates a highly conserved heart “kernel”
in conjunction with BMP2/4 signaling. F1, H1, H2, and K1 represent hypothetical target genes. Figure is based on Woznica et al. and Cota et al. [41, 42]. d Simplified tunicate phylogeny based on
DeBiasse et al. (in prep), that is congruent with Delsuc et al. [43]. Backgroundh a–c Regulatory network diagrams for cardiopharyngeal founder lineage cells during three embryonic
stages. Schematics on the left indicate stage and cell lineage. Background colors delineate discrete regulatory modules. Solid lines indicate regulatory connections supported by functional
enhancer analysis, while dashed lines indicate regulatory connections supported by expression data. Circles represent signal dependent activation and double slanted lines represent signal
transduction. a Initial specification of the cardiopharyngeal founder cells (pink) through exclusive up-regulation of Mesp and subsequent expression of Ets1/2. b Signal-dependent regulation
of early trunk ventral cell genes by Ets1/2 and an unknown ATTA-binding co-factor. Ets1/2 activation in the TVCs is dependent on FGF9/16/20 signaling transduced by the MapK pathway. c
Presumptive modules differentially regulated by FoxF, Hand-like, or GATAa. FoxF is portrayed as the primary regulator of TVC migration, while GATAa regulates a highly conserved heart “kernel”
in conjunction with BMP2/4 signaling. F1, H1, H2, and K1 represent hypothetical target genes. Figure is based on Woznica et al. and Cota et al. [41, 42]. d Simplified tunicate phylogeny based on
DeBiasse et al. (in prep), that is congruent with Delsuc et al. [43]. Background colors represent sub-clades, Phlebobranchia (yellow), Stolidobranchia (red), or Appendicularia (blue) alu
rts
a
G
4
1
-
2
1
t
S
alu
r
u
e
N
7
1
-
5
1
t
S
d
u
blia
r
T
1
2
-
8
1
t
S
Founder cells
Trunk ventral cells
Migratory trunk
ventral cells
Cardiopharyngeal
founder cell genes
Primary trunk ventral
cell genes
Hand-like
target genes
FoxF target
genes
Heart “kernel”
genes
Mesp
Ets1/2
ATTA binding co-factor
FGF9/16/20
signal
FoxF
Hand-like
GATAa
Other genes
RhoD/F
ASB
F1
H1
H2
K1
Nk4
BMP2/4
signal
Tbx6b + Lhx3
//
MapK
//
a
b
c
d
Phallusia mammillata
Corella inflata
Corella willmeriana
Ciona robusta
Ciona savignyi
Botryllus leachi
Botryllus schlosseri
Halocynthia roretzi
Molgula oculata
Molgula occidentalis
Oikopleura dioica
Branchiostoma floridae
Homo sapiens
Phlebobranchia
Stolidobranchia
Appendicularia
~270 mya
~390 mya
Fig. 1 Ciona robusta cardiopharyngeal gene regulatory network and tunicate phylogeny. a–c Regulatory network diagrams for cardiopharyngeal founder lineage cells during three embryonic
stages. Schematics on the left indicate stage and cell lineage. Background colors delineate discrete regulatory modules. Solid lines indicate regulatory connections supported by functional
enhancer analysis, while dashed lines indicate regulatory connections supported by expression data. Circles represent signal dependent activation and double slanted lines represent signal
transduction. Backgroundh Founder cell-specific expression of Mesp is mediated by
two upstream transcription factors: a T-Box family tran-
scription factor, TBX6b, and a LIM homeobox family
transcription factor, LHX3, which are expressed in over-
lapping maternally specified domains [51, 53, 54]. During
gastrulation, the founder cells divide once, forming a pair
of cells on each side of the embryo, and express the tran-
scription factor Ets1/2 (Fig. 1a). The four resulting cells
then divide asymmetrically, creating two distinct cell lin-
eages: the anterior tail muscle cells (ATMs) and the trunk
ventral cells (TVCs). The TVCs are bi-potential progeni-
tors, giving rise to pharyngeal muscle and cardiac line-
ages (Fig. 1b). TVC specification is dictated by fibroblast
growth factor (FGF)/Map Kinase (MapK)-dependent
activation of Ets1/2 [55–57]. Ets1/2 in conjunction with
an unknown ATTA-binding co-factor then upregulates
a set of 218 primary genes which include the conserved
cardiac transcription factors FoxF, Hand-like, and GATAa
(Fig. 1b) [41, 58, 59]. These three transcription factors are
thought to regulate distinct modules in the C. robusta
cardiopharyngeal GRN (Fig. 1c) [42, 60–63]. Tunicates, or urochordates, are a powerful system for
studying developmental systems drift (Fig. 1). They are
closely related to vertebrates but diverged prior to ver-
tebrate genome duplications, so they have a single copy
of many important developmental genes [27, 28]. Tuni-
cates also have relatively compact genomes, enabling easy
identification of cis-regulatory elements through phy-
logenetic footprinting or detection of clustered binding
motifs [29–32]. In addition, some tunicate species can be
electroporated en masse, enabling high-throughput test-
ing of cis-regulatory elements with transgenic reporters
[33]. These techniques have been successfully employed
to intensively characterize developmental gene regulatory
networks in Ciona robusta (formerly known as Ciona
intestinalis, type A), including the network underlying
heart and pharyngeal development (Fig. 1a–c). Further-
more, tunicate embryos employ similar, deeply conserved
patterning mechanisms for early development. Remarka-
bly, species in two major tunicate clades, Phlebobranchia
and Stolidobranchia, have nearly identical embryonic
fate maps and employ similar programs for specification
and morphogenesis, despite having diverged ~ 390 mil-
lion years ago (Fig. 1d) [10, 34–36]. These similarities in
developmental patterning are even more striking when
the extreme rate of genomic divergence between tunicate
species is taken into consideration [37–40]. The unique Page 3 of 17 Colgan et al. EvoDevo (2019) 10:24 Fig. 1 Ciona robusta cardiopharyngeal gene regulatory network and tunicate phylogeny. Backgroundh Background colors represent sub-clades, Phlebobranchia (yellow), Stolidobranchia (red), or Appendicularia (blue) d
Phallusia mammillata
Corella inflata
Corella willmeriana
Ciona robusta
Ciona savignyi
Botryllus leachi
Botryllus schlosseri
Halocynthia roretzi
Molgula oculata
Molgula occidentalis
Oikopleura dioica
Branchiostoma floridae
Homo sapiens
Phlebobranchia
Stolidobranchia
Appendicularia
~270 mya
~390 mya d alu
rts
a
G
4
1
-
2
1
t
S
alu
r
u
e
N
7
1
-
5
1
t
S
d
u
blia
r
T
1
2
-
8
1
t
S
Founder cells
Trunk ventral cells
Migratory trunk
ventral cells
Cardiopharyngeal
founder cell genes
Primary trunk ventral
cell genes
Hand-like
target genes
FoxF target
genes
Heart “kernel”
genes
Mesp
Ets1/2
ATTA binding co-factor
FGF9/16/20
signal
FoxF
Hand-like
GATAa
Other genes
RhoD/F
ASB
F1
H1
H2
K1
Nk4
BMP2/4
signal
Tbx6b + Lhx3
//
MapK
//
a
b
c
d Colgan et al. EvoDevo (2019) 10:24 Page 4 of 17 c
b
d
a
8H
10H
11H
20H
CiHandLike:lacZ
in Corella
e
U0126 7.5H
f
U0126 10-11H
CiFoxF:GFP
in Corella
Mesp-GFP
Mesp-GFP
h
i
n
o
it
a
r
g
i
m
C
V
T
o
N
%
100
75
50
25
0
7.5H 10/11H
g
*
Fig. 2 Conserved founder cell lineage behavior and TVC induction in Corella embryos. a–c Representative Corella embryos expressing Cirobu.Mesp
−1916:Esconsin-3XGFP in presumptive founder lineage cells. Note labeling of mitotic spindle in 8H embryo (a). Hours post-fertilization indicated at
the lower right of each panel. d Representative Corella embryo expressing Cirobu.Mesp −1916:H2B:GFP to track founder lineage cell divisions in later
stages. e, f Transgenic Cirobu.Mesp 1916:GFP Corella embryos treated with the Map Kinase inhibitor U0126 at 7.5 HPF, e immediately prior to founder
cell division or f ~ 2 h after division at 10-11 HPF. Arrow points to migrated TVCs. g Summary of results for U0126 treatments. Data spans 6 trials,
N > 70 for each condition, Student’s T test, p value < 0.0005. Note that the levels of migration defects in the 10-11HPF treatment samples were similar
to basal levels seen in untreated, transgenic embryos (data not shown). h, i Representative embryos illustrating TVC expression for the Cirobu. FoxF-3052:GFP and Cirobu.Hand-like-2954/− 445:− 296:lacZ reporters d
20H c
11H d c a b e
U0126 7.5H
f
U0126 10-11H
Mesp-GFP
Mesp-GFP i g CiFoxF:GFP
in Corella
h
H CiHandLike:lacZ
in Corella
i e e CiHandLike:lacZ
in Corella Fig. Backgroundh 2 Conserved founder cell lineage behavior and TVC induction in Corella embryos. a–c Representative Corella embryos expressing Cirobu.Mesp
−1916:Esconsin-3XGFP in presumptive founder lineage cells. Note labeling of mitotic spindle in 8H embryo (a). Hours post-fertilization indicated at
the lower right of each panel. d Representative Corella embryo expressing Cirobu.Mesp −1916:H2B:GFP to track founder lineage cell divisions in later
stages. e, f Transgenic Cirobu.Mesp 1916:GFP Corella embryos treated with the Map Kinase inhibitor U0126 at 7.5 HPF, e immediately prior to founder
cell division or f ~ 2 h after division at 10-11 HPF. Arrow points to migrated TVCs. g Summary of results for U0126 treatments. Data spans 6 trials,
N > 70 for each condition, Student’s T test, p value < 0.0005. Note that the levels of migration defects in the 10-11HPF treatment samples were similar
to basal levels seen in untreated, transgenic embryos (data not shown). h, i Representative embryos illustrating TVC expression for the Cirobu. FoxF-3052:GFP and Cirobu.Hand-like-2954/− 445:− 296:lacZ reporters Comparative analysis of the C. robusta cardiopharyn-
geal GRN has been initiated in two species, Ciona savi-
gnyi and Molgula occidentalis. Regulatory elements and
upstream trans-factors appear to be highly conserved in
C. robusta and C. savignyi despite ~ 100 million years of
rapid genomic divergence [29, 64]. In M. occidentalis and
C. robusta, which diverged ~ 390 million years ago, cardi-
opharyngeal founder lineages still exhibit nearly identical
patterns of cell division and transcription factor expres-
sion [10]. However, there have been partial or complete
losses of intelligibility between cardiopharyngeal cis-reg-
ulatory elements in these two species, indicating that sig-
nificant developmental systems drift has occurred both
in cis and in trans [10].l cis-regulatory elements within this GRN exhibit different
levels of conservation. These differences correspond to
different structural and functional constraints. The FoxF TVC enhancer is highly conserved between C.
inflata and C. robusta l
To further explore drift of the FoxF-regulatory ele-
ment, we attempted to identify a candidate orthologous
enhancer in Corella using mVISTA multi-sequence align-
ment [66]. This alignment revealed a small region of
sequence conservation in C. inflata at the position of the
previously characterized C. robusta FoxF TVC enhancer
(Fig. 3a) [58]. Strikingly, this 183 bp region contained a
set of three conserved Ets1/2 and two conserved ATTA-
binding motifs that precisely matched the number, spac-
ing, and arrangement of the characterized binding sites in
the orthologous Ciona FoxF enhancer, while intervening
DNA was poorly conserved (Fig. 3b). Reporter constructs
containing this conserved element in C. inflata were
able to drive TVC-specific expression in both C. inflata
(Fig. 3c) and C. robusta (Fig. 3d). Thus, cross-species test-
ing demonstrated mutual intelligibility of a remarkably
well-conserved FoxF TVC enhancer (Figs. 2h, 3c, d). C. inflata and C. robusta share a conserved TVC
specification program robusta and Molgula
occidentalis embryos, treatment with the MEK inhibitor
U0126 just prior to B7.5 founder cell division (late gas-
trula stage) blocked induction of the heart progenitor lin-
eage (as indicated by lack of TVC migration, Fig. 2e, g),
while treatment at a later time point had no effect (Fig. 2f,
g) [10, 55]. We also began to examine conservation of
the heart gene network downstream of FGF-dependent
induction. In C. robusta, a small group of transcrip-
tion factors including FoxF, Hand-like, and GATAa are
upregulated directly downstream of FGF/MapK induc-
tion (Fig. 1) [41]. Through in situ hybridization in tailbud
stage embryos, we found that C. inflata FoxF is expressed
in the trunk epidermis and TVCs, mirroring similar
expression in C. robusta embryos at this stage (Fig. 3f). This initial analysis indicates that the program for trunk
ventral cell specification and migration in C. inflata and
C. robusta embryos has been conserved. expression immediately after TVC induction and are
co-regulated by Ets1/2 and an ATTA-binding co-factor
[41, 58]. As seen with the Cirobu.Mesp-1916 enhancer
(Fig. 2a–f), both these reporters recapitulated charac-
terized Ciona expression patterns in transgenic Corella
embryos. The FoxF reporter drove expression in the
TVCs and trunk epidermis (Fig. 2h) and the Hand-like
reporter drove expression in the TVCs and trunk endo-
derm along with weak expression in the ATM lineage
(Fig. 2i). The cross-species intelligibility of these three
reporters indicates that TVC specification and migration
in Corella and Ciona embryos rely on a conserved set of
upstream trans-factors. C. inflata and C. robusta share a conserved TVC
specification program To initiate our analysis of the Corella cardiopharyn-
geal GRN, we tested the activity of a characterized
C. robusta reporter construct for the heart founder
lineage
transcription
factor,
Mesp
(Cirobu.Mesp-
1916:Ensconsin:3XGFP) [56]. Fortunately, electroporation
protocols developed for C. robusta [30] were also effec-
tive for C. inflata embryos. As observed in Ciona, the
Cirobu.Mesp enhancer drove robust activity in Corella
B7.5 founder lineage cells, including both TVC and ATM
lineages. The Ensconsin:GFP reporter labels microtubules
[56, 65], allowing us to deploy this construct to track
founder cell lineage position and division in developing
C. inflata embryos. As seen previously in both Molgulid
and Cionid species, bilateral pairs of C. inflata heart
founder cells divide asymmetrically at the early neu-
rula stage (~ 8HPF) to produce the heart progenitor and
anterior tail muscle lineages (Fig. 2a, b). Further analysis
will be required to determine if this division is unequal
and whether differential induction involves receptor
localization as characterized in C. robusta [57]. Dur-
ing tailbud stages, C. inflata heart progenitors undergo To explore how evolutionary constraints influence drift
in developmental programs, we have begun comparative
studies of the cardiopharyngeal GRN in Corella inflata,
a phlebobranch that diverged from C. robusta ~ 270 mil-
lion years ago (Fig. 1d) (DeBiasse et al. 2019, in prep)
[43]. C. inflata is experimentally tractable, as synchro-
nized C. inflata embryos can be electroporated en masse
to test reporter constructs, and we recently sequenced
its genome and transcriptome (DeBiasse et al. 2019, in
prep). We used this genome to characterize enhancers
for key genes in the cardiopharyngeal GRN, including
Mesp, FoxF, and Hand-like. We show that the trans-reg-
ulatory architecture of the cardiopharyngeal GRN is
largely conserved between C. robusta and C. inflata, but Colgan et al. EvoDevo (2019) 10:24 Page 5 of 17 Page 5 of 17 a conserved anterior migration along the epidermis into
the ventral trunk region (Fig. 2c), where they undergo
an unequal cleavage to form smaller medial and larger
lateral daughters (Fig. 2d). Whether this represents an
asymmetric division to produce pharyngeal muscle and
heart precursors as seen in C. robusta will require fur-
ther analysis [62]. We also used the Cirobu.Mesp reporter
to examine whether TVC specification (as marked by
anterior migration) is dependent on FGF/MapK signal-
ing. As seen previously in both C. C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata To further explore developmental systems drift in the
cardiopharyngeal gene regulatory network, we began
to perform cross-species testing of regulatory elements. Since C. inflata and C. robusta shared a common ances-
tor more recently than C. robusta and M. occidentalis
(Fig. 1d) [43], we hypothesized that there would be con-
servation in the trans-regulatory architecture despite
divergence of cis-regulatory elements. Based on this
hypothesis, we expected the C. inflata and C. robusta
cardiopharyngeal GRN enhancers to display mutual
intelligibility in cross-species testing but not to align or
exhibit similar binding site arrangements. Alternatively,
it is possible that both cis-regulatory elements and trans-
regulatory architecture have been conserved, as seen in
comparisons between C. savignyi and C. robusta [29,
41, 50, 58], or that there has been divergence of both
the cis-regulatory elements and trans-regulatory archi-
tecture, as seen in comparisons between M. occidentalis
and C. robusta [10]. To begin exploring these hypoth-
eses, we tested two well-characterized C. robusta TVC
enhancers, Cirobu.FoxF-3052:GFP and Cirobu.Hand-
Like-2954/−445:−296:lacZ, in Corella embryos. In C. robusta, both of these enhancer elements mediate TVC To further evaluate whether the conserved region
upstream of Corella FoxF represented a functionally
constrained regulatory element, we cloned a 146 bp
fragment containing the full set of conserved binding
motifs. We then fused this minimal region to a 255 bp
basal promoter that had no independent reporter expres-
sion (data not shown). The resulting construct (Coinfl. FoxF −547/−401::−255) drove reporter expression in
Corella B7.5 lineage cells, including the TVCs and ATM
precursors (Fig. 3e, g). We then individually knocked out
the five conserved binding motifs in this minimal ele-
ment through site-directed mutagenesis and visualized
reporter expression in C. inflata embryos. While the dis-
ruption of the first Ets1/2 (E1) or first ATTA (A1) binding
motifs significantly reduced TVC reporter expression,
knocking out the other binding motifs had no discernible
impact (Fig. 3g). These results mirrored the results from a
similar analysis of the C. robusta FoxF TVC enhancer [41,
58] with the exception of the second Ets1/2 (E2)-binding
motif which was required in the C. robusta enhancer Colgan et al. EvoDevo (2019) 10:24 Page 6 of 17 Colgan et al. EvoDevo a
100%
50%
100%
50%
0k
1k
2k
3k
4k
5k
6k
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Alignment with C. robusta
C. savignyi
C. C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata inflata
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FoxF
Conserved
TVC enhancer
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%TVC expression in C. inflata
C. inflata FoxF
TVC enhancer
*
CoFoxF 547-401
+ 255
CoFoxF 547-401
+ 255 A1 KO
CoFoxF 547-401
+ 255 E1 KO
CoFoxF 547-401
+ 255 A2 KO
CoFoxF 547-401
+ 255 E2 KO
CoFoxF 547-401
+ 255 E3 KO
X
X
X
X
X
*
g
E1
A1
E2 E2C
A2
E3
C. robusta
C. savignyi
C. inflata
C. robusta
C. savignyi
C. inflata
956
1005
556
Ets1/2 binding site
ATTA binding site
TGTT binding site
Required
No functionality
T1
TVCs
Coinfl.FoxF 547-401:255 in C. inflata
Coinfl.FoxF -2622 in C. inflata Coinfl.FoxF -2622 in C. robusta
50 µm
E2
TVC
TVC
c
D
e
TVC
C. inflata FoxF
f
TVC
X
ΔE
(GGAW -> GCTW)
(ATTA->ATCT)
X
ΔA
d
Fig. 3 Characterization of the C. inflata FoxF TVC enhancer. a mVISTA alignments depict sequence conservation between C. robusta and C. savignyi and between C. robusta and C. inflata for the FoxF gene and 5′ intergenic region (LAGAN alignment, conservation across 100 bp window,
conservation > 70% highlighted). There is increased conservation associated with the FoxF coding region (orange) and conserved TVC enhancer
(purple). b ClustalW alignment of the 183 bp conserved TVC enhancer with Ets1/2 (red), ATTA (blue), and TGTT (orange)-binding motifs highlighted. Dark-shaded-binding motifs were required for reporter expression and boxed-binding motifs exhibited no functionality. C. robusta FoxF-binding
motif knockout data come from Beh et al. and Woznica et al. [41, 58]. c–e Representative embryos showing the activity of Coinfl.FoxF −2622 GFP
reporter constructs in C. inflata and C. robusta (arrows indicate expression in TVCs, and scale bar is 50 μm). f Representative C. inflata mid-tailbud
stage embryo displaying expression of Coinf.FoxF in TVCs (arrow) and epidermis. g Effect of Ets1/2 and ATTA-binding motif knockouts (Δ) on
reporter expression driven by the C. inflata 146 bp minimal TVC enhancer fused to a 255 bp basal promoter (Coinfl.FoxF −547/−401::−255). Names
of binding motifs correspond to the names in b. LacZ reporter constructs are diagramed on the left with X indicating a binding motif knockout. C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata The
h d
i
%TVC
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100%
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0k
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4k
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Alignment with C. robusta
C. savignyi
C. inflata
d
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FoxF
Conserved
TVC enhancer
b
E1
A1
E2 E2C
A2
E3
C. robusta
C. savignyi
C. inflata
C. robusta
C. savignyi
C. inflata
956
1005
556
Ets1/2 binding site
ATTA binding site
TGTT binding site
Required
No functionality
T1
TVCs
Coinfl.FoxF 547-401:255 in C. inflata
Coinfl.FoxF -2622 in C. inflata Coinfl.FoxF -2622 in C. robusta
50 µm
E2
TVC
TVC
c
D
e
TVC
C. inflata FoxF
f
TVC
d Alignment with C. robusta a b Conserved
TVC enhancer Coinfl FoxF -2622 in C inflata
Coinfl FoxF -2622 in C robusta
50 µm
E2
TVC
TVC
c
Dd Dd e c Coinfl.FoxF 547-401:255 in C. inflata
a Coinfl.FoxF -2622 in C. robusta %TVC expression in C. inflata
C. inflata FoxF
TVC enhancer
*
CoFoxF 547-401
+ 255
CoFoxF 547-401
+ 255 A1 KO
CoFoxF 547-401
+ 255 E1 KO
CoFoxF 547-401
+ 255 A2 KO
CoFoxF 547-401
+ 255 E2 KO
CoFoxF 547-401
+ 255 E3 KO
X
X
X
X
X
*
g
X
(ATTA
ATCT)
X g %TVC expression in C. inflata X
ΔE
(GGAW -> GCTW)
(ATTA->ATCT)
X
ΔA Fig. 3 Characterization of the C. inflata FoxF TVC enhancer. a mVISTA alignments depict sequence conservation between C. robusta and C. savignyi and between C. robusta and C. inflata for the FoxF gene and 5′ intergenic region (LAGAN alignment, conservation across 100 bp window,
conservation > 70% highlighted). There is increased conservation associated with the FoxF coding region (orange) and conserved TVC enhancer
(purple). b ClustalW alignment of the 183 bp conserved TVC enhancer with Ets1/2 (red), ATTA (blue), and TGTT (orange)-binding motifs highlighted. Dark-shaded-binding motifs were required for reporter expression and boxed-binding motifs exhibited no functionality. C. robusta FoxF-binding
motif knockout data come from Beh et al. and Woznica et al. [41, 58]. c–e Representative embryos showing the activity of Coinfl.FoxF −2622 GFP
reporter constructs in C. inflata and C. robusta (arrows indicate expression in TVCs, and scale bar is 50 μm). C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata inflata Hand-like (HL) upstream genomic fragment to
test two predicted enhancers. LacZ reporter constructs are diagramed on the left. The graph depicts %TVC expression in C. robusta (number of
trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). Significance relative to Coinfl.HL −1737 was determined with a Student t test
(p < 0.001 indicated by ***). The second predicted enhancer is both necessary and sufficient for reporter expression in the TVCs. b–e Representative
embryos showing the expression of LacZ reporter constructs that contain the second predicted enhancer (Coinfl.HL −1048) or lack the second
predicted enhancer (Coinfl.HL −899) in both C. robusta and C. inflata (arrows indicate expression in TVCs, and scale bar is 50 μm). f Effect of Ets1/2
and ATTA-binding motif knockouts (Δ) on the expression of a C. inflata Hand-like::LacZ reporter construct containing a 1048 bp upstream genomic
fragment (Coinfl.HL −1048). Names of binding motifs correspond to the names in panel B. LacZ reporter constructs are diagramed on the left with
X indicating a binding motif knockout. The graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 25, and error bars indicate
standard deviation). Significance relative to Coinfl.HL −1048 was determined with a Student t test (p < 0.01 indicated by ** and p < .001 indicated
by ***). g Comparison of Hand-like TVC enhancer structure in C. robusta and C. inflata. Darkly shaded binding motifs were required for reporter
expression. Lightly shaded binding motifs exhibited ‘limited” functionality as assessed by mutagenesis of multiple sites in the minimal Cirobu. FoxF enhancer [41] or by a non-significant reduction in reporter expression following mutagenesis (this study). Boxed binding motifs exhibited no
functionality. C. robusta Hand-like binding motif knockout data comes from Woznica et al. [41]
(See figure on next page.) candidate elements in the 5ʹ intergenic region (Addi-
tional file 1: Figure S1B). The distal element (prediction
1) was located 1737–1587 bp upstream of the gene, in
a similar position to the previously characterized C. robusta enhancer. The proximal element (prediction 2)
was located 1048–898 bp upstream of the gene. Both
predicted elements contained Ets1/2 and ATTA-binding
motifs and exhibited some structural similarity to the
previously characterized TVC enhancer of C. robusta
Hand-like (Additional file 1: Figure S1B) [41]. (Fig. 3g). C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata f Representative C. inflata mid-tailbud
stage embryo displaying expression of Coinf.FoxF in TVCs (arrow) and epidermis. g Effect of Ets1/2 and ATTA-binding motif knockouts (Δ) on
reporter expression driven by the C. inflata 146 bp minimal TVC enhancer fused to a 255 bp basal promoter (Coinfl.FoxF −547/−401::−255). Names
of binding motifs correspond to the names in b. LacZ reporter constructs are diagramed on the left with X indicating a binding motif knockout. The
graph depicts %TVC expression in C. inflata (number of trials ≥ 2, total N ≥ 150, and error bars indicate standard deviation). Significance relative to
Coinfl.FoxF −547/−401::−255 was determined with a Student’s t test, p < 0.05 indicated by * Fig. 3 Characterization of the C. inflata FoxF TVC enhancer. a mVISTA alignments depict sequence conservation between C. robusta and C. savignyi and between C. robusta and C. inflata for the FoxF gene and 5′ intergenic region (LAGAN alignment, conservation across 100 bp window,
conservation > 70% highlighted). There is increased conservation associated with the FoxF coding region (orange) and conserved TVC enhancer
(purple). b ClustalW alignment of the 183 bp conserved TVC enhancer with Ets1/2 (red), ATTA (blue), and TGTT (orange)-binding motifs highlighted. Dark-shaded-binding motifs were required for reporter expression and boxed-binding motifs exhibited no functionality. C. robusta FoxF-binding
motif knockout data come from Beh et al. and Woznica et al. [41, 58]. c–e Representative embryos showing the activity of Coinfl.FoxF −2622 GFP
reporter constructs in C. inflata and C. robusta (arrows indicate expression in TVCs, and scale bar is 50 μm). f Representative C. inflata mid-tailbud
stage embryo displaying expression of Coinf.FoxF in TVCs (arrow) and epidermis. g Effect of Ets1/2 and ATTA-binding motif knockouts (Δ) on
reporter expression driven by the C. inflata 146 bp minimal TVC enhancer fused to a 255 bp basal promoter (Coinfl.FoxF −547/−401::−255). Names
of binding motifs correspond to the names in b. LacZ reporter constructs are diagramed on the left with X indicating a binding motif knockout. The
graph depicts %TVC expression in C. inflata (number of trials ≥ 2, total N ≥ 150, and error bars indicate standard deviation). Significance relative to
Coinfl.FoxF −547/−401::−255 was determined with a Student’s t test, p < 0.05 indicated by * Page 7 of 17 Colgan et al. EvoDevo (2019) 10:24 Fig. 4 Characterization of the C. inflata Hand-like TVC enhancer. a Minimization of the C. C. robusta cardiac gene enhancers drive TVC reporter
expression in C. inflata This apparent divergence in enhancer structure
may reflect the presence of a third (presumably supple-
mental) Ets1/2-binding motif in C. inflata immediately
adjacent to the second Ets1/2 motif (E2C), potentially
creating redundancy. These results suggest that selection
has stringently constrained FoxF TVC enhancer struc-
ture, preventing any major shifts in the order, number,
or spacing of binding sites over nearly 300 million years
of rapid genomic divergence between C. robusta and C. inflata. i
We tested these computational predictions through
sequential minimization of the C. inflata Hand-like
5′ intergenic region using LacZ reporter constructs
(Fig. 4a). The full-length construct (Coinfl.HL −1737)
containing both candidate elements had strong TVC
expression in C. robusta, demonstrating that the Hand-
like TVC enhancer is intelligible by C. robusta. We
employed C. robusta for further minimization experi-
ments, because this species is more readily available than
C. inflata. Deletions that removed the first candidate
cis-regulatory element (Coinfl.HL −1615) or the region
between the candidate cis-regulatory elements (Coinfl.HL
−1048) did not affect TVC reporter expression (Fig. 4a,
b), but removing the second candidate cis-regulatory ele-
ment (Coinfl.HL −899) eliminated TVC reporter expres-
sion (Fig. 4a, c). A minimal 208 bp region encompassing
the second candidate cis-regulatory element fused to a
299 bp basal promoter (Coinfl.HL −1048/−844::−299)
drove strong TVC expression along with some ectopic
expression in the mesenchyme, a hotspot for ectopic
reporter expression [67], demonstrating that this region
is both necessary and sufficient for Hand-like TVC
expression (Fig. 4a). Coinfl.HL −1048 had strong TVC
reporter expression (Fig. 4b) and Coinfl.HL −899 had no
TVC reporter expression (Fig. 4c). Similar results were
obtained in C. inflata (Fig. 4d, e). Thus, the Hand-like
TVC enhancer is mutually intelligible in cross-species FoxF functions upstream of Hand‑like
in the cardiopharyngeal GRN To further investigate levels of drift across the cardi-
opharyngeal GRN, we characterized the regulatory ele-
ment for founder cell expression of Mesp in C. inflata. In
C. robusta, Mesp is expressed in the B7.5 cardiopharyn-
geal founder cell lineage downstream of TBX6b and
LHX3 (Fig. 1) [50–53]. Sequence alignments did not
reveal a conserved region in C. inflata associated with
the characterized Mesp enhancer in C. robusta (Addi-
tional file 1: Figure S4A) [66]. We therefore computation-
ally predicted candidate C. inflata Mesp enhancers based
on binding site clustering. This approach yielded one
candidate cis-regulatory element that aligned with the
known cis-regulatory element for C. robusta (Additional
file 1: Figure S4B) [51]. However, this candidate was a
poor match, as it was missing the first two TBX6-binding
motifs which were previously shown to be required in C. robusta [51]. We therefore started a sequential minimi-
zation analysis upstream of the candidate cis-regulatory
element. The full-length construct (Coinfl.Mesp −866)
drove strong expression in the founder lineage (ATMs
and TVCs) in both C. inflata and C. robusta, demonstrat-
ing mutual intelligibility (Figs. 2, 5a, b, e). This reporter
construct displayed almost no background expression
(Fig. 5a, b, e). Two shorter constructs (Coinfl.Mesp −651
and Coinfl.Mesp −576) still drove strong expression in
the founder lineage, but also produced ectopic expres-
sion in the primary trail muscle lineage (Fig. 5a, c). This
result suggests that there is a silencer element 866–
576 bp upstream of Mesp that represses tail muscle line-
age expression. A slightly shorter construct (Coinfl.Mesp
−421) drove no expression in the founder lineages or pri-
mary trail muscle lineages (Fig. 5a, d), indicating that the When we aligned the FoxF and Hand-like TVC enhanc-
ers for C. robusta, C. savignyi, and C. inflata, we noticed
a conserved TGTT-binding motif in both enhancers
across all three species (Figs. 3b and Additional file 1: Fig-
ure S1B). TGTT is part of the consensus binding motif of
Forkhead transcription factors such as FoxF (Additional
file 1: Figure S2A) [15]. Prior studies noted the enrich-
ment of this motif in Cionid TVC enhancer elements
[41] and a recent study also detected a significant enrich-
ment of putative FoxF-binding sites in the predicted cis-
regulatory elements of a wider range of primary TVC
genes [68]. Differential divergence of the Hand‑like vs. FoxF TVC
enhancer elements To determine if the rigorous conservation of the FoxF
TVC enhancer was unique or reflected generally high
levels of constraint in the cardiopharyngeal GRN, we
characterized the C. inflata TVC enhancer for Hand-like. Hand-like and FoxF occupy very similar positions in the
C. robusta cardiopharyngeal GRN [42]. Both these genes
are expressed shortly after TVC induction. They are both
regulated by Ets1/2 and an ATTA-binding co-factor and
they encode key transcription factors for TVC progenitor
fate (Fig. 1b). Based on the proposition that the hierarchi-
cal position of a gene within a GRN correlates with the
level of selective constraint on its regulatory elements [4],
we hypothesized that Hand-like and FoxF-regulatory ele-
ments would exhibit a similar level of conservation. Sequence alignments did not reveal a conserved region
in C. inflata associated with the characterized Hand-
like TVC enhancer in C. robusta (Additional file 1: Fig-
ure S1A) [66]. However, this analysis did not exclude the
presence of a conserved enhancer that may have shifted
position relative to the Hand-like gene and thus failed to
align globally. We, therefore, searched more broadly for
the C. inflata Hand-like TVC enhancer based on bind-
ing motif clustering and organization (see methods for
further details). This approach identified two strong Colgan et al. EvoDevo (2019) 10:24 Page 8 of 17 Colgan et al. EvoDevo a
b
c
d
e
f
g a c d e b e c d b f f g g We next began to functionally characterize the binding
sites in the C. inflata Hand-like TVC enhancer through
site-directed mutagenesis (Fig. 4f). This enhancer con-
tains two Ets1/2 and four ATTA-binding motifs (Fig. 4g). assays (Figs. 2i, 4c) while exhibiting substantially more
divergence in binding motif organization in comparison
with the FoxF TVC enhancer. Colgan et al. EvoDevo (2019) 10:24 Colgan et al. EvoDevo (2019) 10:24 Page 9 of 17 Knocking out the second or third ATTA motif (A2, A3)
or the second Ets1/2 motif (E2) significantly reduced
TVC reporter expression, while knocking out the
remaining motifs did not significantly alter TVC reporter
expression (Fig. 4f). In contrast, published mutational
analysis of the C. robusta Hand-like element indicated
that both Ets sites along with the first and second ATTA
sites were required for full reporter activity (dark shading
indicates functionally required binding motifs, Fig. 4f)
[41]. Differential divergence of the Hand‑like vs. FoxF TVC
enhancer elements In summary, our analysis indicates that trans-regu-
lation of Hand-like expression in the TVCs by Ets1/2 and
an ATTA-binding co-factor has been conserved between
these two species, while the cis-regulatory element has
undergone substantial divergence, including changes in
the number, order, orientation, and spacing of binding
motifs. Thus, the cis-regulatory elements for FoxF and
Hand-like appear to have experienced distinct levels of
functional constraint, despite occupying similar positions
in the cardiopharyngeal GRN. C. robusta GATAa TVC enhancer [61]. A minimal
223 bp region of the intron containing this candidate ele-
ment fused to a C. robusta Hand-like minimal promoter
(Coinfl.GATAa
+642/+820::Cirobu.Hand-like
−299)
was able to drive reporter expression in the TVCs (Addi-
tional file 1: Figure S3). Although the C. inflata GATAa
enhancer diverged substantially from the C. robusta ele-
ment, it still contains a conserved TGTT-binding motif
(Additional file 1: Figure S2C). This finding suggests that
GATAa is also regulated by FoxF. Taken together, these
results suggest that FoxF plays a central role in TVC
specification, responding rapidly to FGF-dependent
Ets1/2 activation, and contributing to the up-regulation
of other primary TVC genes including Hand-like, while
also maintaining its own expression. The putative role of
FoxF upstream of Hand-like also suggests that the more
stringent conservation of the FoxF-regulatory element
may reflect this more critical functional role. FoxF functions upstream of Hand‑like
in the cardiopharyngeal GRN robusta and C. inflata (number of trials ≥ 2, total N ≥ 75,
and error bars indicate standard deviation). Significance relative to Coinfl.Mesp −576 or the minimal −576/−421 enhancer was determined with
a Student t test (p < 0.05 indicated by * and p < 0.01 indicated by **). g Structure of the C. inflata Mesp founder cell enhancer. Darkly shaded green
TBX6 motifs were required for reporter expression, and lightly shaded pink LHX binding motifs exhibited some functionality, as determined by
mutagenesis of multiple motifs. There is no conservation of functional binding motifs in the aligned upstream genomic region of C. robusta. h
Representative C. robusta embryo showing the founder lineage and primary tail muscle lineage expression for Coinfl.Mesp −576/−421::−138. i–k
Representative C. robusta embryos showing lack of reporter expression for i Coinfl.Mesp ΔT1, and j Coinfl.Mesp ΔL1,2,3,4 and k reporter expression in
the primary tail muscle lineage, but not the founder lineage for Coinfl.Mesp ΔL4,5,6 (Fig. 5f, k). Thus, trans-activation of Mesp by TBX6
and LHX3 appears to be conserved in C. inflata and C. robusta, while cis-regulatory elements have undergone
substantial divergence. computationally predicted cis-regulatory element was
not sufficient for reporter expression. Instead, we found
that a region 576–421 bp upstream of Mesp fused to a
138 bp basal promoter (Coinfl.Mesp −576/−421::−138)
drove strong founder lineage expression, demonstrat-
ing that this 155 bp region is both necessary and suffi-
cient for founder lineage expression (Fig. 5a). Strikingly,
this 155 bp minimal enhancer bears almost no sequence
similarity to the characterized C. robusta element (Addi-
tional file 1: Figure S4C) and is also a very poor match
to the globally aligned region 426–261 bp upstream of C. robusta Mesp (Fig. 5g) Thus, our analysis reveals substan-
tial divergence between the minimal Mesp founder cell
enhancers of these two species. In summary, our data indicate that upstream tran-
scription factors dictating FoxF, Hand-like, and Mesp
expression in the cardiopharyngeal GRN are conserved
between C. robusta and C. inflata. However, the cis-
regulatory elements that control the expression of these
genes exhibit distinct levels of conservation between C. robusta and C. inflata. The FoxF TVC enhancer is highly
conserved, with identical organization of binding motifs,
while the Hand-like and Mesp enhancers exhibit exten-
sive divergence. These distinct levels of cis-regulatory
conservation do not appear to reflect GRN hierarchy,
as Mesp functions at the top of the GRN. FoxF functions upstream of Hand‑like
in the cardiopharyngeal GRN The conservation of this motif suggests that
FoxF works to maintain its own expression and acti-
vate other primary TVC genes such as Hand-like in the
C. robusta cardiopharyngeal GRN. As predicted by this
hypothesis, mutation of the TGTT motif (T1) in the
minimal C. robusta Hand-like TVC enhancer (Cirobu.HL
−1914/−1314::−299) abrogated TVC reporter expres-
sion (Additional file 1: Figure S2B). In addition, mutation
of the TGTT motif (T1) in the minimal C. robusta FoxF
TVC enhancer (Cirobu.FoxF −1072/−847::pFkh) did not
impact TVC expression, as predicted by the hypothetical
role of this site in maintaining rather than initiating FoxF
expression (Additional file 1: Figure S2B). Based on these
results, we sought to determine if the TVC enhancer
for GATAa also contains a conserved TGTT-binding
motif. Using our script to computationally predict TVC
enhancers for C. inflata GATAa, we identified one strong
candidate element in the first intron (Additional file 1:
Figure S2C), similar to the position of the characterized Colgan et al. EvoDevo (2019) 10:24 Page 10 of 17 (See figure on next page.)
Fig. 5 Characterization of the C. inflata Mesp founder lineage enhancer. a Minimization of the C. inflata Mesp 5′ intergenic region to identify the
B7.5 founder lineage enhancer. LacZ reporter constructs are diagramed on the left. The graphs depict % founder lineage (TVC + ATM) expression
or % founder lineage + primary tail muscle lineage expression in C. robusta and C. inflata (number of trials ≥ 2, total N ≥ 25, and error bars indicate
standard deviation). Significance relative to Coinfl.Mesp −651 was determined with a Student t test (p < 0.01 indicated by ** and p < 0.001 indicated
by ***). b Representative C. robusta embryo showing founder lineage-specific expression of Coinfl.Mesp −866 (arrows indicate TVCs and ATMs, and
scale bar is 50 μm). c Representative C. robusta embryo showing the founder lineage and primary tail muscle lineage expression for Coinfl.Mesp
−576. d Representative C. robusta embryo showing the lack of founder lineage expression for Coinfl.Mesp −421. e Representative C. inflata embryo
showing the founder lineage-specific expression for Coinfl.Mesp −866. f Effect of TBX6 and LHX3 binding motif knockouts (Δ) on the expression of
the C. inflata Mesp founder cell enhancer. Binding motifs designated as shown in g. LacZ reporter constructs are diagramed on the left with an X
indicating a binding motif knockout. The graphs depict % founder lineage expression in C. FoxF functions upstream of Hand‑like
in the cardiopharyngeal GRN Therefore,
we began to explore alternative hypotheses regarding
the exceptional conservation of the FoxF TVC enhancer
over ~ 270 million years of rapid evolutionary divergence. To begin investigating trans-regulation of Mesp in C. inflata, we mutagenized putative binding sites in the
minimal reporter construct and assayed the impact on
reporter expression in both C. robusta and C. inflata
(Fig. 5f–k). The minimal C. inflata Mesp founder cell
enhancer contains two TBX6-binding motifs and six
LHX3-binding motifs (Fig. 5g). Knocking out either
TBX6-binding motif (T1 or T2) completely eliminated
founder lineage reporter expression in both C. robusta
and C. inflata (Fig. 5f, i). In contrast, knocking out indi-
vidual LHX3-binding motifs did not affect founder line-
age reporter expression (data not shown). This result
could reflect redundancy in the LHX3-binding sites, so
we knocked out combinations of LHX3-binding motifs. When we knocked out the first four LHX3-binding
motifs (L1, L2, L3, and L4), founder lineage and tail mus-
cle lineage expression were lost in both C. robusta and C. inflata (Fig. 5f, j). When we knocked out the last three
LHX3-binding motifs (L4, L5, and L6), founder line-
age expression was almost completely eliminated, but
primary tail muscle lineage expression was maintained Precise binding site spacing is required for FoxF TVC
enhancer functionh There are a number of possible explanations for the rela-
tively stringent conservation of the FoxF TVC enhancer
between C. inflata and C. robusta. The first is that a spe-
cific organization of binding sites is required for physical
interactions between transcription factors [3, 14]. Alter-
natively, the enhancer may be constrained to ensure pre-
cise temporal or spatial expression [69]. To distinguish
between these hypotheses, we displaced the first Ets1/2-
binding motif (E1) in the C. robusta FoxF TVC enhancer
and examined the impact on reporter expression. We
chose this binding site because it is required for strong
TVC expression in both C. robusta and C. inflata (Fig. 3b,
g). Moreover, the ten base-pair spacing between this Page 11 of 17 Colgan et al. EvoDevo (2019) 10:24 Colgan et al. EvoDevo a
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%TVC expression in C. robusta
Cirobu.FoxF -1072/-827
::pFkh
E1
E1
E1
A1
A1
A1
A1
Cirobu.FoxF -1072/-827
::pFkh
Cirobu.FoxF -1072/-827
::pFkh E1 Move 1
Cirobu.FoxF -1072/-827
::pFkh E1 Move 2
C. robusta FoxF
TVC enhancer
C. robusta
Fkh promoter
*
*
*
Cirobu.FoxF -1072/-827::pFkh
c
d
Cirobu.FoxF -1072/-827::pFkh
Cirobu.FoxF -1072/-827::pFkh
E1 Move 1
TVC
b
0.0 10.0 20.0 30.0 40.0 50.0
Fig. 6 Functional constraint on binding site spacing in the C. robusta FoxF TVC enhancer. a The first Ets1/2-binding site was moved by knocking out
the endogenous binding site (GGAT ⟶ GCTT) and introducing a new binding site using site-directed mutagenesis. Reporter constructs contained
the 245 bp minimal C. robusta FoxF TVC enhancer fused to the C. robusta Fkh basal promoter (Cirobu.FoxF −1072/−827::pFkh). The sequence of
the enhancer region containing this first Ets1/2-binding site is shown on the left with Ets1/2 (red) and ATTA (blue)-binding sites highlighted. The
graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). b–d Representative
C. robusta embryos showing reporter expression for b Corobu.FoxF −1072/−827::pFkh, c Corobu.FoxF −1072/−827::pFkh ∆E1, or d Corobu.FoxF
−1072/−827::pFkh Move 1. Arrows point to normal position of TVCs in the trunk region. Precise binding site spacing is required for FoxF TVC
enhancer functionh Note substantial ectopic expression in the anterior tail
muscle lineage (ATM) and in other muscle and mesenchyme lineage cells a
%TVC expression in C. robusta
Cirobu.FoxF -1072/-827
::pFkh
E1
E1
E1
A1
A1
A1
A1
Cirobu.FoxF -1072/-827
::pFkh
Cirobu.FoxF -1072/-827
::pFkh E1 Move 1
Cirobu.FoxF -1072/-827
::pFkh E1 Move 2
C. robusta FoxF
TVC enhancer
C. robusta
Fkh promoter
*
*
*
0.0 10.0 20.0 30.0 40.0 50.0 a C. robusta FoxF
TVC enhancer d
Cirobu.FoxF -1072/-827::pFkh
E1 Move 1 Cirobu.FoxF -1072/-827::pFkh
TVC
b b c
Cirobu.FoxF -1072/-827::pFkh b d c Fig. 6 Functional constraint on binding site spacing in the C. robusta FoxF TVC enhancer. a The first Ets1/2-binding site was moved by knocking out
the endogenous binding site (GGAT ⟶ GCTT) and introducing a new binding site using site-directed mutagenesis. Reporter constructs contained
the 245 bp minimal C. robusta FoxF TVC enhancer fused to the C. robusta Fkh basal promoter (Cirobu.FoxF −1072/−827::pFkh). The sequence of
the enhancer region containing this first Ets1/2-binding site is shown on the left with Ets1/2 (red) and ATTA (blue)-binding sites highlighted. The
graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). b–d Representative
C. robusta embryos showing reporter expression for b Corobu.FoxF −1072/−827::pFkh, c Corobu.FoxF −1072/−827::pFkh ∆E1, or d Corobu.FoxF
−1072/−827::pFkh Move 1. Arrows point to normal position of TVCs in the trunk region. Note substantial ectopic expression in the anterior tail
muscle lineage (ATM) and in other muscle and mesenchyme lineage cells Fig. 6 Functional constraint on binding site spacing in the C. robusta FoxF TVC enhancer. a The first Ets1/2-binding site was moved by knocking out
the endogenous binding site (GGAT ⟶ GCTT) and introducing a new binding site using site-directed mutagenesis. Reporter constructs contained
the 245 bp minimal C. robusta FoxF TVC enhancer fused to the C. robusta Fkh basal promoter (Cirobu.FoxF −1072/−827::pFkh). The sequence of
the enhancer region containing this first Ets1/2-binding site is shown on the left with Ets1/2 (red) and ATTA (blue)-binding sites highlighted. The
graph depicts %TVC expression in C. robusta (number of trials ≥ 2, total N ≥ 75, and error bars indicate standard deviation). b–d Representative
C. robusta embryos showing reporter expression for b Corobu.FoxF −1072/−827::pFkh, c Corobu.FoxF −1072/−827::pFkh ∆E1, or d Corobu.FoxF
−1072/−827::pFkh Move 1. Arrows point to normal position of TVCs in the trunk region. Precise binding site spacing is required for FoxF TVC
enhancer functionh Note substantial ectopic expression in the anterior tail
muscle lineage (ATM) and in other muscle and mesenchyme lineage cells hypothesis that binding site organization is constrained
by required interactions between trans-factors. binding motif (E1) and the first ATTA-binding motif (A1)
is conserved between C. robusta and C. inflata. A ten
base-pair increment between binding sites corresponds
to a single helical turn and is often observed in enhanceo-
some-like cis-regulatory elements [14]. We displaced this
first Ets1/2-binding site by knocking out the endogenous
site and introducing a new site either 16 or 24 base pairs
from the first ATTA site. We conducted this analysis in a
LacZ reporter construct containing the minimal 245 bp
C. robusta FoxF TVC enhancer fused to the basal Fork-
head promoter (Cirobu.FoxF −1072/−827::pFkh:lacZ). This is a slightly longer construct than the previously
characterized 232 bp minimal reporter (Cirobu.FoxF
−1072/−840::pFkh:lacZ) [58]. When the first Ets1/2-
binding motif (E1) was knocked out in the context of
the 245 bp minimal element, TVC reporter expression
was significantly reduced (Fig. 6a, c). The introduc-
tion of new Ets1/2-binding sites 6 bp (Move 1), or 14 bp
(Move 2) upstream of the original position failed to res-
cue TVC reporter expression (Fig. 6a, d). The fact that
this reorganization reduced expression rather than alter-
ing temporal or spatial expression patterns supports the Developmental systems drift within the tunicate
cardiopharyngeal GRN Mutual intelligibility in our cross-species assays sug-
gests that the trans-regulatory architecture of the cardi-
opharyngeal GRN is largely conserved between C. inflata
and C. robusta. These findings are in contrast to previous
comparisons between M. occidentalis and C. robusta that
revealed numerous instances of enhancer incompatibility
caused by extensive trans drift in the cardiopharyngeal
GRN [10]. Both these studies are based on functional
analysis of minimal regulatory elements and thus may
not encompass the full range of cis-regulatory function
(as mentioned in the introduction, our use of the term
drift in this instance and throughout the discussion is
speculative, because observed changes in GRN structure
may have undetected impacts on expression and thus
may not be independent of selection). However, these
studies still provide a robust framework for developing Colgan et al. EvoDevo (2019) 10:24 Colgan et al. EvoDevo (2019) 10:24 Page 13 of 17 models regarding the rate and nature of developmental
systems drift. In particular, these findings are congruent
with two alternative models for the emergence of trans
drift in developmental GRNs. Trans drift may arise at a
steady rate, so that the amount of drift roughly correlates
with the absolute evolutionary distance between two spe-
cies and is not influenced by other taxonomic considera-
tions. Alternatively, the rate of trans drift may vary due
to factors independent of evolutionary distance. In par-
ticular, increased drift may occur during the divergence
of major clades, such as that between phlebobranchs and
stolidobranchs, in association with shifts in morphology
or rewiring of underlying developmental gene networks. According to the first model, the differential occurrence
of trans drift between M. occidentalis and C. robusta can
be attributed to the longer period of divergence between
these species, ~ 390 million years, in comparison with
C. inflata, which diverged from C. robusta ~ 270 million
years ago [43]. According to the second model, differ-
ential trans drift may have arisen during GRN rewiring
associated with changes in body plan or divergence of
developmental programs between Phlebobranchs and
Stolidobranchs. A broader cross-species analysis is
required to distinguish between these models. additional transcription factor required for Mesp activa-
tion. Overall, our results provide preliminary support for
the hypothesis that heterogeneous levels of constraint on
trans-regulatory inputs reflect directive rather than per-
missive functional contributions. Clearly, further analysis
is required to solidify our understanding of Mesp regula-
tion and further test this general hypothesis. Developmental systems drift within the tunicate
cardiopharyngeal GRN Our findings provide more robust insights into cis-
regulatory drift. Sequence alignments and functional
enhancer analysis reveal highly variable levels of diver-
gence for cis-regulatory elements within the cardi-
opharyngeal GRN. The minimal FoxF TVC enhancer is
highly conserved, with identical organization and spac-
ing of binding motifs. In contrast, the minimal Hand-
like TVC enhancer is poorly conserved and the minimal
Mesp founder cell lacks any apparent structural conser-
vation. These findings do not align with models in which
differential constraints associated with the position or
function of a gene in a GRN dictate relative levels of cis-
regulatory drift. Rather, our findings suggest that drift is
dictated by distinct structural and functional constraints
that are unique to each cis-regulatory element. Our find-
ings have also begun to illuminate the specific structural
and functional constraints that dictate conservation of
the FoxF enhancer, as discussed in the following section. q
g
Our analysis of the Mesp founder cell enhancer also
provides an alternative perspective on differential diver-
gence between trans-regulatory inputs [70]. The acti-
vation of Mesp by TBX6b is conserved between M. occidentalis, C. inflata, and C. robusta, while its activa-
tion by LHX3 is only conserved between C. inflata and
C. robusta. Our results suggest that differential levels of
constraint on these trans-factor inputs reflect a primary
directive role for TBX6b, while LHX3 plays a more sec-
ondary, permissive role. When we removed the 300 bp
genomic region upstream of the C. inflata Mesp founder
cell enhancer, we observed ectopic primary tail muscle
lineage reporter expression. A similar result has been
observed during deletion analysis of the C. robusta Mesp
enhancer (Brad Davidson, unpublished results). Ectopic
tail muscle expression is likely caused by TBX6b, which
is expressed in a broad domain encompassing the B7.5
founder cells and neighboring tail muscle lineages [53]. According to this model, regions’ upstream of the mini-
mal Mesp element may contain a silencer bound by a tail
muscle specific repressor. Thus, in tail muscle lineages,
TBX6 may be able to activate Mesp expression indepen-
dently of LHX3, which is expressed only in the endo-
derm/founder lineage cells. We are unsure why one set
of LHX3 binding motif knockouts eliminated primary
tail muscle and founder lineage expression, while another
set only eliminated founder lineage expression. It is pos-
sible that mutagenesis of the first four LHX3-binding
motifs accidentally impacted the binding motif of an Model for the constraints on the FoxF TVC enhancerl ode o t e co st a
ts o t e o
C e
a ce
Highly conserved enhancers generally reflect cooperative,
position-specific interactions between bound transcrip-
tion factors [14]. This type of highly conserved enhancer
is known as an enhanceosome and is distinguished by
conservation of the number, order, orientation, and spac-
ing of binding motifs [3, 14]. The prototypical enhan-
ceosome is the interferon-β cis-regulatory element [71]. Although relatively rare, additional enhanceosome-like
cis-regulatory elements have subsequently been char-
acterized [14, 17–19, 72]. However, general principles
regarding the deployment of enhanceosomes within
developmental GRNs have not been delineated. Muta-
tions that disrupt the relative position of binding sites
generally disable enhanceosome elements, presumably
because they disrupt protein–protein interactions [16]. We show that displacing the first Ets1/2-binding motif in
the C. robusta FoxF TVC enhancer significantly reduces
reporter expression. This result suggests that the FoxF
TVC enhancer is an enhanceosome-like cis-regulatory
element, in which Ets1/2, the ATTA-binding co-factor,
and possibly other proteins must physically interact to
activate FoxF expression. However, further experimenta-
tion will be required to provide more definitive support
for this hypothesis. In particular, the use of a wider range
of mutations will help determine whether the specific
mutations we introduced had unintended impacts, such Colgan et al. EvoDevo (2019) 10:24 Page 14 of 17 a
b
FoxF TVC enhancer
Early TVC gene enhancers
A repressor blocks precocious FoxF expression. FoxF acts as a pioneer factor, opening chromatin
Ets1/2 & ATTA co-factor binding promotes expression
FoxF helps maintain its own expression. n
oitc
u
d
ni-
e
r
P
c
Ets1/2 & ATTA co-factor complex evicts repressor,
permitting FoxF expression. P
P
n
oitc
u
d
n
I
n
oitc
u
d
ni-ts
o
P
Condensed chromatin blocks
precocious expression. promoter
promoter
P
P
P
P
Ets1/2
ATTA binding
co-factor
Hypothetical co-activator
Hypothetic repressor
FoxF
Nucleosome
RNA Pol II
Fig. 7 Model for the differential constraint on FoxF vs. other early TVC enhancers. a Before FGF induction, the chromatin around early TVC gene
enhancers is condensed preventing aberrant expression. In contrast, chromatin is decondensed at the FoxF TVC enhancer locus, suggesting that a
repressor (purple) is required to prevent precocious expression. b FGF/MapK-signaling phosphorylates Ets1/2 in the TVCs, permitting recruitment of
a co-factor (green) that serves to lift repression. The cooperative recruitment of this co-factor constrains binding site position and orientation. Model for the constraints on the FoxF TVC enhancerl FoxF
(orange) then accumulates in the TVC nuclei, where it acts as a pioneer factor opening the chromatin around other TVC enhancers. c Once early
TVC gene enhancers are open, the binding of Ets1/2, ATTA, and FoxF activates transcription in a non-cooperative fashion, as reflected by a lack of
constraint on binding site position. FoxF also binds the FoxF TVC enhancer helping to maintain its own expression a
b
FoxF TVC enhancer
Early TVC gene enhancers
A repressor blocks precocious FoxF expression. FoxF acts as a pioneer factor, opening chromatin
Ets1/2 & ATTA co-factor binding promotes expression
FoxF helps maintain its own expression. n
oitc
u
d
ni-
e
r
P
c
Ets1/2 & ATTA co-factor complex evicts repressor,
permitting FoxF expression. P
P
n
oitc
u
d
n
I
n
oitc
u
d
ni-ts
o
P
Condensed chromatin blocks
precocious expression. promoter
promoter
P
P
P
P
Ets1/2
ATTA binding
co-factor
Hypothetical co-activator
Hypothetic repressor
FoxF
Nucleosome
RNA Pol II
Fig 7 Model for the differential constraint on FoxF vs other early TVC enhancers a Before FGF induction the chromatin around early TVC gene FoxF TVC enhancer Early TVC gene enhancers Condensed chromatin blocks
precocious expression. Ets1/2 & ATTA co-factor complex evicts represso
permitting FoxF expression. RNA Pol II Fig. 7 Model for the differential constraint on FoxF vs. other early TVC enhancers. a Before FGF induction, the chromatin around early TVC gene
enhancers is condensed preventing aberrant expression. In contrast, chromatin is decondensed at the FoxF TVC enhancer locus, suggesting that a
repressor (purple) is required to prevent precocious expression. b FGF/MapK-signaling phosphorylates Ets1/2 in the TVCs, permitting recruitment of
a co-factor (green) that serves to lift repression. The cooperative recruitment of this co-factor constrains binding site position and orientation. FoxF
(orange) then accumulates in the TVC nuclei, where it acts as a pioneer factor opening the chromatin around other TVC enhancers. c Once early
TVC gene enhancers are open, the binding of Ets1/2, ATTA, and FoxF activates transcription in a non-cooperative fashion, as reflected by a lack of
constraint on binding site position. FoxF also binds the FoxF TVC enhancer helping to maintain its own expression further supports the hypothesis that FoxF acts as a pio-
neer factor during TVC specification and also suggests
that FoxF maintains its own expression. as the creation or elimination of cryptic binding sites. Model for the constraints on the FoxF TVC enhancerl In
addition, by further varying binding site displacement,
we can test whether presumed cooperativity is depend-
ent on relative position on the helix. Furthermore, it will
be interesting to analyze whether the conserved dis-
tances between other binding motifs in the FoxF minimal
enhance also reflect functional constraints. Conclusion Taken together, these results allow us to formulate a
model that explains the specific deployment of a highly
constrained, enhanceosome-like element for the regu-
lation of FoxF (Fig. 7). Before FGF induction, the chro-
matin around the enhancers of most early TVC genes is
condensed, which prevents aberrant expression (Fig. 7a). One exception is the FoxF enhancer, which remains
decondensed, so it can mediate a rapid, primary response
to FGF/MapK-dependent activation of Ets1/2 (Fig. 7a). Since chromatin condensation does not constrain aber-
rant expression of FoxF, another mechanism is required. We propose that this alternate mechanism involves the
occupation of a silencer element located near the FoxF
enhancer. Indeed, ectopic reporter expression through-
out the B7.5 founder lineage in our 245 bp minimal FoxF
enhancer construct (Fig. 6b) suggests that a silencer
element serves to block precocious FoxF expression, l
The deployment of an enhanceosome for regulation
of FoxF may be associated with its role as a pioneer fac-
tor. This hypothesis arises from the recent findings of
Racioppi et al., who found that FoxF promotes TVC
specification by changing chromatin accessibility [68]. In particular, the binding of FoxF to the enhancers of
other early TVC genes, including Hand-like and GATAa,
appears to increase the accessibility of these cis-regu-
latory elements by decondensing chromatin, thereby
enabling activation of these genes by Ets1/2, and the
ATTA-binding co-factor [68]. Racioppi et al. also showed
that CRISPR/Cas9 knockdown of FoxF led to down-reg-
ulation of several early TVC genes, including Hand-like
[68]. Our mutational analysis of the FoxF-binding motif
in the C. robusta Hand-like and FoxF TVC enhancer Colgan et al. EvoDevo (2019) 10:24 Colgan et al. EvoDevo (2019) 10:24 Page 15 of 17 possibly mediated by unphosphorylated Ets. According
to our model, FGF/MapK-dependent phosphorylation
of Ets1/2 leads to the formation of a complex with the
ATTA-binding factor and the recruitment of a presump-
tive, non-DNA binding co-factor that is able to lift base-
line repression (Fig. 7b). Once the FoxF gene is expressed,
FoxF maintains its own expression and opens the chro-
matin around other TVC enhancers (Fig. 7c). This model
may reflect a general principle for the seemingly sporadic
occurrence of enhanceosomes. Namely, enhanceosomes
may be specifically deployed for pioneer trans-factors,
ensuring precise temporal or spatial expression despite a
lack of chromatin-dependent regulation. Embryological techniques
Fertilization and dechorionationl p
p
The enhancers for C. inflata Hand-like, GATAa, and
Mesp were computationally predicted based on struc-
tural similarity to the previously characterized enhanc-
ers in C. robusta [50, 51, 61]. A custom Python (version
2.7.13) script was used to slide a 150 bp window over
the C. inflata 5′ intergenomic region for each of these
genes in 25 bp increments (https://github.com/colganwi/
CRMFinder). Each window position was scored with a
linear combination of four features [1]: the number of
oligomers ≥ 4 bp which were present in both the window
and the C. robusta enhancer, allowing for reverse com-
plements, [2] similarity in oligomer ordering—the num-
ber of steps needed to transform one ordering into the
other normalized by the number of conserved oligomers
[3], similarity in enhancer position—the difference in the
distance to the start codon normalized by the size of the
5′ intergenic region, and [4] the presence of specific con-
served motifs, Ets1/2 (GGAW) for Hand-like and GATAa
and TBX6 (GGNG) for Mesp. Fertilization and dechorionation
Adult C. inflata were harvested from docks on Lopez
or San Juan Island, WA. M_REP (Carlsbad, CA) sup-
plied adult C. robusta from multiple collection locations
along the coast of San Diego, CA. C. robusta fertiliza-
tion, dechorionation, electroporation, and staging were
carried out as previously described [30, 56, 73]. For C. inflata, similar protocols were used with the follow-
ing modifications. Sperm and then eggs were dissected
from 4 to 6 gravid, freshly collected adults. Concen-
trated sperm from all adults was mixed in a 10 ml dish
of FNSW (filtered natural sea water). Eggs were dis-
sected from each individual into a separate small dish of
FNSW, and then, all eggs were rinsed once using 70 μm
mesh. Sperm was added to rinsed eggs, and after 12 min,
zygotes were passed through six rinse dishes. The zygotes
were then transferred to a 10 ml dish, and excess water
was removed and replaced with a dechorionation solu-
tion (10 ml FNSW + a 200 μl freshly thawed aliquot of 5%
protease in FSW Streptomyces griseus, Sigma P8811-1G). After 4 min, zygotes were pipetted gently and checked for
dechorionation every minute. After ~ 9–11 min, decho-
rionated zygotes were rinsed sequentially in six 10 ml
dishes of FNSW. Electroporation was as described for C. robusta except that only 50 μl of total mannitol + DNA
solution was used. Conclusion temperature (Tm) of ≥ 78 °C, the mutation placed in
the exact center of the primer with 10–30 bp of correct
sequence on both sides, and a minimum GC content of
40%. Primers were diluted to 125 ng/μl and PCR run
with 5–50 ng of template, Pfu ultra II taq polymerase
(Agilent). If template was > 5 kb, we added 3 μl DMSO,
and the reaction was run for 12–30 cycles based on the
extent of the mutagenesis (12 for point mutations, 16
for 2–3 bp mutations, up to 30 for larger mutations). The PCR reaction was then cut with 1–2 μl of DpnI at
37 °C for 1 h and incubated at 70 °C for 20 min prior to
transformation of competent cells according to stand-
ard protocols. LacZ reporter constructs Molecular cloning was performed according to estab-
lished protocols [51]. C. inflata genomic regions used for
enhancer analysis were amplified with sequence-specific
primers carrying appropriate restriction sites (Additional
file 1: Table S1). Cloning of C. robusta FoxF and Hand-
like minimal enhancers was described by Beh et al. and
Woznica et al. [41, 58]. Embryological techniques
Fertilization and dechorionationl Embryos were transfected with
100–300 μg of DNA. Higher time constants (~ 20 ms)
appeared to give the best incorporation and did not
hinder development. Embryos were cultured in gelatin-
coated dishes with 10 ml of FNSW on a floating platform
in a sea table (~ 14–16 °C) with the lids upside down to
ensure that sea table water did not enter the cultures. Embryos were transferred after 2–4 h (4–16 cell stage) to
a fresh dish of FNSW to ensure proper development. Authors’ contributions 9. Combs PA, Fraser HB. Spatially varying cis-regulatory divergence
in Drosophila embryos elucidates cis-regulatory logic. PLoS Genet. 2018;14(11):e1007631. 9. Combs PA, Fraser HB. Spatially varying cis-regulatory divergence
in Drosophila embryos elucidates cis-regulatory logic. PLoS Genet. 2018;14(11):e1007631. Computational prediction of Ciinf.Mesp and Ciinf.Hand-like enhancer elements
along with the design and implementation of cross-species testing of these
elements was primarily conducted by WC. WC also designed and conducted
the FoxF motif and enhancer spacing experiments and wrote the manuscript. BD conceived and oversaw much of the experimental design and conducted
some of the experiments. JFR and MBD assembled the C. inflata genome and
generated a revised phylogeny. AH helped to develop and refine protocols
for transgenesis of C. inflata embryos along with conducting the Map Kinase
inhibitor assays. AL conducted the initial analysis of the FoxF enhancer. IL
and DR conducted the Ciinf.Mesp and Ciinf.Hand-like reporter assays in C. inflata, analyzed the resulting data, and helped generate relevant figures. The
remaining student co-authors worked as a lab group associated with their
Developmental Biology class (Bio24, 2017) to conduct Ciinf.Mesp and Ciinf. Hand-like reporter assays in C. robusta, analyze the resulting data and generate
relevant figures. All authors read and approved the final manuscript. 10. Stolfi A, Lowe EK, Racioppi C, Ristoratore F, Brown CT, Swalla BJ, et al. Divergent mechanisms regulate conserved cardiopharyngeal devel‑
opment and gene expression in distantly related ascidians. Elife. 2014;3:e03728. 10. Stolfi A, Lowe EK, Racioppi C, Ristoratore F, Brown CT, Swalla BJ, et al. Divergent mechanisms regulate conserved cardiopharyngeal devel‑
opment and gene expression in distantly related ascidians. Elife. 2014;3:e03728. 11. Haag ES. The same but different: worms reveal the pervasiveness of
developmental system drift. PLoS Genet. 2014;10(2):e1004150. 11. Haag ES. The same but different: worms reveal the pervasiveness of
developmental system drift. PLoS Genet. 2014;10(2):e1004150. 12. True JR, Haag ES. Developmental system drift and flexibility in evolution‑
ary trajectories. Evol Dev. 2001;3(2):109–19. 12. True JR, Haag ES. Developmental system drift and flexibility in evolution‑
ary trajectories. Evol Dev. 2001;3(2):109–19. 13. Long HK, Prescott SL, Wysocka J. Ever-changing landscapes:
transcriptional enhancers in development and evolution. Cell. 2016;167(5):1170–87. 14. Arnosti DN, Kulkarni MM. Transcriptional enhancers: intelligent enhance‑
osomes or flexible billboards? J Cell Biochem. 2005;94(5):890–8. 15. Khan A, Fornes O, Stigliani A, Gheorghe M, Castro-Mondragon JA, van
der Lee R, et al. JASPAR 2018: update of the open-access database of
transcription factor binding profiles and its web framework. References 1. Levine M, Davidson EH. Gene regulatory networks for development. Proc
Natl Acad Sci USA. 2005;102(14):4936–42. 1. Levine M, Davidson EH. Gene regulatory networks for development. Proc
Natl Acad Sci USA. 2005;102(14):4936–42. 1. Levine M, Davidson EH. Gene regulatory networks for development. Proc
Natl Acad Sci USA. 2005;102(14):4936–42. Funding
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f Funding for BD was provided by the Swarthmore College Department of Biol‑
ogy and by NIH Grant Number R15HD080525-01. JFR and MBD acknowledge
funding through the National Science Foundation under Grant Number
1542597. 16. Thanos D, Maniatis T. Virus induction of human IFN beta gene expression
requires the assembly of an enhanceosome. Cell. 1995;83(7):1091–100. 17. Srivastava V, Dey I, Leung P, Chadee K. Prostaglandin E2 modulates IL-8
expression through formation of a multiprotein enhanceosome in
human colonic epithelial cells. Eur J Immunol. 2012;42(4):912–23. Received: 3 June 2019 Accepted: 13 September 2019 Stage 22–23 embryos were fixed with 0.175% glutaral-
dehyde and then stained with X-gal to visualize LacZ
reporter expression as previously described [51]. Acknowledgements We wish to acknowledge Christina Cota for her technical assistance, mentor‑
ing, and training efforts throughout this project. We also wish to acknowledge
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Evaluation of coupling coordination development between digital economy and green finance: Evidence from 30 provinces in China
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OPEN ACCESS Citation: Liu Z, Zhang X, Wang J, Shen L, Tang E
(2023) Evaluation of coupling coordination
development between digital economy and green
finance: Evidence from 30 provinces in China. PLoS ONE 18(10): e0291936. https://doi.org/
10.1371/journal.pone.0291936 Editor: Rita Yi Man Li, Hong Kong Shue Yan
University, HONG KONG University, HONG KONG
Received: April 22, 2023
Accepted: September 10, 2023
Published: October 13, 2023 Received: April 22, 2023
Accepted: September 10, 2023
Published: October 13, 2023 Copyright: © 2023 Liu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. In addtion, other data is from
CSMAR database(http://www.csmar.com/
channels/31.html) RESEARCH ARTICLE
Evaluation of coupling coordination
development between digital economy and
green finance: Evidence from 30 provinces in
China Zebin LiuID1, Xiaoheng Zhang2*, Jingjing Wang1*, Lei Shen1*, Enlin Tang1*
1 School of Finance and Mathematics, Huainan Normal University, Huainan, Anhui Province, China,
2 School of Economics and Management, Anhui University of Science & Technology, Huainan, Anhui
Province, China Zebin LiuID1, Xiaoheng Zhang2*, Jingjing Wang1*, Lei Shen1*, Enlin Tang1*
1 School of Finance and Mathematics, Huainan Normal University, Huainan, Anhui Province, China,
2 School of Economics and Management, Anhui University of Science & Technology, Huainan, Anhui
Province, China a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * zhang.xiaoheng@foxmail.com (XZ); wjjwjj1985@163.com (JW); shenl@hnnu.edu.cn (LS);
tangenlin2046@163.com (ET) Abstract The convergence of China’s digital economy and green finance holds great significance for
fostering a sustainable and high-quality developmental path. However, existing studies
have not explored the coupling coordination development between these two crucial sub-
systems. To bridge this gap, this paper employs a modified coupling coordination degree
(CCD) model to assess and affirm the coupling coordination degree between the digital
economy and green finance across 30 provinces in China from 2015–2021. Based on
degree results, provinces are classified into three clusters by using K-means and hierarchi-
cal clustering algorithm. Our findings unveil that the current level of coupling coordination
development in China is at a primary coordination stage. Although regional disparities signif-
icantly exist, the overall level of coordination remains steadily increasing, with the eastern
region outperforming the western region. Additionally, we determine that the COVID-19 pan-
demic’s disruption on the coupling coordination development of these systems has been
limited. This research sheds light on the evolution of coupling systems and offers practical
recommendations for strengthening the coordinated development of the digital economy
and green finance. PLOS ONE PLOS ONE 1 Introduction In recent times, the development of the digital economy has significantly contributed to pro-
moting economic growth and optimizing and upgrading the economic structure [1]. For
instance, China’s digital economy expanded to 39.2 trillion yuan in 2020, contributing signifi-
cantly to its GDP [2]. Additionally, China’s 14th Five-Year Plan emphasized the significance of
green development and acknowledged green finance as a crucial component of the economy
[3, 4]. Green finance is an effective mechanism for mitigating carbon emissions and promoting
the progression of a low-carbon economy [5–7]. However, the development of green finance Funding: This study was financially supported by
Department of Education of Anhui Province in the
form of a grants (KJ2021A0966; 2022AH051585)
received by J.W, a grant (2023AH051512) received
by ZL, and a grant (2023AH051508) received by
LS. This study was also financially supported by 1 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance Anhui University of Science & Technology in the
form of a grant (QNSK202001) received by XZ. This study was also financially supported by
Mining Enterprise Safety Management of
Humanities and Social Science Key Research Base
in Anhui Province in the form of a grant
(MF2022006) received by XZ. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. encounters challenges such as inadequate technology and insufficient data [8]. The integration
of the digital economy with green finance mitigates these challenges and enhances contribu-
tions toward sustainable development [9]. Therefore, studying the coupling coordination
development between the digital economy and green finance is a crucial research topic that
contributes to China’s transition towards high-quality sustainable development. With the growing integration of the digital economy and green finance, they are progres-
sively coupling to foster high-quality economic growth. The digital economy’s digital platforms
and technological tools find widespread application in the realm of green finance, primarily
focusing on green technology innovation, decision-making in green finance, and management
of green supply chain. For instance, digital technology enables financial institutions to provide
innovative services rooted in big data and artificial intelligence, including risk assessment, green
bond issuance, and carbon emission trading, which serves as the groundwork for the innovation
of green finance [10]. 1 Introduction In addition, Digital platforms, such as MSPs, can facilitate research, devel-
opment, and promotion of green technologies through a range of services, including funding,
sales channel management, and business intelligence solutions [11]. Competing interests: The authors have declared
that no competing interests exist. g
,
g
[
]
The relationship between the digital economy and green finance has emerged as a promi-
nent area of research, with scholars exploring two key aspects. Some scholars considered that
the digital economy played a significant role in enhancing the efficiency of green finance. Tian
et al. (2022) [12] and Han et al. (2023) [13] believed that the digital economy had the potential
to enhance the efficiency of output, resource allocation, and resource utilization in green
finance. Zhang et al. (2022) [14] suggested that the development level of the digital economy
and the total factor productivity index of the green economy had a positive correlation, show-
ing the potential of the digital economy for promoting and enhancing the efficiency of green
finance. Guo et al. (2022) [15] believed that the digital economy was also an great platform for
applying green finance, which could more accurately allocate funds to the fields of environ-
mental protection and sustainable development, thereby optimizing resource allocation and
improving the efficiency of green finance. Some scholars had researched how the digital econ-
omy stimulated innovation in green finance. They primarily argued that digital technology
such as digital finance could enhance the efficiency and quality of green innovation. Lin et al. (2022) [16] considered that digital technology could mitigate financing constraints for enter-
prises, upgrade industrial structure, consequently enhancing the capacity for regional green
technology innovation. The empirical research conducted by Sun and Lin (2022) [17] revealed
that the utilization of digital technology has a substantial impact on the advancement of green
innovation in enterprises. Digital technology could reduce internal and external expenses,
leading to increased profitability, and thus facilitating the acquisition of financial resources for
green innovation. Zhao and Qian (2023) [18] discovered the heterogeneity in the impact of
digital technology on green innovation in terms of human capital, R&D intensity, and envi-
ronmental regulation. The study brought to light that regions with high-level talents, high
R&D intensity, and stringent environmental regulations experience a significantly higher
improvement effect on green innovation performance. 1 Introduction In addition to promoting green inno-
vation through digital technology, some scholars asserted digital transformation could facili-
tate green innovation. Xue et al. (2022) [19] contended that a company’s digital
transformation could significantly drive innovation in green technology by improving the dig-
itization level of its data and accessibility to information, as well as enhancing the dissemina-
tion of external information. According to Zhang et al. (2023) [20], empirical research
revealed that companies could increase their green dynamic capability, which enabled them to
respond more quickly to market demands and environmental changes via digital transforma-
tion, leading to advancements in green technological innovation. The relationship between the digital economy and green finance has emerged as a promi-
nent area of research, with scholars exploring two key aspects. Some scholars considered that
the digital economy played a significant role in enhancing the efficiency of green finance. Tian
et al. (2022) [12] and Han et al. (2023) [13] believed that the digital economy had the potential
to enhance the efficiency of output, resource allocation, and resource utilization in green
finance. Zhang et al. (2022) [14] suggested that the development level of the digital economy
and the total factor productivity index of the green economy had a positive correlation, show-
ing the potential of the digital economy for promoting and enhancing the efficiency of green
finance. Guo et al. (2022) [15] believed that the digital economy was also an great platform for
applying green finance, which could more accurately allocate funds to the fields of environ-
mental protection and sustainable development, thereby optimizing resource allocation and
improving the efficiency of green finance. Some scholars had researched how the digital econ-
omy stimulated innovation in green finance. They primarily argued that digital technology
such as digital finance could enhance the efficiency and quality of green innovation. Lin et al. (2022) [16] considered that digital technology could mitigate financing constraints for enter-
prises, upgrade industrial structure, consequently enhancing the capacity for regional green
technology innovation. The empirical research conducted by Sun and Lin (2022) [17] revealed
that the utilization of digital technology has a substantial impact on the advancement of green
innovation in enterprises. Digital technology could reduce internal and external expenses,
leading to increased profitability, and thus facilitating the acquisition of financial resources for
green innovation. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 1 Introduction The coupling coordination degree (CCD),
nevertheless, considers not only the degree of interaction between systems, but also their coor-
dination and balance. For instance, Gan et al. (2020) [25] examined the degree of coupling
coordination between urbanization and industrial development in China through CCD
model. Li et al. (2022) [26] utilized the CCD model to assess the ecological-economic coupling
coordination index in northern China while delving into the driving factors that impact its
coordination. However, the coupling coordination values calculated using the traditional CCD
model may have an uneven distribution, which is unable to resolve the issue of the gap
between system reliability and effectiveness. Thus, this paper utilizes a modified CCD model
to measure the coupling and coordination between digital economy and green finance in
order to more reasonably assess the level of coupling coordination and system development
for each system [27]. ever, the coupling degree only characterizes the degree of interaction between systems,
ignoring the capacity level of each system [23, 24]. The coupling coordination degree (CCD),
nevertheless, considers not only the degree of interaction between systems, but also their coor-
dination and balance. For instance, Gan et al. (2020) [25] examined the degree of coupling
coordination between urbanization and industrial development in China through CCD
model. Li et al. (2022) [26] utilized the CCD model to assess the ecological-economic coupling
coordination index in northern China while delving into the driving factors that impact its
coordination. However, the coupling coordination values calculated using the traditional CCD
model may have an uneven distribution, which is unable to resolve the issue of the gap
between system reliability and effectiveness. Thus, this paper utilizes a modified CCD model
to measure the coupling and coordination between digital economy and green finance in
order to more reasonably assess the level of coupling coordination and system development
for each system [27]. With the gradual infusion of digital elements into the green industry, digital technology
and digital platforms of the digital economy have emerged as the core elements of the develop-
ment of green finance. By enhancing the digital foundation of green finance development, dig-
ital technology makes up for its technological limitations. However, current researches have
mainly examined the significance and influence of the relationship between the digital econ-
omy and green finance. There are few studies discerning their logical nexus and regional dis-
parities from a coupling perspective. 1 Introduction Zhao and Qian (2023) [18] discovered the heterogeneity in the impact of
di i l
h
l
i
i
i
f h
i l R&D i
i
d
i leading to increased profitability, and thus facilitating the acquisition of financial resources for
green innovation. Zhao and Qian (2023) [18] discovered the heterogeneity in the impact of
digital technology on green innovation in terms of human capital, R&D intensity, and envi-
ronmental regulation. The study brought to light that regions with high-level talents, high
R&D intensity, and stringent environmental regulations experience a significantly higher
improvement effect on green innovation performance. In addition to promoting green inno-
vation through digital technology, some scholars asserted digital transformation could facili-
tate green innovation. Xue et al. (2022) [19] contended that a company’s digital transformation could significantly drive innovation in green technology by improving the dig-
itization level of its data and accessibility to information, as well as enhancing the dissemina-
tion of external information. According to Zhang et al. (2023) [20], empirical research
revealed that companies could increase their green dynamic capability, which enabled them to
respond more quickly to market demands and environmental changes via digital transforma-
tion, leading to advancements in green technological innovation. 2 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance The phrase coupling usually refers to the interconnectedness between two entities that
exhibit mutual influence and interaction [21]. The theory of coupling portrays system relation-
ships distinctively and quantifies the degree of relationship, known as the coupling degree
(CD). The coupling degree represents the consistency and agreement between systems, dem-
onstrating how the system changes from chaos to order following system contact [22]. How-
ever, the coupling degree only characterizes the degree of interaction between systems, The phrase coupling usually refers to the interconnectedness between two entities that
exhibit mutual influence and interaction [21]. The theory of coupling portrays system relation-
ships distinctively and quantifies the degree of relationship, known as the coupling degree
(CD). The coupling degree represents the consistency and agreement between systems, dem-
onstrating how the system changes from chaos to order following system contact [22]. How-
ever, the coupling degree only characterizes the degree of interaction between systems,
ignoring the capacity level of each system [23, 24]. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 2 Literature review The integration of the digital economy and green finance contributes to promoting sustainable
and high-quality economic development. Digital technology innovation enables the interac-
tion and connection between green finance and digital technology, leading to a transformation
from a traditional risk management approach into a value enhancer that fosters sustainable
development. Digital technology enhances data analysis and evaluation methods for green
finance, thereby promoting the development of green industries. Consequently, the coordi-
nated development of the digital economy and green finance has become a hot research topic
within the academic community. The existing literature has studied the relationship between digital economy and green
finance from various perspectives, which can be organized into two major themes through
sorting and summarizing, as follows. Regarding the significance of digital economy to the development of green finance, some
scholars suggested that the efficiency, transparency, and feasibility of green finance can be
improved through the application of digital technology of digital economy, which can further
promote sustainable development and the rational utilization of resources. The advancement
of digital technology has substantially improved the efficiency and transparency of green
finance. Wang et al. (2022) [28] claimed that the utilization of digital economic technology, for
instance, employing big data and artificial intelligence to evaluate environmental risks and
opportunities, could enhance the efficiency, feasibility, and transparency of green finance. Dig-
ital technology can help promote the financing of green finance. Li et al. (2021) [29] and Ozili
(2022) [30] pointed out that the digital economy provided various new financing methods and
platforms, such as green bonds, crowdfunding, and digital currencies that could facilitate the
financing of green projects and enterprises. In addition, the digital economy significantly pro-
motes the innovation of green financial products and services. Han and Liu (2022) [31]
pointed out that the development of the digital economy offered opportunities for innovating
green finance. For instance, the application of smart contracts and blockchain technology
could improve the traceability and verifiability of green financial products. Likewise, by
leveraging technologies such as artificial intelligence and blockchain, the processes involved in
green finance could also be optimized, ensuring information transparency, risk control, and
efficiently managing investment and financing, as well as improving fund utilization [32]. What’s more, Zhuang et al. (2022) [33] and Liu et al. 1 Introduction Therefore, elucidating the coupling and coordination
relationship between the digital economy and green finance has become a significant topic. Specifically, this study aims to answer the following questions: 1. Based on theoretical analysis, how does the coupled system comprising the digital economy
subsystem and the green finance subsystem operate? 1. Based on theoretical analysis, how does the coupled system comprising the digital economy
subsystem and the green finance subsystem operate? 2. By establishing a modified CCD model, whether it can be validated that digital economy
subsystem and green finance subsystem have a coupling and coordination relationship? 3. Has the evolution of the coupled system reached a definitive conclusion? If not, are there
different stages of coupling coordination and, if so, what types of variation are present? In order to answer these questions, this study establishes evaluation metrics and constructs
a modified CCD model to evaluate the CD and CCD. On one hand, by utilizing coupling the-
ory as the analytical framework, a novel coupled system is constructed, elucidating the main
logical relationships and mechanism. On the other hand, based on the analysis results of CD
and CCD, examinations are conducted regarding the trends of change and regional disparities. Furthermore, a clustering analysis of the 30 provinces is conducted based on the CCD over the
past few years. Ultimately, recommendations are proposed concerning the coupling and coor-
dination development of the digital economy and green finance. The structure of this paper is as follows: Section 2 provides a review of the existing research
on the relationship between the digital economy and green finance, examining it from two
perspectives. Section 3 presents a theoretical analysis of the coupling mechanism, focusing on
the digital economy subsystem and the green finance subsystem. Section 4 establishes a com-
prehensive evaluation index system to assess the CD and the CCD. It also introduces the main
models utilized in this study. Section 5 discusses and analyzes the empirical results derived PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 3 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance from the evaluation index system. Section 6 proposes countermeasures and suggestions for
promoting the coupling coordination development between the digital economy and green
finance. Section 7 summarizes the key findings and draws three main conclusions. Addition-
ally, it discusses the innovation and limitations of this paper. 2 Literature review (2023) [34] proposed that regulatory
agencies and financial institutions played critical roles in the era of the digital economy,
because they were responsible for establishing digital financial standards and guidelines, in
addition to offering digital financial products and services to promote the growth of green
finance. Scholars have extensively examined the influence of digital finance on green finance. As a
pivotal component of the digital economy, digital finance has redefined the conventional
financial framework by means of tools such as online payment, mobile payment, electronic
banking, and digital currencies [35]. This transformation holds profound implications for
green finance, particularly concerning resource utilization and innovation. Digital finance
substantially enhances resource utilization efficiency while concurrently curbing environmen-
tal pollution. The application of digital technology and innovative approaches facilitated by PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 4 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance digital finance, as underscored by Liu et al. (2022) [36], facilitates intelligent management of
energy and water resources, leading to reduced consumption and the advancement of green
energy solutions. Wu et al. (2023) [37] emphasize the imperative adoption of digital finance
technologies by green financial institutions, broadening user scenarios and expanding the
range of green financial services, thereby steering resources towards sustainable industries. Moreover, the symbiotic relationship between digital finance and innovations in green tech-
nology enhances the efficiency of carbon emission reduction efforts [38]. However, the impact
of digital finance on green efficiency is multifaceted. Excessive advancements can potentially
introduce risks that impede energy efficiency [39]. In summary, scholars have predominantly examined the significance and relationship of
the digital economy and green finance. However, research is limited on the coordinated rela-
tionship between them and regional development variations. In addition, limited research
exists on the pathways of their coordinated development and the related quantitative assess-
ments. What’s more, there is no study that have conducted in-depth research on the logical
relationship and regional variations surrounding the coupled coordination development of the
digital economy and green finance. Therefore, this study has established an evaluation index system and constructed relevant
models to assess the coupling and coordination relationship between the digital economy and
green finance, as well as to further examine the provincial development disparities in China. 2 Literature review To achieve this, we first constructed an analytical framework based on system theory and cou-
pling theory to clarify the logical relationship and mechanism of the coupled system. Subse-
quently, by utilizing the analysis results of the coupling degree (CD) and coupling
coordination degree (CCD), our study has examined trend changes and regional variability,
providing valuable recommendations for the coupled development of the digital economy and
green finance. 3 Theoretical analysis of coupled system The relationship between the development of the digital economy and green finance is not
entirely independent, parallel, contradictory, or incompatible. Instead, it represents a fusion
and mutually reinforcing connection. This study builds upon system theory [40] and coupling
theory [41] to examine the larger system as a coupled system consisting of the subsystems of
the digital economy and green finance. It is a coupled entity formed through the interaction of
elements and forces within the system. To unveil the coupling relationship between the digital economy and green finance, it is
essential to elucidate their interactions and the pathways that connect them. The digital econ-
omy represents a novel economic form, with data resources at its core, which are primarily dis-
seminated and applied through modern information networks; Information and To unveil the coupling relationship between the digital economy and green finance, it is
essential to elucidate their interactions and the pathways that connect them. The digital econ-
omy represents a novel economic form, with data resources at its core, which are primarily dis-
seminated and applied through modern information networks; Information and
communication technologies (ICTs) play a crucial role in driving the development of the digi-
tal economy [42]. Existing researches have outlined three key aspects of the digital economy:
digital industrialization, industrial digitalization, and digitized infrastructure. Consequently,
the coupling and coordination between the digital economy and green finance can be attained
through three primary dimensions: industry coupling, technology coupling, and mindset cou-
pling, as shown in Fig 1. This synergistic coupling has the potential to advance high-quality
and sustainable economic development. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 3.1 Industry coupling The similarities in the developmental processes of the digital economy and green finance sig-
nify the possibility for synergistic development between the digital industry and the green 5 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 1. Framework of the coupled system. https://doi.org/10.1371/journal.pone.0291936.g001 Fig 1. Framework of the coupled system. https://doi.org/10.1371/journal.pone.0291936.g001 industry [15]. Firstly, both the digital economy and green finance undergo development by
transforming and integrating traditional industries. The digital economy utilizes digital infor-
mation and communication technologies to enhance efficiency and optimize economic struc-
tures; Meanwhile, green finance centers on financial activities and investments that support
environmentally sustainable projects and initiatives. Secondly, the development of both the
digital economy and green finance relies on the construction of their respective infrastruc-
tures. The digital economy necessitates infrastructure construction such as fiber optic cables,
mobile phone networks, and communication base stations. Similarly, the development of
green finance also requires infrastructure, including renewable energy facilities, green power
generation plants, and the establishment of green transportation systems. The digital economy, through digital industrialization, brings new technologies, business
models, and dynamics to traditional industries [43]. On the other hand, green finance, through
green industrialization, introduces new concepts, pathways, and models to traditional indus-
tries [44]. These two aspects synergistically work together to promote the upgrading and trans-
formation of traditional industry structures. The digital economy and green finance optimize
the allocation of traditional production factors such as capital, labor, land, and knowledge
technology, while promoting the integration between different industries through 6 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance technological and innovative models [45]. This enables traditional industries to not only have
technological support, data support, and algorithmic empowerment but also possess character-
istics of being greener, more rational, and higher-end. The development of the digital economy
and green finance has given rise to a new type of IT industry based on digital technology that
is pollution-free and environmentally friendly. It has also driven the development of emerging
green industries such as new energy vehicles. The development demands of these new indus-
tries further stimulate digital and green innovation, creating a virtuous cycle and an economic
multiplier effect [46]. 3.2 Technology coupling Through the process of industrial digitalization, digital technology has the potential to enhance
the efficiency of traditional industries, reduce product costs, and address the problem of infor-
mation asymmetry. Simultaneously, the adoption of green technology has played a vital role in
industrial transformation by boosting energy efficiency, curbing pollution emissions, and facil-
itating resource allocation through sustainable practices [47, 48]. These advancements have
brought about substantial transformations in industrial development and have had a positive
impact on sustainable growth. The synergistic development of digital technology and green
technology has the potential to enhance resource utilization efficiency, optimize production
processes, and facilitate intelligent and sustainable solutions [49]. For instance, the utilization
of Internet of Things (IoT) sensors can enable monitoring and control of energy consumption,
waste management systems, and emissions. This, in turn, allows for effective resource utiliza-
tion and reduction of environmental impacts. In addition, the integration of digital technology
and green technology has resulted in a notable decrease in environmental pollution during
product manufacturing. Furthermore, digital services have replaced resource-intensive activi-
ties in various domains. For instance, the adoption of cloud services has the potential to sub-
stantially reduce energy consumption and carbon emissions. By hosting business applications
on remote servers, energy consumption can be reduced by up to 80%, while the use of renew-
able energy sources can cut carbon emissions by as much as 96%. Emerging technologies like
3D printing, agricultural robots, and smart irrigation have significantly enhanced resource uti-
lization efficiency and sustainability in production and operations [50]. They have also con-
tributed to the replacement and reduction of harmful gas emissions, along with improved
waste recycling and utilization. What’s more, big data and artificial intelligence technologies
can be extensively utilized to acquire ecological and environmental information, addressing
the issue of information asymmetry among polluters, victims, and regulators [51]. Moreover,
these technologies can accurately align consumers’ demand for green products, optimize the
allocation of product resources, facilitate the integration of market supply and demand, and
unlock the potential purchasing power of consumers [52]. 3.3 Mindset coupling The coupling of the digital mindset and the green mindset is beneficial for achieving high-
quality and sustainable economic development. As these mindsets intertwine, they place
higher demands on economic progress. The green mindset acknowledges the interdependence
between humans and nature, perceiving the environment and humans as integral parts of a
symbiotic system. Within this system, growth and development hold distinct meanings;
Growth implies a quantitative increase, while development emphasizes the enhancement of
quality [53]. Secondly, green technology is playing a pivotal role in shifting the focus of the dig-
ital economy from a ‘price-oriented’ mindset to a more holistic ‘value-oriented’ approach,
with sustainability at its core. As we navigate the path towards a sustainable digital economy, a 7 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance triangular dilemma emerges, encompassing the crucial aspects of “security, speed, and energy
consumption”. However, attaining optimal levels of all three factors simultaneously proves to
be an unattainable goal. In most cases, the resolution lies in finding a delicate equilibrium
between security and speed, even if it results in higher energy consumption [54]. Within the
realm of the digital economy, green technology assumes a pivotal role as it seamlessly incorpo-
rates environmentally conscious practices. Unlike the conventional price-oriented perspective
typically adopted by the economic system, green technology acknowledges the critical impor-
tance of the natural environment in which the system operates. By considering the ecosystem
as a whole, this approach surpasses the limitations associated with a purely market-driven inte-
gration of technology, thus overcoming its inherent drawbacks. Thirdly, as the green paradigm
permeates various aspects of corporate supply chains, a significant shift is underway, transi-
tioning from the conventional “production rationality” to an emerging “ecological rationality”
in the realm of production [55]. The notion of green development has gained substantial
momentum, leading to an increased environmental consciousness and literacy among individ-
uals. Consequently, the concept of “greenness” is evolving into a broader and more encom-
passing “green perspective”, reflecting a deeper understanding and commitment to
environmentally sustainable practices. 4 Indicators system and research methods
4.1 Construction of the index system To explore the coupling and coordination relationship between the digital economy subsystem
and the green finance subsystem, it is essential to establish an evaluation index system. Taking
into account the research conducted by Su et al. (2022) [56] and Wang et al. (2021) [57] and
adhering to principles of scientific rigor, rationality, comprehensiveness, and operability, we
have developed an evaluation index system for the selection of indicators. Currently, there is no unified standard in the academic community to measure the level of
digital economic development. However, several studies have proposed evaluation indicator
systems. For example, Liu et al. (2020) [58] decomposed the digital economy index into three
dimensions: informatization development indicators, internet development indicators, and
digital transaction development indicators, using a total of 14 measurement indicators. Li et al. (2022) [59] guided by the new development concept of “innovation, coordination, greenness,
openness, and sharing”, selected 55 indicators to evaluate the level of digital economic develop-
ment from these five aspects. Yang et al. (2022) [60] compiled a digital economy input-output
table based on the “Classification of Digital Economy and its Core Industries (2021)” pub-
lished by the National Bureau of Statistics, using it as an evaluation indicator system for the
digital economy. Xu et al. (2023) [61] constructed a digital economic evaluation indicator sys-
tem from five aspects: digital infrastructure, digital innovation capability, and digital coverage
breadth. Yang et al. (2023) [62] constructed an evaluation indicator system for the digital econ-
omy based on eight aspects: digital economic development carriers, digital economic develop-
ment environment, and digital economic development benefits. This paper utilizes established
evaluation indicator systems and incorporates the CSMAR digital economic research module
to formulate a comprehensive evaluation indicator framework for the digital economy. The
framework is built upon three dimensions: digital industrialization [63], industrial digitaliza-
tion [64], and technological innovation foundation, as shown in Table 1. The subsystem of dig-
ital economy development constructed in this paper includes seven sub-criterion layer and 32
indicators. Currently, there is no unified standard for evaluating indicators in the field of green
finance. Jiang et al. (2020) [65] constructed an evaluation system for green finance by selecting PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 8 / 31 coupling coordination development between Digital Economy and Green Finance PLOS ONE PLOS ONE Table 1. Evaluation index system of the digital economy. Subsystem
Criterion Layer
Sub-Criterion Layer
Calculated Metrics
Order
Parameter
Index
Attribute
Digital economy
development (U1)
Digital Industrialization
Communication Industry
Penetration rate of fixed-line
telephones
X1
+
Penetration rate of Mobile
phones
X2
+
Total telecommunications
services revenue
X3
+
Number of SMS messages
X4
+
Number of Mobile phone users
X5
+
Number of cellular Base Stations
X6
+
Length of fiber optic cable line
X7
+
Number of personal computers
per 100 people
X8
+
Internet Services
Number of websites per 100
companies
X9
+
Number of internet users
X10
+
Number of internet broadband
access port
X11
+
Number of Mobile internet
users
X12
+
Number of Mobile internet
traffic
X13
+
Number of users with Internet
broadband access
X14
+
Software service revenue
X15
+
Software and Information Technology
Services
Software product revenue
X16
+
Service revenue from
information technology
X17
+
Software export revenue
X18
+
industrial digitalization
Major economic indicators of industrial
enterprises above designated size
Total assets
X19
+
Operating revenue
X20
+
Total income
X21
+
Digital finance
the coverage breadth of digital
finance
X22
+
the usage depth of digital
finance
X23
+
Digitalization degree of digital
finance
X24
+
online mobile payment level
X25
+
foundation of the scientific and
technological innovation
sci-tech input
R&D personnel FTE
X26
+
R&D expenditure
X27
+
expenditure of new product
development
X28
+
sci-tech outputs
number of patent applications
granted
X29
+
number of new product
development projects
X30
+
sales revenue of new product
X31
+
export revenue of new product
X32
+
https://doi.org/10.1371/journal.pone.0291936.t001 Table 1. Evaluation index system of the digital economy. https://doi.org/10.1371/journal.pone.0291936.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 9 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance relevant indicators from three dimensions: economic, financial, and social. Yu et al. (2021)
[66] developed an evaluation system to comprehensively measure the level of green finance
development, focusing on four green financial instruments: green credit, green bonds, green
investment, and carbon finance. Qiao et al. (2021) [67] and Zhou et al. (2022) [68] divided
green finance into five subcategories: green credit, green securities, green insurance, green
investment, and carbon finance, and constructed indicator systems accordingly. PLOS ONE Wang (2022)
[69] and Zheng (2022) [70] constructed evaluation indicator systems for green finance based
on three macro aspects: environment, finance, and society. Zeng et al. (2022) [71] utilized met-
rics such as carbon intensity as indicators for evaluating the performance of green supply
chains, effectively measuring the manifestation of environmentally sustainable development. Zhang et al. (2023) [72], taking into account the current status of green finance and ecological
civilization construction in Shandong Province, selected eight indicators from four dimen-
sions: green investment, green credit, green insurance, and green securities to measure the
level of green finance development. Therefore, this study draws on the research of previous scholars and considers data avail-
ability. This paper selects five indicators from three dimensions: green securities, green invest-
ment market, and carbon market for constructing the evaluation indicators, as shown Table 2. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE Table 2. Evaluation index system of the green finance. Subsystem
Criterion Layer
Sub-Criterion
Layer
Calculated Metrics
Order
Parameter
Index
Attribute
Green finance development
(U2)
green securities
green credit
High energy-consuming industrial interest share
X33
-
green stock
High energy-consuming industry market
capitalization ratio
X34
-
green investment
market
green investment
Proportion of investment in environmental
pollution
X35
+
green insurance
The proportion of agricultural insurance scale
X36
+
Carbon Market
carbon finance
carbon intensity
X37
-
https://doi.org/10.1371/journal.pone.0291936.t002 Table 2. Evaluation index system of the green finance. into dimensionless index measures. The formula is as follows: into dimensionless index measures. The formula is a into dimensionless index measures. The formula is as follows: into dimensionless index measures. The formula is as follows: Zij ¼ xij xj
si
; i ¼ 1; 2; . . . ; n; j ¼ 1; 2; . . . ; p
ð1Þ ð1Þ 2. Calculate the correlation coefficient matrix R for variables
The formula is as follows: 2. Calculate the correlation coefficient matrix R for variables
The formula is as follows: rij ¼
Xn
k¼1~xki ~xkj
n 1
; ði; j ¼ 1; 2; ; pÞ
ð2Þ ð2Þ Where rii = 1, rij = rij, and rij represents the correlation coefficient between the i-th and j-th
indicators in the formula. Calculate the elementary loading matrix. 3. Calculate the elementary loading matrix. Compute the eigenvalues λ1 λ2 . . . λp 0 of the correlation coefficient matrix R,
along with their corresponding eigenvectors u1, u2, ,up, where u1, u2, ,up, uj = (u1j,
u2j, , unj)T. The elementary loading matrix is represented byfififififififififififififififi 3. Calculate the elementary loading matrix. Compute the eigenvalues λ1 λ2 . . . λp 0 of the correlation coefficient matrix R,
along with their corresponding eigenvectors u1, u2, ,up, where u1, u2, ,up, uj = (u1j,
u2j, , unj)T. The elementary loading matrix is represented byfififififififififififififififi A ¼
ffiffiffiffiffi
l1
p
u1
ffiffiffiffiffi
l2
p
u2
ffiffiffiffiffi
lp
q
up
h
i
. A ¼
ffiffiffiffiffi
l1
p
u1
ffiffiffiffiffi
l2
p
u2
ffiffiffiffiffi
lp
q
up
h
i
. fifififififififififi
4. Extract common factors fifififififififififi
4. Extract common factors fififififififififififififififi
4. Based on the rotation, factor model is constructed. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 4.2 Research methods Various methods exist for constructing indices, including AutoML [73], TOPSISS [74], and
the relative importance index [75], etc.. In this paper, the factor analysis model and entropy
weight method are utilized to calculate the digital economy index and green finance index. Factor analysis is a classical statistical method extensively employed to extract latent factors
that underlie the data. This method enables the identification of the most pertinent and repre-
sentative factors from a vast array of indicators, thereby enhancing the effectiveness of captur-
ing key characteristics of the digital economy [76]. The entropy weight method is a multi-
indicator weight determination approach specifically designed to tackle the uncertainties and
subjectivity associated with indicators [77]. Digital economy index and green finance index serve as the basis for assessing the coupling
coordination degree between the digital economy and green finance, employing a modified
CCD model. Furthermore, the K-means clustering analysis algorithm is applied to classify and
analyze the development level of coupling coordination among 30 provinces in China, based on
the results of the coupling coordination degree analysis. The analytical process is shown in Fig 2. 4.2.1 Factor analysis model. Considering the large number of evaluation indicators in the
field of digital economy and taking into account the advantages of factor analysis models, this
paper selects the factor analysis model to calculate the digital economic index (U1). Factor
analysis is a statistical technique employed to streamline and scrutinize datasets characterized
by high dimensionality. By effectively transforming numerous observed variables, also known
as indicators, into a reduced set of latent factors, this method facilitates a more concise repre-
sentation of the data. Such dimensionality reduction serves to simplify the subsequent analysis
and interpretation processes, while also mitigating the computational complexity associated
with subsequent analytical procedures [78]. The specific calculation steps are as follows: 1. Data standardization
In this paper, the Z-score method is used to standardize the raw data to transform them 1. Data standardization
In this paper, the Z-score method is used to standardize the raw data to transform them 10 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 coupling coordination development between Digital Economy and Green Finance PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE Extract common factors We need to calculate the contribution rate (w) of each common factor based on the elemen-
tary loading matrix and extract m principal factors. This paper employs probabilistic princi-
pal component analysis (PPCA) as a method to extract common factors. In comparison to
traditional principal component analysis (PCA), PPCA presents several advantages, includ-
ing enhanced flexibility, interpretability, and robustness [79, 80]. Rotate the extracted factor loading matrix 5. Rotate the extracted factor loading matrix 5. Rotate the extracted factor loading matrix
To better comprehend the connotations conveyed by the common factors, the component
matrix is orthogonally rotated using Kaiser’s normalized maximum variance method [81],
f
l
f ll
d 5. Rotate the extracted factor loading matrix
To better comprehend the connotations conveyed by the common factors, the component
matrix is orthogonally rotated using Kaiser’s normalized maximum variance method [81],
formula as followed: To better comprehend the connotations conveyed by the common factors, the component
matrix is orthogonally rotated using Kaiser’s normalized maximum variance method [81],
formula as followed: RVARIMAX¼arg maxð
X
k
j¼1
X
p
i¼1
ðLRÞ
4
ij g
p
X
k
j¼1
ð
X
k
j¼1
ðLRÞ
2
ijÞ
2Þ
ð3Þ ð3Þ Based on the rotation, factor model is constructed. Based on the rotation, factor model is constructed. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 11 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 2. Analytical process. https://doi.org/10.1371/journal.pone.0291936.g002 Fig 2. Analytical process. https://doi.org/10.1371/journal.pone.0291936.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 12 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance 6. Calculate factor scores
Factor scores are calculated by regression method, formula as shown: 6. Calculate factor scores Factor scores are calculated by regression method, formula as shown: F ¼ w1f1 þ w2f2 þ þ wkfk
ð4Þ ð4Þ 4.2.2 Entropy weight method (EWM). Due to the limited number of evaluation indica-
tors for green finance, this paper utilizes the EWM to calculate the green finance index (U2). EWM is an objective way to give weights based on the entropy value provided by the standard-
ized values, specifically by measuring the degree of differentiation to assess the indicator’s
value [82]. EWM is calculated as follows: 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE Standardize data with the following formula: For positive indicators: For positive indicators: Sij ¼
ðAij minij
1jn
Þ
ðmax Aij
1jn
min Aij
1jn
Þ
ð5Þ ð5Þ For negative indicators: Sij ¼
ðmax Aij
1jn
AijÞ
ðmax Aij
1jn
min Aij
1jn
Þ
ð6Þ ð6Þ In this paper, the standardized data is shifted to prevent the zero value from making the log-
arithm meaningless. Namely Zij = Sij × 0.998 + 0.002. j
j
2. Measure the weight Yij of indicator j in year i by the following formula: Yij ¼
Zij
X
m
i¼1
Zij
ð7Þ ð7Þ 3. The following equation is used to calculate the information entropy of the jth indicator: 3. The following equation is used to calculate the information entropy of the jth indicator: ej ¼ 1
lnm
X
m
i¼1
Yij lnYij
ð8Þ ð8Þ 4. Use this formula to get the entropy redundancy of the jth indicator: dj ¼ 1 ej
ð9Þ ð9Þ PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023
13 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 13 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance 5. The following formula is used to calculate the weight of the jth indicator: wj ¼
dj
X
n
j¼1
dj
ð10Þ ð10Þ 6. The standardized data of each indicator is multiplied and summed with the weights: Uij¼1;2 ¼
X
p
j¼1
wijSij;
X
p
j¼1
wij ¼ 1
ð11Þ ð11Þ 4.2.3 Modified coupling coordination degree (CCD) model. The interpretation of the
coupling degree C relies on its interval distribution, and the values calculated by the traditional
coupling model may exhibit uneven distribution, thereby diminishing its validity. To address
this issue, this paper follows the methodology proposed by Wang [27] and establishes the mod-
ified CCD model as outlined below:fififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififififi C ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 X
n
i>j;j¼1
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Fi Uj
2
r
X
n 1
m¼1
m
2
66664
3
77775
Y
n
i¼1
Fi
maxFi
! 5 Empirical results and discussion In order to ensure statistical consistency and data availability, this study focuses on 30 prov-
inces in China (excluding Tibet, Hong Kong, Macau, and Taiwan) as the research subjects. The analysis primarily relies on various data sources, including the “China Statistical Year-
book”, “China Third Industry Statistical Yearbook”, “China Electronic Information Industry
Yearbook”, “China Industrial Statistical Yearbook”, “China Science and Technology Statistical
Yearbook”, provincial statistical yearbooks and bulletins, EPS Global Statistical Database,
CSMAR Database, among others. To address any missing data for specific years, trend extrap-
olation or interpolation methods are employed to supplement the gaps and maintain data
continuity. PLOS ONE coupling coordination development between Digital Economy and Green Finance PLOS ONE Table 3. Evaluation standard of CCD. CCD Degree
Level
CCD Degree
Grade
(0.0, 0.2]
Serious disorders
(0.4, 0.6]
Primary coordination
(0.2, 0.3]
Slight disorders
(0.6, 0.8]
Intermediate coordination
(0.3, 0.4]
Barely coordination
(0.8, 0.10]
Senior coordination
https://doi.org/10.1371/journal.pone.0291936.t003 Table 3. Evaluation standard of CCD. CCD Degree
Level
CCD Degree
Grade
(0.0, 0.2]
Serious disorders
(0.4, 0.6]
Primary coordination
(0.2, 0.3]
Slight disorders
(0.6, 0.8]
Intermediate coordination
(0.3, 0.4]
Barely coordination
(0.8, 0.10]
Senior coordination
https://doi.org/10.1371/journal.pone.0291936.t003 Table 3. Evaluation standard of CCD. Therefore, two algorithms, namely the K-means algorithm and hierarchical clustering algo-
rithm, are adopted. The K-means clustering algorithm is specifically designed to partition data
into K distinct clusters, optimizing the similarity within each cluster while minimizing the sim-
ilarity between different clusters [87]. It is particularly suitable for distance-based data cluster-
ing problems. Moreover, the K-means algorithm offers flexibility in adjusting the number of
clusters (K) to meet specific requirements. By increasing or decreasing the value of K, the gran-
ularity of clustering results can be controlled, thus catering to practical needs [88]. On the
other hand, the hierarchical clustering algorithm provides an effective visualization of data and
is applicable to various data types. The results of hierarchical clustering can be visually repre-
sented through a dendrogram, facilitating a clear understanding of the similarity and hierar-
chical structure among data points. This visualization aids in the interpretation and
explanation of clustering outcomes. PLOS ONE 1
n 1
v
u
u
u
u
u
u
u
t
ð12Þ
T ¼ a U1 þ b U2
ð13Þ
D ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffi
C T
p
ð14Þ ð12Þ T ¼ a U1 þ b U2
ð13Þ
D ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffi
C T
p
ð14Þ ð13Þ D ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffi
C T
p D ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffi
C T
p
ð14Þ ð14Þ fififififififififififififi
where T represents the combined development of green finance and the digital economy, U1
represents the digital economy index, and U2 is green finance index. D is the CCD. Since the
digital economy and green finance complement each other, it is considered α = 0.5, β = 0.5. According to the study of Liu [83], Table 3 outlines the six levels that make up the CCD. 4.2.4 Clustering algorithm of machine learning. Cluster analysis is a type of unsuper-
vised learning in machine learning, where the objective is to provide an explanation for the
underlying nature of the data and the patterns being seen by gaining knowledge from unla-
beled training examples. Common clustering analysis algorithms commonly employed in vari-
ous fields include the K-means algorithm, hierarchical clustering algorithm, and Modularity
clustering algorithm, among others. These algorithms find extensive applications in diverse
domains. For example, Feng et al. (2020) [84] introduced a non-parametric K-means algo-
rithm specifically tailored for analyzing economic data. In another study, Govender et al. (2020) [85] utilized both the K-means algorithm and hierarchical clustering algorithm to clas-
sify and analyze air pollution data. Li et al. (2022) [86] employed the modularity clustering
analysis technique to identify clusters of research articles pertaining to prefabricated building,
construction management, economic development, and ESG attributes. This paper aims to perform a clustering analysis on the level of coupling coordination
development across 30 provinces in China, based on the outcomes of CCD. The clustering
analysis in this study falls under the realm of traditional numerical data clustering problems. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 14 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 Results of digital economy index for 30 provinces from 2015 to 2021. 5.1 Evaluation of digital economy index Based on the calculation process outlined in section 4.2.1 on factor analysis, Python is utilized
in this study to calculate the digital economy index for each province spanning the years 2015
to 2021, as shown in Fig 3. The results of its descriptive statistics are shown as Table 4. From
the analysis of Table 4, several observations can be made regarding the digital economy index
in different years. From 2015 to 2021, the digital economy index displayed a consistent upward
trajectory, with the average increasing gradually from 1.02 to 1.04. Similarly, the median exhib-
ited progress from 0.80 in 2015 to 0.89 in 2021, signifying an incremental rise in the majority
of provinces. Concurrently, the standard deviation decreased progressively from 0.67 in 2015
to 0.55 in both 2020 and 2021, indicating a diminishing level of volatility within the digital
economy index throughout this period. However, there is a notable difference between the
minimum and maximum values of the digital economy index, indicating significant disparities
in digital economic development among the provinces. This reflects regional variations and
disparities in terms of economic foundations, technological innovation capabilities, and levels
of informatization. In summary, the digital economy index demonstrated an overall upward
trend within this timeframe, although notable disparities persisted among different provinces. Our findings are similar to the research conducted by Zhang et al. (2021) [42] and Jiang et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 15 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 3. Results of digital economy index for 30 provinces from 2015 to 2021. h
//d i
/10 1371/j
l
0291936 003 https://doi.org/10.1371/journal.pone.0291936.g003 Table 4. Descriptive statistics of digital economy index for 30 provinces from 2015–2021. Statistic
2015
2016
2017
2018
2019
2020
2021
Mean
1.02
1.01
1.03
1.02
1.04
1.03
1.04
Median
0.80
0.84
0.79
0.87
0.84
0.88
0.89
Standard deviation
0.67
0.61
0.72
0.63
0.76
0.55
0.55
Min
0.35
0.32
0.32
0.25
0.18
0.35
0.34
Max
3.31
2.66
3.34
2.92
3.55
2.77
2.77 Table 4. Descriptive statistics of digital economy index for 30 provinces from 2015–2021. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 16 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance (2022) [89], who also mentioned that the overall development of digital economy in China is
steady. 5.1 Evaluation of digital economy index Moreover, their research findings indicate that the growth of China’s digital economy
makes a substantial contribution to high-quality economic development. In Fig 3, the depth of color corresponds to a higher level of the digital economy index. In
terms of regional distribution, provinces located in the eastern coastal areas, such as Beijing,
Shanghai, Jiangsu, and Zhejiang, exhibit higher digital economy indexes. These provinces ben-
efit from their advanced economies, robust technological innovation ecosystems, and exten-
sive implementation of information technology. On the other hand, provinces in the western
regions, such as Guizhou, Yunnan, and Qinghai, have lower digital economy indexes. This dis-
crepancy can be attributed to their relatively weaker economic foundations, limited technolog-
ical innovation capacities, and comparatively lower levels of informatization. In a similar
study, Tang et al. (2021) [90] and Li et al. (2022) [91] explored the digital economy index in
China and identified comparable regional disparities, with coastal provinces displaying higher
digital economy indexes in contrast to inland provinces. The consistency between their find-
ings and ours enhances the validity of our results and emphasizes the importance of regional
disparities in China’s digital economic development. Additionally, our analysis of Fig 3 and Table 4 indicates that the influence of the COVID-19
on the overall growth of China’s digital economy has been relatively minimal. The consistent
stability of the digital economy index over different years suggests that the digital sector has
demonstrated resilience and adaptability, even in the face of the challenges posed by COVID-
19.This finding aligns with the research conducted by Xu et al. (2022) [92], who also proposed
that the rapid advancement of China’s digital economy had mitigated the severity of the
impact of COVID-19 on the Chinese economy. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 5.2 Evaluation of green finance index According to section 4.2.2 on entropy weight method, the results of green finance index and
its descriptive statistics are shown as in Fig 4 and Table 5. From 2015 to 2021, the mean of the
green finance index demonstrated a notable degree of stability, hovering around 0.30, thus
depicting a state of sustained equilibrium. Conversely, the median experienced a minor
decrease from 0.27 in 2015 to 0.26 in 2021, implying a potential marginal reduction in the
green finance index within specific provinces. Additionally, the standard deviation exhibited a
gradual increase from 0.15 in 2017 to 0.16 in 2021, hinting at a slight rise in the volatility of the
green finance index during this timeframe. In conclusion, the green finance index exhibited a
state of relative stability throughout this period, although divergences among different prov-
inces continued to persist. Regions with high levels of green finance development are primarily situated in the north-
eastern coastal areas, such as Shanghai, Beijing, Guangdong, and Zhejiang provinces. These
regions boast higher levels of economic development, abundant economic resources, and
stronger technological capabilities, which provide them with a significant advantage in green
industries and sustainable development. They actively promote the advancement of environ-
mentally friendly industries, facilitate the transition towards a low-carbon economy, and pro-
actively introduce and nurture green financial institutions and projects. To contextualize our
findings, we conducted a comparative analysis with the research conducted by He et al. (2020)
[93] and Lin et al. (2023) [94], who also examined the development of green finance in China. Their study highlighted the concentration of green finance development in the northeastern
coastal areas, aligning with our own findings. This consistency in results strengthens the reli-
ability and validity of our research, further underscoring the significance of these regions in
leading green finance initiatives. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 17 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 4. Results of green finance index for 30 provinces from 2015 to 2021. https://doi org/10 1371/journal pone 0291936 g004 5.3 Evaluation of the CCD According to the section 4.2.3 on modified CCD model, the calculation results of CCD and its
descriptive statistics are shown in Tables 6 and 7. Table 7 presents a comprehensive compila-
tion of descriptive statistical data concerning the coupling coordination degree among the 30
provinces spanning the years 2015 to 2021. It is evident that within this dataset, a discernible
trend emerges. Specifically, there is a gradual increase in the mean from 0.403 to 0.421 over
the specified time frame. This progression strongly implies a noteworthy enhancement in the
overall coupling coordination degree among the distinct provinces. Concurrently, the medians
exhibit marginal annual fluctuations within the range of 0.369 to 0.405, aligning with the tra-
jectory observed in the mean. Furthermore, across the 2015–2021 period, the standard Table 5. Descriptive statistics of green finance index for 30 provinces from 2015 to 2021. Statistic
2015
2016
2017
2018
2019
2020
2021
Mean
0.30
0.29
0.30
0.31
0.31
0.31
0.31
Median
0.27
0.25
0.25
0.28
0.28
0.28
0.26
Standard deviation
0.12
0.13
0.15
0.12
0.13
0.13
0.16
Min
0.16
0.17
0.13
0.17
0.17
0.15
0.14
Max
0.76
0.69
0.76
0.68
0.74
0.68
0.69
htt
//d i
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l
0291936 t005 Table 5. Descriptive statistics of green finance index for 30 provinces from 2015 to 2021. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 18 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Table 6. Results of coupling coordination degree for 30 provinces from 2015–2021. 5.3 Evaluation of the CCD Region
2015
2016
2017
2018
2019
2020
2021
The Eastern Region
Beijing
0.588
0.605
0.629
0.637
0.660
0.645
0.623
Tianjin
0.420
0.415
0.409
0.421
0.436
0.443
0.459
Hebei
0.428
0.482
0.462
0.449
0.441
0.416
0.409
Shanghai
0.659
0.652
0.677
0.596
0.625
0.627
0.648
Jiangsu
0.567
0.581
0.602
0.585
0.621
0.632
0.649
Zhejiang
0.540
0.522
0.644
0.615
0.621
0.651
0.670
Fujian
0.493
0.487
0.540
0.526
0.492
0.461
0.440
Shandong
0.467
0.490
0.503
0.497
0.507
0.528
0.556
Guangdong
0.623
0.652
0.607
0.637
0.720
0.720
0.752
Hainan
0.300
0.331
0.308
0.352
0.312
0.297
0.290
The Central Region
Shanxi
0.347
0.347
0.356
0.362
0.356
0.362
0.361
Anhui
0.404
0.428
0.440
0.431
0.438
0.451
0.453
Jiangxi
0.337
0.356
0.350
0.402
0.395
0.404
0.399
Henan
0.431
0.411
0.430
0.432
0.448
0.468
0.496
Hubei
0.373
0.426
0.397
0.438
0.413
0.411
0.402
Hunan
0.409
0.385
0.398
0.407
0.408
0.406
0.397
The Northeastern Region
Liaoning
0.407
0.400
0.398
0.383
0.394
0.403
0.418
Jilin
0.332
0.334
0.329
0.334
0.311
0.307
0.299
Hei Longjiang
0.354
0.405
0.344
0.330
0.326
0.317
0.302
The Western Region
Chongqing
0.357
0.366
0.374
0.385
0.375
0.374
0.367
Sichuan
0.432
0.429
0.441
0.481
0.472
0.471
0.453
Guizhou
0.296
0.268
0.297
0.317
0.314
0.309
0.316
Yunnan
0.333
0.313
0.290
0.313
0.319
0.308
0.308
Shaanxi
0.366
0.382
0.404
0.419
0.420
0.418
0.425
Gansu
0.272
0.261
0.281
0.284
0.289
0.272
0.269
Qinghai
0.253
0.267
0.285
0.270
0.270
0.283
0.290
Ningxia
0.299
0.282
0.264
0.263
0.247
0.245
0.243
Guangxi
0.335
0.317
0.321
0.322
0.328
0.327
0.321
Xinjiang
0.352
0.393
0.331
0.348
0.367
0.334
0.328
Inner Mongolia
0.349
0.317
0.337
0.380
0.336
0.323
0.320
https://doi.org/10.1371/journal.pone.0291936.t006 deviation demonstrates a progressive rise, ascending from 0.101 to 0.130. This phenomenon
indicates an expanding diversity in coupling coordination across provinces, thus implying a
progressively broader divergence among provinces over these years. Within the same temporal Table 7. Descriptive statistics of coupling coordination degree for 30 provinces from 2015–2021. Descriptive Statistics
2015
2016
2017
2018
2019
2020
2021
Mean
0.403
0.410
0.414
0.419
0.420
0.419
0.421
Median
0.369
0.393
0.396
0.405
0.402
0.405
0.401
Standard deviation
0.101
0.105
0.115
0.105
0.119
0.123
0.130
Min
0.253
0.261
0.264
0.263
0.247
0.245
0.243
Max
0.659
0.652
0.677
0.637
0.720
0.720
0.752
htt
//d i
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0291936 t007 Descriptive statistics of coupling coordination degree for 30 provinces from 2015–2021. Table 7. Descriptive statistics of coupling coordination degree for 30 provinces from 2015–2021. 5.3 Evaluation of the CCD PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 19 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance interval, the minimum value fluctuates between 0.253 and 0.243, while the maximum value
experiences variations between 0.659 and 0.752. These fluctuations underscore substantial dis-
parities in coupling coordination degree across diverse provinces and years. In summary, a
comprehensive assessment reveals a discernible ascending trend in coupling coordination
among provinces from 2015 to 2021. However, this trend is concurrently accompanied by a
growing year-to-year variance. This phenomenon might indicate that developmental discrep-
ancies among provinces have progressively widened over this specific time span, thereby insti-
gating changes in coupling coordination degree. From the Table 6 and Fig 5, it is apparent that the level of coordinated development
between the digital economy and green finance varies significantly among different provinces. In the eastern region, provinces like Beijing, Shanghai, Zhejiang, and Guangdong stand out as
top performers, surpassing the regional average and displaying steady growth in their level of
coordination between the digital economy and green finance. Other provinces in this region,
including Tianjin, Jiangsu, and Shandong, also exhibit favorable performance, albeit with
some fluctuations in certain years. The central region presents a more diverse performance,
with Henan showcasing significant progress in recent years, leading to noticeable improve-
ment in its coordination level. Provinces like Anhui and Hunan have also shown some
improvement, although their overall level remains relatively low. In the northeastern region,
the performance is generally average. Liaoning province consistently falls below the regional
average, while Heilongjiang and Jilin display less satisfactory performance in terms of coordi-
nation between the digital economy and green finance. The western region demonstrates vary-
ing levels of performance. Provinces like Sichuan and Shaanxi exhibit higher levels of
coordination, while Gansu, Qinghai, and Xinjiang demonstrate relatively average
performance. In addition, based on the results, it seems that the COVID-19 has had a modest influence
on the coupling coordination development between China’s digital economy and green
finance. The analysis reveals a slight decline in CCD across each region, indicating that the
COVID-19 has impacted the level of coordination between these two sectors. The reasons
behind this decline in CCD are likely multifaceted. The pandemic has disrupted economic
activities, presenting challenges for the digital economy and green finance sectors. 5.3 Evaluation of the CCD Factors
such as reduced investments, shifts in consumer behavior, and disruptions in supply chains
may have affected the coordinated development between these sectors. It is important to note that although the impact of COVID-19 on CCD is evident in the
results, the decline appears to be relatively minor. This suggests that the digital economy and
green finance sectors have demonstrated resilience and adaptability in the face of the chal-
lenges posed by COVID-19. Government efforts to mitigate the impact and facilitate recovery
from the disruptions caused by COVID-19 may have contributed to the overall stability and
gradual improvement observed in the coupling coordination development between the digital
economy and green finance. In summary, the descriptive statistical results indicate an increasing trend in the coordi-
nated development between the digital economy and green finance in China’s provinces. Prov-
inces in the eastern region, such as Beijing, Shanghai, Zhejiang, and Guangdong, lead the way
with notable progress. The central region shows a mixed performance, while the northeastern
and western regions demonstrate varying levels of coordination. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 5.4 Clustering analysis According to the information provided in section 4.2.4, this paper employs a combination of
the K-means algorithm and hierarchical clustering algorithm for the classification analysis of PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 20 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 5. Spatial pattern distribution of the average CCD of provinces from 2015 to 2021. https://doi.org/10.1371/journal.pone.0291936.g005 Fig 5. Spatial pattern distribution of the average CCD of provinces from 2015 to 2021. https://doi org/10 1371/journal pone 0291936 g005 Fig 5. Spatial pattern distribution of the average CCD of provinces from 2015 to 2021. https://doi.org/10.1371/journal.pone.0291936.g005 https://doi.org/10.1371/journal.pone.0291936.g005 the coordinated development between China’s digital economy and green finance. To evaluate
the quality of the clustering results, the paper conducts silhouette analysis. Silhouette analysis
calculates the silhouette coefficient for each sample, which quantifies the closeness of a sample
to its own cluster compared to other clusters [95]. A silhouette coefficient approaching 1 indi-
cates that a sample has a small distance within its own cluster and a significant distance from
other clusters, suggesting a favorable clustering result. Based on the computational results pre-
sented in the paper, when the number of clusters is set to 3, the analysis yields a good clustering
outcome, as illustrated in Fig 6. The average silhouette coefficient reaches its highest value of
0.537, indicating a relatively strong clustering result. This suggests that the samples within
each cluster are closely related to one another, while there are notable differences between dif-
ferent clusters. Therefore, the clustering outcome at this particular setting is considered PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 21 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance Fig 6. Silhouette analysis for K-Means clustering on 30 provinces with n_clusters = 3,4,5. https://doi.org/10.1371/journal.pone.0291936.g006 Fig 6. Silhouette analysis for K-Means clustering on 30 provinces with n_clusters = 3,4,5. Fig 6. Silhouette analysis for K-Means clustering on 30 provinces with n_clusters = 3,4,5. https://doi.org/10.1371/journal.pone.0291936.g006 desirable. However, as the number of clusters increases beyond 3, the average silhouette coeffi-
cient gradually decreases. This implies a decline in the quality of the clustering result. It sug-
gests that the samples within clusters become less similar to one another, and the separation
between different clusters becomes less distinct. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 5.4 Clustering analysis Fig 7 illustrates the results of a hierarchical clustering algorithm, providing a visual repre-
sentation of the level of coupling and coordination in the development of digital economy and
green finance among different provinces. The first cluster consists of Guangdong, Shanghai,
Beijing, Zhejiang, and Jiangsu. These provinces are currently in an intermediate stage of coor-
dination, focusing on fostering robust digital economies. They actively promote the establish-
ment of digital technology centers and clusters for digital economy, while also integrating
plans for the development of green finance. They are committed to practicing the concept of
green development and actively developing the green finance industry. Additionally, they are
constructing a solid foundation by implementing the dual-carbon policy, aiming to maximize
the benefits of both the real economy and industrial research. For instance, Beijing has
launched the construction of the Beijing Economic and Technological Development Zone as a
demonstration area for digital economic innovation, aiming to further improve the policy
environment for the digital economy. Additionally, Beijing has established a Green Finance
Reform and Innovation Pilot Zone, promoting financial innovations such as green credit,
green insurance, and green funds. Shanghai has implemented supportive policies to foster the
development of the industrial internet and facilitate the digitalization upgrade of traditional
industries. The city actively undertakes the Green Credit Project, encouraging banking and
financial institutions to issue green financial bonds. In Guangdong province, digital economy 22 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE
coupling coordination development between Digital Economy and Green Finance PLOS ONE coupling coordination development between Digital Economy and Green Finance innovation and development pilot zones have been established in cities like Guangzhou and
Shenzhen. These zones actively focus on the artificial intelligence industry. Moreover, Guang-
dong province encourages and guides social capital to establish green development funds and
carbon funds to support green and low-carbon projects. Therefore, the level of coupling and
coordination between the digital economy and green finance in these regions is steadily
improving. The provinces included in the second cluster, namely Tianjin, Hebei, Liaoning, Anhui,
Fig 7. Hierarchical clustering results of 30 provinces. https://doi.org/10.1371/journal.pone.0291936.g007 Fig 7. Hierarchical clustering results of 30 provinces. https://doi org/10 1371/journal pone 0291936 g007 Fig 7. Hierarchical clustering results of 30 provinces. Fig 7. Hierarchical clustering results of 30 provinces. https://doi.org/10.1371/journal.pone.0291936.g007 https://doi.org/10.1371/journal.pone.0291936.g007 innovation and development pilot zones have been established in cities like Guangzhou and
Shenzhen. 5.4 Clustering analysis These zones actively focus on the artificial intelligence industry. Moreover, Guang-
dong province encourages and guides social capital to establish green development funds and
carbon funds to support green and low-carbon projects. Therefore, the level of coupling and
coordination between the digital economy and green finance in these regions is steadily
improving. innovation and development pilot zones have been established in cities like Guangzhou and
Shenzhen. These zones actively focus on the artificial intelligence industry. Moreover, Guang-
dong province encourages and guides social capital to establish green development funds and
carbon funds to support green and low-carbon projects. Therefore, the level of coupling and
coordination between the digital economy and green finance in these regions is steadily
improving. The provinces included in the second cluster, namely Tianjin, Hebei, Liaoning, Anhui,
Fujian, Shandong, Henan, Hubei, Hunan, Sichuan, and Shaanxi, exhibit a CCD between their 23 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance digital economy and green finance at approximately 0.4, indicating that they are in the primary
stage of coordination. Despite the relatively low coupling degree, these provinces have demon-
strated a positive trend of stable growth over the past seven years. To facilitate the development
of the digital economy, these provinces prioritize the creation of favorable conditions. They
actively promote digital innovation capabilities and accelerate the digital transformation of
their respective industries. By embracing digital technologies and fostering innovation, these
regions aim to enhance productivity, competitiveness, and overall economic growth. More-
over, these provinces make investments in green finance and closely monitor its development
and challenges. Recognizing the importance of sustainable finance, they establish multi-level
green finance policies and service platforms. These initiatives facilitate the coordinated devel-
opment of green finance across regions, based on unified industry standards. By aligning their
efforts, these provinces foster a synergistic approach to green finance, contributing to sustain-
able economic growth and environmental protection. Overall, while these provinces may have
a relatively lower level of coordination between the digital economy and green finance, they
have shown positive progress and stability in their development. Their focus on creating favor-
able conditions, promoting digital innovation, and investing in green finance demonstrates
their commitment to achieving a more coordinated and sustainable approach to economic
development. 5.4 Clustering analysis The third cluster includes Shanxi, Inner Mongolia, Jilin, Heilongjiang, Jiangxi, Guangxi,
Hainan, Chongqing, Guizhou, Yunnan, Gansu, Qinghai, and Xinjiang provinces. These prov-
inces exhibit a low degree of CCD between the digital economy and green finance, nearly in
the lowest level of coordination. Most of these regions have relatively lower levels of economic
development and lack a foundation for nurturing the digital economy. They face challenges
such as insufficient innovation capacity in key areas, relatively slow digitization of traditional
industries, and the need to overcome the digital divide. Moreover, the governance system for
the digital economy requires improvement. Additionally, the development level of green
finance in these regions is relatively low, with inadequate product innovation capacity and lim-
ited market depth and breadth. The intermediary services for green finance development in
these regions are also relatively weak. PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 6.1 Formulate differentiated regional development policies to narrow the
gap in regional development To rectify the disparity in the coordinated development of the digital economy and green
finance across different regions in China, the policymaker should implement macro-level mea-
sures aimed at regulating and optimizing fiscal intervention. These measures seek to distribute
policy resources more equitably and efficiently. One specific measure involves strategically allocating policy resources to the central and
western regions, which are comparatively less developed. The policymaker should increase
investments in green technology innovation and digital infrastructure construction within
these regions. By directing resources and support towards these areas, the objective is to stimu-
late their rapid development in the digital economy and green finance sectors. This approach
aims to promote sustainable development and bridge the regional development gap within the
country. Through fostering the expansion of the digital economy and green finance in the central
and western regions, the policymaker intends to ensure that all areas can benefit from the
opportunities presented by these sectors. This approach fosters economic inclusivity by
enabling regions with traditionally fewer advantages to participate and contribute to the PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 24 / 31 PLOS ONE coupling coordination development between Digital Economy and Green Finance overall development of the country. Additionally, it seeks to establish a more balanced and
harmonized economic landscape, where the benefits and opportunities of the digital economy
and green finance are distributed more evenly nationwide. In summary, the government’s macro-level measures prioritize leveraging fiscal interven-
tion to address regional disparities in the coordinated development of the digital economy and
green finance in China. By allocating policy resources towards the less developed central and
western regions, increasing investments in green technology innovation and digital infrastruc-
ture, and promoting sustainable development, the policymaker aims to bridge the regional
development gap, foster economic inclusivity, and ensure that all regions can benefit from the
opportunities presented by the digital economy and green finance. 6.2 Promote the deep coordination of green development and digitalization In the realm of production and consumption, businesses are increasingly recognizing the sig-
nificance of utilizing digital technology to expand their range of green consumer products. This enables them to offer a broader selection of high-quality digital intelligent products that
not only cater to but also guide the preferences of environmentally conscious consumers. Gov-
ernments play a crucial role in facilitating this shift towards green consumption. Governments should actively promote industrial digitization by leading efforts to enhance
the integration of front-end and back-end industries. This involves collaborating with
upstream and downstream enterprises to reduce resource consumption during production
and minimize pollution emissions throughout the consumption process. Through active
engagement with stakeholders and the implementation of effective policies, governments can
drive economic development towards technological advancement and encourage the adoption
of environmentally friendly products. However, it is important to acknowledge that while the digital economy brings notable pen-
etrability and significant economies of scale, it also carries environmental implications of its
own. The substantial energy consumption and carbon dioxide emissions associated with the
digital economy must not be overlooked. Therefore, it becomes imperative for us to establish a
green consensus and mitigate redundant consumption resulting from the digital transforma-
tion process in order to ensure sustainable development. By embracing sustainable practices and striving for resource efficiency, both businesses and
governments can effectively address the environmental impact of the digital economy. This
necessitates a comprehensive approach that encompasses the development and promotion of
green consumer products, as well as the implementation of policies and measures to minimize
the ecological footprint of digital technologies. In conclusion, the integration of digital technology with green production and consump-
tion is vital for driving economic growth and achieving sustainability goals. Through digital
innovation, businesses can diversify their product offerings to meet the needs of environmen-
tally conscious consumers. Simultaneously, governments must actively promote industrial dig-
itization and collaborate with industry stakeholders to reduce resource consumption and
pollution emissions. By establishing a green consensus and adopting sustainable practices, we
can ensure that the process of digital transformation aligns with the principles of green devel-
opment and contributes to a more environmentally friendly future. 7 Conclusions In this study, both factor analysis modeling and the entropy weight method were employed to
calculate and analyze the digital economy index and the green finance index of various prov-
inces. Based on the index outcomes, a modified coupling coordination degree model was 25 / 31 PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
October 13, 2023 PLOS ONE coupling coordination development between Digital Economy and Green Finance formulated to assess and validate the disparities in coupling relationships and coordination
levels between the two subsystems. Furthermore, to illuminate spatial and temporal variations
in coupling coordination development across different regions, this study employed two clus-
tering algorithms: K-means and hierarchical clustering. According to the findings presented in
this research, 30 provinces were categorized into three clusters based on their coupling coordi-
nation degree. On a broader perspective, China’s digital economy and green finance are pres-
ently undergoing coupling coordination development at a relatively modest level, positioned
at the primary coordination stage. The coupling coordination development of the two subsys-
tems, in general, demonstrated steady growth; however, there were pronounced regional dis-
parities among provinces. Specifically, the level of coupling coordination development in the
eastern regions was comparatively higher than that in the western regions. Likewise, coastal
areas exhibited higher level of coupling coordination development compared to inland
regions. Remarkably, despite the impact of COVID-19, the coupling coordination develop-
ment of the two subsystems displayed resilience, showcasing an overall upward trend. This study makes significant contributions in both theoretical and practical aspects. Theo-
retical expansion is achieved by extending the research scope of coupling theory to encompass
the digital economy and green finance. In terms of practical implications, the study enriches
the application of the digital economy and green finance through the use of the modified cou-
pling coordination model to analyze the degree, evolutionary stages, and trends of their cou-
pling coordination. Additionally, based on the research findings, we can put forth several
policy recommendations to facilitate the coupling coordination development between digital
economy and green finance in China, such as formulate region-specific development policies,
support green technology innovation and digital infrastructure, promote industrial digitiza-
tion and collaboration and establish a consensus on green practices. While this study demonstrates innovation, there are limitations attributed to factors such as
time constraints and data availability. The evaluation index system may also have limitations,
despite employing a rigorous selection process and a substantial number of evaluation indica-
tors. S1 Raw data.
(ZIP) S1 Raw data. (ZIP) 7 Conclusions Further consideration is necessary to address the issue of indicator deviation, which may
require calibration and improvement in different environments or provinces. Moreover, the
study only analyzes 30 provinces in China due to data availability, posing limitations in terms
of representativeness. Future research could expand the sample range to include a broader
range of regions, such as urban and rural areas, in order to obtain more comprehensive and
accurate research results. Supporting information S1 Raw data. (ZIP) PLOS ONE | https://doi.org/10.1371/journal.pone.0291936
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34208552 31 / 31
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Figure supplementary 2 Figure supplementary 2 A
B
C
PRJNA612966
GEO:GSE147082
−50
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0
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tSNE_1
tSNE_2
CD11b(ITGAM)
−50
−25
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−50
−25
0
25
50
tSNE_1
tSNE_2
1.0
1.5
2.0
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3.0
CD16 (FCGR3A)
−50
−25
0
25
50
−50
−25
0
25
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tSNE_1
tSNE_2
CD163
−50
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0
25
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−50
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0
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tSNE_1
tSNE_2
1
2
3
4
CD206 (MRC1)
B−cells
Fibroblasts
CD4+ T−cells
CD8+ T−cells
Endothelial cells
HSC
DC
Chondrocytes
Epithelial cells
B−cells
Epithelial cells
−50
−25
0
25
−50
−25
0
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tSNE_1
tSNE_2
Macrophages
Fibroblasts
Figure supplementary 2
No metastasis
Metastasis
CD163
CD68
CD206
Merge
ISH
0
50
100
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CD68+CD163+CD206+ cells
Fluorescence intensity value
***
No metastasis
Metastasis A
No metastasis
Metastasis
CD163
CD68
CD206
Merge
ISH
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100
150
200
250
CD68+CD163+CD206+ cells
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No metastasis
Metastasis A Metastasis 0
20
40
60
80
100
120
0.0
0.2
0.4
0.6
0.8
1.0
Months
Overall survival
M0 low score n = 237
M0 high score n = 141
log-rank p = 0.188
0
20
40
60
80
100
120
0.0
0.2
0.4
0.6
0.8
1.0
Months
M1 low score n = 185
M1 high score n = 193
log-rank p = 0.012
TCGA-OV
Cibersort score of M0
TCGA-OV
Cibersort score of M1
B
C
D
PRJNA612966
GEO:GSE147082
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tSNE_2
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1.5
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3.0
CD16 (FCGR3A)
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0
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50
−50
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0
25
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tSNE_1
tSNE_2
CD163
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tSNE_1
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CD206 (MRC1)
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CD4+ T−cells
CD8+ T−cells
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HSC
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Chondrocytes
Epithelial cells
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Fibroblasts
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Overall survival B
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PRJNA612966
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Fibroblasts
CD4+ T−cells
CD8+ T−cells
Endothelial cells
HSC
DC
Chondrocytes
Epithelial cells
B−cells
Epithelial cells
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CD16 (FCGR3A) −50
−25
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25
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−25
0
25
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tSNE_2
CD11b(ITGAM) −50
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0
25
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tSNE_1
tSNE_2
CD11b(ITGAM)
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0
25
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−50
−25
0
tSNE_1
tSNE_2
CD16 (FCGR3A)
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0
25
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−50
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25
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tSNE_1
tSNE_2
CD163
−50
−25
0
25
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−50
−25
0
tSNE_1
tSNE_2
CD206 (MRC1) B PRJNA612966
GEO:GSE147082
B−cells
Fibroblasts
CD4+ T−cells
CD8+ T−cells
Endothelial cells
HSC
DC
Chondrocytes
Epithelial cells
B−cells
Epithelial cells
−50
−25
0
25
−50
−25
0
25
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tSNE_2
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120
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Overall survival
M0 low score n = 237
M0 high score n = 141
log-rank p = 0.188
0
20
40
60
80
100
120
0.0
0.2
0.4
0.6
0.8
1.0
Months
M1 low score n = 185
M1 high score n = 193
log-rank p = 0.012
TCGA-OV
Cibersort score of M0
TCGA-OV
Cibersort score of M1
D
Overall survival 0
20
40
60
80
100
120
0.0
0.2
0.4
0.6
0.8
1.0
Months
Overall survival
M0 low score n = 237
M0 high score n = 141
log-rank p = 0.188
TCGA-OV
Cibersort score of M0
D D TCGA-OV
Cibersort score of M1
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The Critical Role of Potassium in Plant Stress Response
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International journal of molecular sciences
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Min Wang, Qingsong Zheng, Qirong Shen and Shiwei Guo * Agricultural Ministry Key Lab of Plant Nutrition and Fertilization in Low-Middle Reaches of the
Yangtze River, Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization,
Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, China; Agricultural Ministry Key Lab of Plant Nutrition and Fertilization in Low-Middle Reaches of the
Yangtze River, Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization,
Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, China; * Author to whom correspondence should be addressed; E-Mail: sguo@njau.edu.cn;
Tel.: +86-25-8439-6393. * Author to whom correspondence should be addressed; E-Mail: sguo@njau.edu.cn;
Tel.: +86-25-8439-6393. Received: 12 December 2012; in revised form: 23 February 2013 / Accepted: 21 March 2013 /
Published: 2 April 2013 Received: 12 December 2012; in revised form: 23 February 2013 / Accepted: 21 March 2013 /
Published: 2 April 2013 Abstract: Agricultural production continues to be constrained by a number of biotic and
abiotic factors that can reduce crop yield quantity and quality. Potassium (K) is an essential
nutrient that affects most of the biochemical and physiological processes that influence plant
growth and metabolism. It also contributes to the survival of plants exposed to various biotic
and abiotic stresses. The following review focuses on the emerging role of K in defending
against a number of biotic and abiotic stresses, including diseases, pests, drought, salinity,
cold and frost and waterlogging. The availability of K and its effects on plant growth,
anatomy, morphology and plant metabolism are discussed. The physiological and molecular
mechanisms of K function in plant stress resistance are reviewed. This article also evaluates
the potential for improving plant stress resistance by modifying K fertilizer inputs and
highlights the future needs for research about the role of K in agriculture. Keywords: biotic stress; abiotic stress; potassium; physiological and molecular mechanisms;
plant resistance The Critical Role of Potassium in Plant Stress Response The Critical Role of Potassium in Plant Stress Response
Min Wang, Qingsong Zheng, Qirong Shen and Shiwei Guo *
Agricultural Ministry Key Lab of Plant Nutrition and Fertilization in Low-Middle Reaches of the
Yangtze River, Jiangsu Key Lab and Engineering Center for Solid Organic Waste Utilization,
Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, China;
E-Mails: 2010203034@njau.edu.cn (M.W.); qszheng@njau.edu.cn (Q.Z.); shenqirong@njau.edu.cn (Q. * Author to whom correspondence should be addressed; E-Mail: sguo@njau.edu.cn;
Tel.: +86-25-8439-6393. Received: 12 December 2012; in revised form: 23 February 2013 / Accepted: 21 March 2013 /
Published: 2 April 2013 Int. J. Mol. Sci. 2013, 14, 7370-7390; doi:10.3390/ijms14047370 Int. J. Mol. Sci. 2013, 14, 7370-7390; doi:10.3390/ijms14047370 International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
OPEN ACCESS 1. Introduction The world population is expanding rapidly and will pass from its current number of 7.0 billion to
9.4 billion by the year 2050 [1]. To provide enough food for an expanding world population, a massive
increase in crop production is required to meet the food demands of future generations, while preserving Int. J. Mol. Sci. 2013, 14 7371 the ecological and energy-related resources of our planet. However, agricultural production continues to
be constrained by a variety of biotic (e.g., pathogens, insects and weeds) and abiotic (e.g., drought,
salinity, cold, frost and waterlogging) factors that can significantly reduce the quantity and quality of
crop production. Evidence indicates that biotic stress can cause a 28.2% yield loss of wheat, 37.4% loss
of rice, 31.2% loss of maize, 40.3% loss of potatoes, 26.3% loss of soybeans and 28.8% loss of cotton [2]. Meanwhile, yield losses from abiotic stress were estimated at 65.8% for maize, 82.1% for wheat, 69.3%
for soybeans and 54.1% for potatoes [3]. During their evolution, plants have developed a wide range of mechanisms to resist a variety of
stressed conditions. Increasing evidence suggests that mineral nutrients play a critical role in plant stress
resistance [4–8]. Out of all the mineral nutrients, potassium (K) plays a particularly critical role in plant
growth and metabolism, and it contributes greatly to the survival of plants that are under various biotic
and abiotic stresses. The importance of K fertilizer for the formation of crop production and its quality is
known. As a consequence, potash consumption has increased dramatically in most regions of the world [9]. A strong positive relationship between K fertilizer input and grain yield has been shown [10]. K is an essential nutrient and is also the most abundant cation in plants. The concentration of K+ in
the cytoplasm has consistently been found to be between 100 and 200 mM [11], and apoplastic K+
concentration may vary between 10 and 200 or even reach up to 500 mM [12]. K plays essential roles in
enzyme activation, protein synthesis, photosynthesis, osmoregulation, stomatal movement, energy
transfer, phloem transport, cation-anion balance and stress resistance [4]. This review is focused on the effects of K on plant resistance to various biotic (pathogen and insects)
and abiotic (drought, salinity, cold and frost and waterlogging) stresses. K availability for plant growth,
anatomy and morphology, as well as plant metabolism are discussed. 1. Introduction This review also discusses the
roles of K in stress-resistant mechanisms and evaluates the potential for improving plant resistance by
modifying K fertilizer input and selecting appropriate plant species or varieties. 2. The Role of Potassium in Biotic Stress Resistance Increased evidence has shown that crop production is significantly restricted by biotic stresses. Oerke and Dehne [13] estimated that weeds produce the highest potential loss (32%), followed by
animal pests (18%), fungi and bacteria (15%) and viruses (3%) from 1996 to 1998. These numbers
reflect the total attainable production for eight major crops (wheat, rice, maize, barley, potatoes,
soybeans, sugar beets and cotton). In many cases, K-deficient plants tend to be more susceptible to
infection than those with an adequate supply of K. For example, the rate of rice borer infestation was
greatest when there was no supply of K, but decreased rapidly as the K concentration increased [14]
(Table 1). Similar results were found with a Discula destructiva Redlin infection in Cornus florida L. [15]. Williams and Smith [16] also reported that increased K fertilizer significantly reduced the disease
incidence of stem rot and aggregate sheath spot (AgSS), and negative correlations were found between
the percentage of K in leaf blades and disease severity. K fertilizer is widely reported to decrease insect
infestation and disease incidence in many host plants. Perrenoud [17] reviewed 2449 references and
found that the use of K significantly decreased the incidence of fungal diseases by 70%, bacteria by
69%, insects and mites by 63%, viruses by 41% and nematodes by 33%. Meanwhile, K increased the Int. J. Mol. Sci. 2013, 14 7372 yield of plants infested with fungal diseases by 42%, bacteria by 57%, insects and mites by 36%, viruses
by 78% and nematodes by 19%. 1. Impacts of soil potassium build-up on rice stem borers and grain yield within a
eld [14]. Table 1. Impacts of soil potassium build-up on rice stem borers and grain yield within a
rice field [14]. Table 1. Impacts of soil potassium build-up on rice stem borers and grain yield within a
rice field [14]. Serial
number
Potassium treatments
(kg ha−1)
Stem borer infestation (%)
Yield/plot
(g/3 m2)
Yield
(kg ha−1)
Dead heart
White heads
1
40 kg
3.05 b
5.37 b
1913.00 b
6376.66
2
50 kg
2.64 bc
3.58 c
2287.00 a
7623.33
3
60 kg
2.40 c
3.37 c
2317.00 a
7723.33
4
Control
4.33 a
7.12 a
1690.00 c
5633.33
LSD value
0.619
0.561
219.4
The means in each column are followed by at least one letter in common and are not significantly different at
the 5% level of probability (p ≥ 0.05). 2. The Role of Potassium in Biotic Stress Resistance LSD, least significant difference. K fertilizer application decreased the incidence of diseases in most cases, but sometimes had no effect
or even the opposite effect. Prabhu et al. [18] noted that the effect of K on disease incidence can be
classified as “increased”, “decreased” and having “no effect” or “variable effect” (Table 2). The variable
effects of K on disease incidence could be affected by the amount and source of K, plant and pathogen
species and trial type. Nam et al. [19] found that strawberries that were grown with excess K were very
susceptible to infection by the anthracnose pathogen, Colletotrichum gloeosporioides, but its resistance
was greatly enhanced when no K was supplied. This result was observed because the low plant K status
induces the synthesis of molecules, including reactive oxygen species (ROS) and phytohormones, such
as auxin, ethylene and jasmonic acid (JA), as a result of its enhanced plant stress tolerance [5,20]. Table 2. Number of published papers reporting the effects of potassium on disease [18]. Decrease in disease
Increase in disease
No effect
Total
Fungi
89
33
8
130
Bacteria
18
5
0
23
Viruses
9
5
3
17
Nematode
3
6
1
10 able 2. Number of published papers reporting the effects of potassium on disease [18]. The mechanistic influences of K on plant disease resistance have been reported by several
researchers. Higher K+ concentrations decreased the internal competition of pathogens for nutrient
resources [15]. This nutritional status enables plants to allocate more resources to developing stronger
cell walls for preventing pathogen infection and insect attack and to obtain more nutrients to be used for
plant defense and damage repair [21]. DeDatta and Mikkelson [22] reported that the culm and stalk
strength of rice were increased in the presence of adequate K concentrations as a result of increasing
plant resistance. During airborne pathogen infections (especially from bacteria and viruses), the stomata
were able to function properly when there was sufficient K, thus preventing pathogen invasion by rapid
stomata closing [23,24]. K is also essential to the performance of multiple plant enzyme functions, and it regulates the
metabolite pattern of higher plants, ultimately changing metabolite concentrations [4,21]. In a Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2. The Role of Potassium in Biotic Stress Resistance 2013, 14 7373 K-sufficient plant, the synthesis of high-molecular-weight compounds (such as proteins, starches and
cellulose) was markedly increased, thereby depressing the concentrations of low-molecular-weight
compounds, such as soluble sugars organic acids, amino acids and amides, in the plant tissues. These
low-molecular-weight compounds are important for the development of infections and insect
infestations, so lower concentrations, thereby, leave plants less vulnerable to disease and pest attacks in
K-sufficient plants [4]. Adequate K increases phenol concentrations, which play a critical role in plant
resistance [25]. Furthermore, Sarwar [14] concluded that less pest damage in higher K plants can be
attributed to a lack of pest preference under sufficient nutrient concentrations, as well as the synthesis of
defensive compounds leading to higher pest mortality. Figure 1 summarizes the main roles of K in plant
resistance to biotic stress. Figure 1. Role of K under biotic stress. 3. The Role of Potassium in Abiotic Stress Resistance Figure 1. Role of K under biotic stress. 3. The Role of Potassium in Abiotic Stress Resistance 3.1.1. Cell Elongation and Cell Membrane Stability 3.1.1. Cell Elongation and Cell Membrane Stability It is worthwhile to try to improve crop tolerance to stress in low-moisture soils by inducing deeper
rooting, larger absorption surfaces and greater water retention in plant tissues. Deeper rooting could be
achieved by deep placement of K fertilizer that is associated with other mineral nutrients, such as P and
N, which both have root signaling functions [27]. Adequate amounts of K can enhance the total dry mass
accumulation of crop plants under drought stress in comparison to lower K concentrations [28]. This
finding might be attributable to stomatal regulation by K+ and corresponding higher rates of
photosynthesis [4]. Furthermore, K is also essential for the translocation of photoassimilates in root
growth [6]. Root growth promotion by increased appropriate K supply under K-deficient soil was found
to increase the root surface that was exposed to soil as a result of increased root water uptake [6]. Lindhauer [29] reported that fine K nutrition not only increased plant total dry mass and leaf area, but
also improved the water retention in plant tissues under drought stress. Increased evidence shows that the maintenance of membrane integrity and stability under drought
stress is also essential for plant drought tolerance [30]. Cell membrane stability was significantly
declined under drought stress [31]. In a study by Premachandra et al. [32], maize plants with higher K
applications showed greater adaptation to water stress. This improvement was mainly attributed to the
role of K in improving cell membrane stability and osmotic adjustment ability. An adequate K supply
is essential to enhancing drought resistance by increasing root elongation and maintaining cell
membrane stability. 3.1. Potassium and Drought Stress 3.1. Potassium and Drought Stress The major limitation for plant growth and crop production in arid and semi-arid regions is soil water
availability. Plants that are continuously exposed to drought stress can form ROS, which leads to leaf
damage [7,13,26] and, ultimately, decreases crop yield. During drought stress, root growth and the rates
of K+ diffusion in the soil towards the roots were both restricted, thus limiting K acquisition. The resulting lower K concentrations can further depress the plant resistance to drought stress, as well as
K absorption. Maintaining adequate plant K is, therefore, critical for plant drought resistance. A close Int. J. Mol. Sci. 2013, 14 7374 relationship between K nutritional status and plant drought resistance has been demonstrated. The roles
of K in physiological and molecular mechanisms of plant drought resistance have been explored. 3.1.2. Aquaporins and Water Uptake Aquaporins are channel proteins that are present in the plasma and intracellular membranes of plant
cells. They play a crucial role in plant water relations by regulating the osmotic potential and hydraulic
conductivity of membranes and make changes in plant water permeability [33,34]. Under drought stress
conditions, aquaporin gene expression can be regulated [35,36] to help plants maintain their
water balance [36–38]. During water stress, roots regulated their water and ion uptake capacities by modifying PIPs
(plasma membrane intrinsic proteins) and K+ channel at the transcription level to cope with the water
deficiency [37,39–42]. Liu et al. [41] observed that transcription expression levels of the PIPs and K+
channel-encoding genes was induced by K+ starvation and could be downregulated by a polyethylene
glycol (PEG)-mediated water deficit in rice, which may result in a reduction in the membrane water
permeability and then promote cellular water conservation during drought stress. However, it should be
mentioned that the expression level of some other water channels, such as OsPIP1;1, OsPIP1;2,
AtPIP1;4 and AtPIP2;5, could be induced by a relative long-term water-deficit treatment, which
should result in greater osmotic water permeability and facilitated water flux [40,41]. Recently,
Kanai et al. [43] also observed close coupling between aquaporin activities and K-channel transporters. The initial response of K deficiency was perceived in the form of a change in K-channel activity,
which altered root hydraulic conductance, and transduction of the follow up signal resulted in a shift of Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 7375 aquaporin activity. They found that aquaporin activities might have been suppressed by K deficiency
and result in a reduction of root hydraulic conductance and water supply to the growing stem for
diameter expansion and the leaf for transpiration. In Arabidopsis roots, the transcripts encoding water channels, PIP1;2 (PIP1b), PIP2;2 (PIP2b) and
TIP1;2 (TIP), and the K+ transporter, HAK5, were reduced under treatment of the K+-channel blocker
(CsCl) [44]. Furthermore, water transport in onion roots was sensitive to inhibitors of the water channel
and K+-channel, and the reduction in hydraulic conductivity (Lp) by treatment with a K+-channel
inhibitor suggests that K+ fluxes are linked to water channel activity in the plasma membrane [45]. Water channels and K+ channel/transporters are functionally co-regulated as a part of plant
osmoregulation to maintain appropriate cytosolic osmolarity and acclimate the plant to drought or other
stresses [41]. Guo et al. 3.1.3. Osmotic Adjustment The maintenance of a favorable water status is critical for plant survival under drought stress. Osmotic adjustment is a major trait that is associated with maintaining high cellular turgor potential and
water retention in response to drought stress. Many studies have shown that osmotic adjustment of
leaves is positively correlated with drought tolerance in various plant species [49]. As one of the most
prominent inorganic osmotica in plants, K+ plays a key role in formation of the osmotic adjustment
ability, even under drought conditions [4]. Cell turgor recovery in osmotically-generated stress was
regulated by increasing K+, Cl− and Na+ uptake by root cells, which was partly mediated by
voltage-gated K+ transporters at the cellular plasma membrane [50]. Furthermore, sufficient K induces
solute accumulation, thus lowering osmotic potential and helping to maintain plant cell turgor under
osmotic stress. In summary, an adequate K status may facilitate osmotic adjustment, which maintains
higher turgor pressure, relative water content and lower osmotic potential, thus improving the ability of
plants to tolerate drought stress [8,51]. 3.1.2. Aquaporins and Water Uptake [46] also showed a positive correlation between K absorption and water uptake
in Phaseolus vulgaris plants. The loading of K+ into the xylem most likely mediated the xylem hydraulic
conductance that aided plants in maintaining cell turgor, stomatal aperture and gas exchange rates as part
of their drought adaptations [47,48]. 3.1.4. Stomatal Regulation One of the major functions of the stomata is to control plant water loss via transpiration. During
drought stress, quick stomatal closure and internal moisture preservation are essential for plant
adaptation to drought conditions. K plays a crucial role in turgor regulation within the guard cells
during stomatal movement [4]. As stomatal closure is preceded by a rapid release of K+ from the guard
cells into the leaf apoplast, it is reasonable to think that stomata would be difficult to remain open
under K-deficient conditions. Some studies also stated that K deficiency may induce stomatal closure
and inhibit photosynthetic rates in several crop plants [52,53]. Conversely, many studies suggest that K
had no effect on stomatal conductance and photosynthetic rates under well-watered conditions, but K
starvation could favor stomatal opening and promote transpiration, compared with K sufficiency in
several plants under drought stress [54–56]. Furthermore, photosynthetic rate was decreased under Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 7376 drought stress in K-deficient plants [51,54,57]. This discrepancy may be related to the plant species,
experimental system and environmental factors within the experimental field or interspecific differences. The effects of drought stress on stomata closure in olive trees and sunflower plants were found to be
dependent on the K+ nutrient status [55,56]. When plants were supplied with different K+
concentrations and then subjected to drought stress, their stomatal conductance was more markedly
reduced in normal K plants than in low K plants (Table 3). Benlloch-Gonzalez et al. [56] explained
that the low plant K status could inhibit water-stress-induced stomatal closure via ethylene synthesis,
and stomatal conductance could be significantly reduced in K+-starved plants after the adding of an
ethylene synthesis inhibitor (cobalt). K+ starvation increases the transcription of genes involved in
ethylene production and signaling and stimulates ethylene production [56,58]. Then, the increased
ethylene could inhibit the action of abscisic acid (ABA) on stomata and delay stomata closure [59,60]. During drought stress, the stomata cannot function properly in K+-deficient plants, resulting in greater
water loss. Drought stress did not decrease water use efficiency (WUE), whereas it did increase WUE
by rapid stomata closing during water deficit [51]. Adequate levels of K nutrition enhanced plant
drought resistance, water relations, WUE and plant growth under drought conditions [51]. Table 3. 3.1.4. Stomatal Regulation Effect of K+ concentration in irrigation water (normal versus low K) and water
availability in the growth medium (irrigation versus drought) on K+ accumulation and
stomatal conductance in leaves [56]. Treatment
K+ content in leaves (µmol/gFW)
Stomatal conductance (mmol/m2/s) Treatment
K+ content in leaves (µmol/gFW)
Stomatal conductance (mmol/m2/s)
Normal K: Irrigated
133.6 ± 7.3
456 ± 5.6
Normal K: Drought
119.4 ± 3.8
281 ± 27.9
Low K: Irrigated
36.3 ± 1.4
462 ± 4.0
Low K: Drought
25.7 ± 0.8
351 ± 15.2
3.1.5. Detoxification of Reactive Oxygen Species 3.1.5. Detoxification of Reactive Oxygen Species 3.1.5. Detoxification of Reactive Oxygen Species Stomatal closing in response to drought stress leads to a reduction in photosynthetic efficiency as a
consequence of chloroplast dehydration [7]. Photosynthesis inhibition can further disturb the balance
between ROS production and antioxidant defense [61–63], resulting in ROS accumulation. The ROS
have a dual action in biotic and abiotic stresses that depends on their cellular concentration [64]. Low levels of ROS could be involved in the stress-signaling pathway by triggering stress
defense/acclimation responses [64,65]. However, ROS became extremely injurious to cellular
membranes and other cellular components when its concentrations reached the point of phytotoxicity,
resulting in oxidative stress and, eventually, cell death [64,66]. Drought stress-induced ROS production can additionally be enhanced in K-deficient plants [7]. Under drought stress, photosynthetic CO2 fixation in K-deficient plants is substantially limited by
impairment in stomata regulation, conversion of light energy into chemical energy and phloem export
of photosynthates from source leaves into sink organs [51]. As the impairment in photosynthetic CO2
fixation occurs, molecular O2 is activated, leading to extensive generation of ROS [67] and, thereby,
oxidative degradation of chlorophyll and membranes. The maintenance of adequate K nutrition is
critical for mitigating or preventing damage by drought stress and controlling the water balance [68]. Egilla [51] suggested that increasing extrachloroplastic K+ concentrations in plant cells with an excess Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 7377 K+ supply could prevent photosynthesis inhibition under drought stress. An adaptive K requirement for
drought-stressed plants could be related to the role of K in enhancing photosynthetic CO2 fixation and
transport of photosynthates into sink organs and inhibiting the transfer of photosynthetic electrons to O2,
thus reducing ROS production [7]. K+ supply could prevent photosynthesis inhibition under drought stress. 3.2. Potassium and Salt Stress Salinity is a major abiotic stress that affects approximately 7% of the world’s total land area. More
than 800 million hectares of land around the world are affected by salinity [73], which results in billions
of dollars in crop production losses. The accumulation of high salt concentrations in the soil makes it
harder for plant roots to take up water and, thereby, disturbs a plant's water balance, while high
concentrations of salts in plant tissue may be toxic. Salinity inhibits seed germination and plant growth,
affects the leaf anatomy and physiology of plants and, thereby, influences their photosynthesis, water
relations, protein synthesis, energy production and lipid metabolism [74]. Plant growth responds to
salinity in the following two phases [75]: a rapid osmotic phase that decreases water availability to plants
and inhibits the growth of young leaves, followed by a slow ionic phase that results in salt toxicity and
accelerates the senescence of mature leaves. Plants have developed diverse strategies to resist salt stress,
such as restricting Na+ uptake, activating Na+ exclusion or cellular compartmentalization of excessive
Na+ into the vacuole [76,77]. Salt-stressed root growth is restricted by osmotic effects and toxic effects of ions, which results in
lower nutrient uptake and inhibits the translocation of mineral nutrients, especially K+. As a result of the
similarities in physicochemical properties between Na+ and K+, Na+ could compete with K+ for major
binding sites in key metabolic processes, including both low-affinity (e.g., non-selective cation channels
(NSCC)) and high-affinity (e.g., KUP and high-affinity K+ transporter (HKT)) transporters and could
also disturb plant metabolism [4,78]. K+ deficiency can usually be observed under salinity stress. First,
high levels of Na+ inhibit K+ activity in the soil solution, resulting in a reduction of K+ availability. Second, Na+ not only interferes with K+ translocation from root to shoot (especially in low K+
status) [79], but also competes with K+ for uptake sites at the plasma membrane, resulting in lower K+
uptake. Third, salinity stress leads to plasma membrane dis-integrity and favors K+ leaking, resulting in
a rapid decline in cytosolic K+ [80]. Also, salinity induces significant membrane depolarization and
favors K+ leaking through depolarization-activated outward-rectifying (KOR) K+ channels [78]. Therefore, keeping cellular K+ content above a certain threshold and maintaining a high cytosolic
K+/Na+ ratio (either by retaining K+ or preventing Na+ from accumulating in the leaves) is critical for
plant growth and salt tolerance. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 7378 3.1.4. Stomatal Regulation An adaptive K requirement for
drought-stressed plants could be related to the role of K in enhancing photosynthetic CO2 fixation and
transport of photosynthates into sink organs and inhibiting the transfer of photosynthetic electrons to O2,
thus reducing ROS production [7]. Beside the photosynthetic electron transport, nicotinamide adenine dinucleotide phosphate
(NADPH)-dependent oxidase activation represents another major source for production of ROS in
plant cells by a number of biotic and abiotic stress factors [65]. NADPH-oxidizing enzymes catalyze
one-electron reduction of O2 to O2
•− by using NADPH as an electron donor [7]. Cakmak [7] reported
that activity of NADPH oxidase was increased in cytosolic fractions of bean roots with increasing
severity of K deficiency, resulting in an increase in NADPH-dependent O2
•− generation. The reason for
the increase of NADPH oxidase by K deficiency is probably that K deficiency induced ABA
accumulation [69]. Furthermore, ABA has also been shown to be effective in increasing H2O2 and O2
•−
accumulations in roots or leaves [70,71], but this point needs to be clarified in future studies. An
improvement in the plant K supply can inhibit ROS production under drought stress by reducing
NADPH oxidase activity and maintaining photosynthetic electron transport [7]. In addition to K, various
micronutrients, including Zn, B, Cu and Mn, have also been shown to be involved in detoxifying oxygen
radicals [72]. The K supply is thus associated with other mineral nutrients and is essential for the
detoxification of active oxygen under drought stress. In brief, a sufficient K status increased cell membrane stability, root growth, leaf area and total dry
mass for plants living under drought conditions and also improved water uptake and water conservation. Maintaining an adequate K nutritional status is critical for plant osmotic adjustment and for mitigating
ROS damage as induced by drought stress. In Figure 2, we summarize the role of K in plants that are
living under drought stress. Figure 2. Role of K under drought stress. Figure 2. Role of K under drought stress. 3.2. Potassium and Salt Stress An increasing K supply corresponded with higher K+ accumulation in
plant tissue, which reduced the Na+ concentration and resulted in a higher K+/Na+ ratio. Members of the
HKT transporter (high-affinity K+ transporter) family that mediate Na+-specific transport or Na+-K+
co-transport play a key role in plant Na+ tolerance mechanisms [81,82]. HKT represents a primary
mechanism in the regulation of Na+ and K+ homeostasis, as well as Na+ exclusion [83,84]. Plant growth and salt tolerance were sharply reduced when exposed to a combination of salt stress
and K-deficiency stress. K+ deficiency significantly increased the negative effects that were induced by
salt in the photosynthesis of barley and was accompanied by an increase in salt sensitivity [85]. Similar
results were found by Qu et al. [86,87], in which K+ deficiency significantly inhibited nitrogen and
photosynthetic carbon assimilation and also impaired the light reaction pathways of PS I and PS II in
maize under salt stress. In a study by Chen et al. [88], K+ flux from barley root in response to NaCl
treatment was highly positively correlated with net CO2 assimilation, plant growth, survival rate,
relative grain yield and tolerance to salt stress (Table 4). Int. J. Mol. Sci. 2013, 14 7379 Table 4. Linear correlation (r values) between various physiological characteristics from 62
barley genotypes in glasshouse and laboratory experiments [88]. Table 4. Linear correlation (r values) between various physiological characteristics from 62
barley genotypes in glasshouse and laboratory experiments [88]. Parameter
Grain yield
K+ flux
Shoot
biomass
Survival
rate
Plant
height
[CO2]ass
TSW
Tillering
K+ flux
0.67 **
-
-
-
-
-
-
-
Shoot biomass
0.96 **
0.69 **
-
-
-
-
-
-
Survival rate
0.65 **
0.70 **
0.74 **
-
-
-
-
-
Plant height
0.70 **
0.69 **
0.61 **
0.51 **
-
-
-
-
[CO2]ass
0.68 **
0.69 **
0.65 **
0.48 **
0.50 **
-
-
-
TSW
0.72 **
0.70 **
0.70 **
0.63 **
0.74 **
0.48 **
-
-
Tillering
0.48 **
0.26 *
0.51 **
0.16
0.23
0.25 *
0.33 *
-
Germination
0.29 *
0.21
0.31 *
0.33 **
0.16
0.02
0.38 **
0.20
** p < 0.01; * p < 0.05; TSW: thousand-seed weight; [CO2]ass: CO2 assimilation. Increased evidence has shown that K can involve osmotic adjustment of salt-stressed plants. 3.2. Potassium and Salt Stress During
salt stress conditions, increased Na+ concentrations were accumulated in the vacuole and a substantial
osmotic potential gradient was established between the vacuole and the cytosol by depressing the
cytosol's water activity. This change requires a coordinated increase in compatible solutes in the cytosol
to balance out the osmotic pressure. Munns and Tester [75] reviewed that plants have a Na+ exclusion
mechanism that maintains a low level of Na+ in the leaves during salt stress; thus, the major osmoticum
in leaves was K+. K+ plays an important role in maintaining cell turgor and osmotic adjustment. The
vacuole and the cytosol are the two major pools of K in plant cells. Cytosolic K+ concentrations are
maintained at a constant level and are essential for plant metabolism, while vacuolar K+ concentrations
may vary dramatically. Under K+-deficient conditions, a constant cytosolic K+ concentration was
attributed to the consumption of vacuolar potassium [89]. Low K+ status might induce the formation of ROS and related cell damage under saline conditions,
which was attributed to the effects of K+ deficiency and/or Na+ toxicity on stomatal closing and the
inhibition of photosynthetic activity and ultimately inhibits plant growth and reduces crop production [90]. Previous articles have shown that salinity-induced ROS formation can lead to programmed cell death
(PCD), and a high cytosolic K+/Na+ ratio is essential for triggering salinity-induced PCD [91]. A
decrease in the cytosolic K+ pool would activate caspase-like proteases and lead to PCD under saline
conditions. The ability of plants to satisfy their metabolic requirements for K+ in the presence of salinity
by using higher K+ fluxes and lower Na+ fluxes that result in a higher K+/Na+ selectivity ratio is essential
for salt tolerance. The addition of K+ to a saline culture solution has been found to increase K+
concentrations in plant tissue that corresponds with a decrease in Na+ content, with a further increase in
plant growth and salt tolerance. Increased evidence shows that it is not the absolute quantity of Na+
per se that influences salt resistance, but rather the cytosolic K+/Na+ ratio that determines plant salt
tolerance [11,78]. Figure 3 summarizes the role of K in plants living under salt stress. 7380 Int. J. Mol. Sci. 2013, 14 Figure 3. Role of K under salt stress. Figure 3. Role of K under salt stress. Figure 3. Role of K under salt stress. 3.3. Potassium and Low-Temperature Stress 3.3. 3.2. Potassium and Salt Stress Potassium and Low-Temperature Stress Cold stress inhibits plant growth and development, which results in limited crop productivity. It affects plants by directly inhibiting metabolic reactions and indirectly influencing cold-induced
osmotic, oxidative and other stresses. The effect of increasing K+ applications on yield and cold
tolerance studied by Devi et al. [92] in Panax ginseng showed that a high K+ concentration activated the
plant's antioxidant system and increased levels of ginsenoside-related secondary metabolite transcripts,
which are associated with cold tolerance. Cold stress may destroy photosynthetic processes and reduce
the effectiveness of antioxidant enzymes, resulting in ROS accumulation [66,93,94]. K improved plant
survival under cold stress by increasing antioxidant levels and reducing ROS production [7,92]. Greater frost damage in K-deficient plants is related to water deficiency from the chilling-induced
inhibition of water uptake and freezing-induced cellular dehydration [95]. A significant negative
correlation was found between frost damage and leaf K concentration, and an adequate K supply can
effectively increase frost resistance [6,8]. Bogdevitch [96] found that oats that were supplied with
sufficient K could survive late frost without obvious damage, whereas much of the crop that was grown
on K-deficient soil did not survive. This finding could be attributed to a regulation of osmotic and water
potential and a reduction of electrolyte leakage caused by cold stress [8,97]. High concentrations of K+
protected against freezing by lowering the freezing point of the plant's cell solution. Furthermore, an
adapted cytosol K+ concentration is also essential for enzyme activities that are involved in regulating
frost resistance [8]. Int. J. Mol. Sci. 2013, 14 Int. J. Mol. Sci. 2013, 14 2013, 14 7382 resulting in severe economic penalties in some area. The important biological consequence of
waterlogging is that the respiration of roots and micro-organisms depletes the residual oxygen and the
environment becomes hypoxic (i.e., oxygen levels limit mitochondrial respiration) and, later, anoxic
(i.e., respiration is completely inhibited) [104,105]. The low energy status under oxygen deficient
conditions results in a substantial depolarization of plasma membrane potential [106], subsequent
impairment of ion transport processes through voltage-gated uptake channels and a decrease of the
uptake of most essential cations (e.g., K+, NH4
+ or Mg2+) [107,108]. Pang et al. [109] reported that
hypoxia-induced K+ flux responses are mediated by both inwardly rectifying potassium (KIR) and
NSCC channels in the elongation zone, while KOR channels in the mature zone are likely to play a
critical role. Avoiding K+ loss during hypoxia or anoxia stress is the key mechanism responsible for
waterlogging resistance in plants [109–112]. Furthermore, as flooding time increases, potentially toxic compounds, such as sulfides, soluble iron
and manganese, ethanol, CO2, ethylene, lactic acid, acetaldehyde and acetic and formic acid, were
accumulated as the result of the reduced soil redox potential [106,113]. Those compounds acted on
cellular membranes, leading to phospholipid oxidation and a subsequent change in membrane integrity
and membrane transport [109,114]. Rapid changes in net K+ were measured in response to the
application of secondary metabolites (various monocarboxylic acids and phenolic acids) produced by
waterlogged soils [115]. Shabala [106] assumed that organic acid uptake across the plasma membrane
results in a net H+ influx and causes a substantial membrane depolarization. Such a depolarization will
significantly affect intracellular K+ homeostasis by reducing K+ uptake via KIR, as well as enhancing
K+ efflux via KOR. Waterlogging is known to block the oxygen supply to the roots, thus inhibiting root respiration,
resulting in a severe decline in energy status of root cells, affecting important metabolic processes of
plants. Under waterlogged conditions, the stomata conductance, photosynthesis rate and root hydraulic
conductivity of plant were hampered [116]. The oxidative damage induced by the generation of reactive
oxygen species affects the integrity of membranes and induces damage to the efficiency of photosystem
II, thereby, causing a considerable decrease in net photosynthetic rates [117]. Exogenous application of
K could effectively ameliorate the adverse effects of waterlogging on plants. Int. J. Mol. Sci. 2013, 14 K supplement under
waterlogging not only increased plant growth, photosynthetic pigments and photosynthetic capacity, but
also improved plant nutrient uptake as a result of higher K+, Ca2+, N, Mn2+ and Fe2+ accumulation [118]. Ashraf et al. [118] also reported that exogenous application of K in soil and as foliar spray alleviated the
adverse effects of waterlogging on cotton plants. Int. J. Mol. Sci. 2013, 14 7381 Because the plasma membrane is the primary site for perceiving changes in temperature, membrane
fluidity can be decreased by cold stress as a result of changes in fatty acid unsaturation and the
lipid-protein composition of the cell membrane [98]. The ratio of unsaturated/saturated fatty acids in the
cell membrane was essential for plant cold tolerance, and the higher the ratio in the cell membrane, the
more tolerant the tissue is to cold stress [99]. A decrease in membrane fluidity could further affect the
transport of ions, water and metabolites. The effects of nitrogen and potassium on spikelet sterility
induced by low temperature at the reproductive stage of rice were studied by Haque [100]. The spikelet
sterility induced by low temperature was decreased with the increase of K+ supply and the increase of the
K/N ratio in the rice leaves. Increasing plant frost resistance by the addition of K is associated with the
increase in phospholipids, membrane permeability and improvement in the biophysical and biochemical
properties of cell [101]. In brief, higher K tissue concentrations reduced chilling damage and increased cold resistance,
ultimately increasing yield production [8,21]. Frost damage was inversely related to K concentration and
was significantly reduced by K fertilization. Figure 4 summarizes the role of K in the plant under low
temperature stress. Figure 4. Role of K under cold and frost stress. 3.4. Potassium and Waterlogging Stress
Waterlogging affects approximately 10% of the global land area [102] and is a serious impediment
for sustainable agriculture development. Yield losses due to waterlogging may vary between 15% and
80%, depending on the crop species and growth stage, soil type and duration of the stress [103], Figure 4. Role of K under cold and frost stress. Figure 4. Role of K under cold and frost stress. 3.4. Potassium and Waterlogging Stress Waterlogging affects approximately 10% of the global land area [102] and is a serious impediment
for sustainable agriculture development. Yield losses due to waterlogging may vary between 15% and
80%, depending on the crop species and growth stage, soil type and duration of the stress [103], Waterlogging affects approximately 10% of the global land area [102] and is a serious impediment
for sustainable agriculture development. Yield losses due to waterlogging may vary between 15% and
80%, depending on the crop species and growth stage, soil type and duration of the stress [103], Int. J. Mol. Sci. 4. Implications The population of the world will exceed 9 billion by the year 2050. It is, therefore, of vital
importance to improve crop yield to match the requirement for food. However, as the environment was
becoming worse, the quantity and quality of crop production were significantly decreased by a variety
of biotic and abiotic stresses. The practice of intensive fertilization to support massive food production
for an increasing global population is a must. However, consumption of excess N fertilization and K
deficiency cause a reduction in crop yields and quality in many regions. Therefore, to enable closing
yield gaps and allow for a much higher productivity in many regions, a significant increase in K Int. J. Mol. Sci. 2013, 14 7383 fertilization application is required. K is an essential plant nutrient that impacts a number of
physiological and biochemical processes that are involved in plant resistance to biotic and abiotic
stresses, as summarized in Figure 5. Figure 5. Roles of K in resisting all stresses. Figure 5. Roles of K in resisting all stresses. Figure 5. Roles of K in resisting all stresses. aintaining an optimum K nutritional status is essential for plant resistance to biotic and abiotic
es. Balanced fertilization and efficient K usage in combination with other nutrients not only
ibute to sustainable crop’s growth, yield and quality, but also influence plant health and reduce
nvironmental risks. However, our understanding about the role of K in whole-plant stress response ing an optimum K nutritional status is essential for plant resistance to biotic and abio Maintaining an optimum K nutritional status is essential for plant resistance to biotic and abiotic
stresses. Balanced fertilization and efficient K usage in combination with other nutrients not only
contribute to sustainable crop’s growth, yield and quality, but also influence plant health and reduce
the environmental risks. However, our understanding about the role of K in whole-plant stress response
mechanisms is limited. In this paper, suggested future needs and prospects for research about the role of
K in agriculture include: 1. Investigating more details about the molecular mechanisms of K in plant stress re 2. Examining the role of K on plant resistance to biotic and abiotic stresses in differentiated cells,
tissues and organs and connecting the data relevantly. 3. 4. Implications Identifying the common or specific response of K to distinct stress and the role of K on
long-term plant responses under multiple stress conditions in nature. 4. Understanding the relationship between K and other nutrients in relation to plant adaptation to
stresses in different agroecological systems. eloping models for better K recommendations based on soil, plant and environmental facto 6. Investigating more researcher on the importance of K on crop production, nutritional quality
and human and animal health. This work was financially supported by the National Basic Research Program of China
(2013CB127403) and the Nature Science Foundation of China (31172020, 31272236). Acknowledgements This work was financially supported by the National Basic Research Program of China
(2013CB127403) and the Nature Science Foundation of China (31172020, 31272236). Int. J. Mol. Sci. 2013, 14 7384 References 1. United States Census Bureau. International Data Base—Total Midyear Population for the World:
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Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene
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Aya BridgelandID☯, Sudip BiswasID☯, Nikolaos Tsakirpaloglou, Michael J. Thomson,
Endang M. SeptiningsihID* Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, United States of America Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, United States of America a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ☯These authors contributed equally to this work. * eseptiningsih@tamu.edu PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Bridgeland A, Biswas S, Tsakirpaloglou N,
Thomson MJ, Septiningsih EM (2023)
Optimization of gene editing in cowpea through
protoplast transformation and agroinfiltration by
targeting the phytoene desaturase gene. PLoS ONE
18(4): e0283837. https://doi.org/10.1371/journal. pone.0283837 Citation: Bridgeland A, Biswas S, Tsakirpaloglou N,
Thomson MJ, Septiningsih EM (2023)
Optimization of gene editing in cowpea through
protoplast transformation and agroinfiltration by
targeting the phytoene desaturase gene. PLoS ONE
18(4): e0283837. https://doi.org/10.1371/journal. pone.0283837 Editor: Valentine Otang Ntui, International Institute
of Tropical Agriculture, KENYA
Received: January 25, 2023
Accepted: March 19, 2023
Published: April 5, 2023
Copyright: © 2023 Bridgeland et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Valentine Otang Ntui, International Institute
of Tropical Agriculture, KENYA Copyright: © 2023 Bridgeland et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This research was supported by the
USDA National Institute of Food and Agriculture
project # 2020-67013-31811 (to EMS and MJT)
and the Texas A&M AgriLife Research (to EMS and
MJT). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. Abstract Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa
and other tropical and sub-tropical regions. Considering projected climate change and
global population increases, cowpea’s adaptation to hot climates, resistance to drought, and
nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due
to its recalcitrance to transformation and long regeneration times. Transient gene expres-
sion assays can provide solutions to alleviate these issues as they allow researchers to test
gene editing constructs before investing in the time and resource- intensive process of
transformation. In this study, we developed an improved cowpea protoplast isolation proto-
col, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing
and validation of gene editing constructs and for gene expression studies. To test these pro-
tocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed
single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transfor-
mation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger
sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves
revealed several large deletions in the target sequences. The protoplast system and agroin-
filtration protocol developed in this study provide versatile tools to test gene editing compo-
nents before initiating plant transformation, thus improving the chance of using active
sgRNAs and attaining the desired edits and target phenotype. Optimization of gene editing in cowpea
through protoplast transformation and
agroinfiltration by targeting the phytoene
desaturase gene Aya BridgelandID☯, Sudip BiswasID☯, Nikolaos Tsakirpaloglou, Michael J. Thomson,
Endang M. SeptiningsihID* Introduction Since the advent of CRISPR-Cas9 technology, genome editing techniques and protocols have
become largely optimized for major crops such as rice, wheat, and maize [1–4]. However,
there remains a gap in optimization for most other crop species, including cowpea. Cowpea
(Vigna unguiculata), is a high-protein, nutritionally dense annual legume species that is grown 1 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Competing interests: The authors have declared
that no competing interests exist. as a staple crop across semi-arid regions of Africa, Asia, and South America [5–7]. Although
cowpea has previously been considered an orphan crop due to a lack of scientific attention, its
superior traits, such as drought tolerance, nitrogen-fixing capabilities, and ability to grow in
marginal soils, have brought it newfound consideration as an archetype of climate-resilience
and sustainability [5, 7]. Despite its many advantages as a crop against abiotic stresses, cowpea
still suffers from low productivity and large yield gaps due to biotic susceptibility to pests, espe-
cially insects [8–10]. Historically, cowpea varietal improvement has been challenging due to a
low-diversity breeding population and sexual incompatibility with wild relatives [8, 11–13]. The recent release of a Maruca pod borer-resistant variety, developed via the transgenesis of a
Bacillus thuringiensis gene with an Agrobacterium vector, shows that a biotech-based approach
can successfully drive crop improvement in ways that were not possible using conventional
means [14]. Nonetheless, only limited CRISPR-Cas9 genome editing applications have been
established in cowpea thus far, based on hairy roots and embryonic axis explant transforma-
tion [15–17]. Competing interests: The authors have declared
that no competing interests exist. Like other legumes, cowpea is notoriously recalcitrant to transformation, which proves to
be a major bottleneck in crop improvement. Even in successful instances, transformation fre-
quency is low, and the long regeneration times make attempting a transformation experiment
a high-investment activity [18, 19]. For these reasons, a major priority of cowpea research is to
improve these bottleneck areas, and one of the examples is by developing transient gene
expression assays for the rapid testing of gene editing constructs. In this regard, protoplast
transformation and leaf infiltration were used as transient assay platforms in this study. Proto-
plasts are often used as a single-guide RNA (sgRNA) activity validation step, as it allows the
transformation and sequencing of live cells with a quick turnaround time. Plant material Cowpea seeds of variety IT97K-499-35 were used in this study [8]. Seeds were multiplied in
the greehouse facility at Texas A&M University (College Station, TX). Plants were grown in
pots containing soils with day/night temperatures of 32/26˚C and a 16/8 h light/dark cycle. Introduction Using protoplasts,
any number of factors, including promoter type, plasmid size, and sgRNA activity, can be
tested, increasing the odds of success in actual transformation events [20, 21]. Protoplasts have
been used for the testing of CRISPR/Cas9 constructs and sgRNA activity in a diverse range of
species, including legumes such as peanut and chickpea, but a protocol has not yet been devel-
oped for cowpea [22–24]. Likewise, Agroinfiltration of leaves has also been used with the
CRISPR-Cas9 system for sgRNA validation and gene expression assays [25–27]. The current
study aims to knock out the cowpea phytoene desaturase (PDS) gene using a CRISPR-Cas9
construct designed with four multiplexed sgRNAs through PEG transformation in cowpea
protoplasts and agroinfiltration in cowpea leaves. Competing interests: The authors have declared
that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 Protoplast isolation by the leaf-cutting method and tape sandwich method The leaf-cutting method was tested following the protocol by Wu and Hanzawa et al. 2018 [28]
with the following modifications (S1 Fig, upper panel). Primary leaves from 6-day-old cowpea
plants were cut from seedlings, sliced into ~0.5 mm strips after removing the midrib, and then
placed in the enzyme solution (S1 Table). Two leaves were used per 10 ml of enzyme solution. The strips were vacuum infiltrated for 15 min and incubated under low-light conditions for
5.5 h with gentle shaking (45 rpm). The tape sandwich method was tested by following the Wu et al. 2009 protocol [29] with
the following modifications (S1 Fig, lower panel). The upper epidermal surface of a 7-day/ 2 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea 12-day-old cowpea primary leaf was fixed to a piece of Fisherbrand Colored Labeling Tape. A
piece of 3 M Magic Tape (standard clear office tape) was gently pressed to the lower epidermal
surface, then peeled off to remove the lower epidermal surface layer. The leaves (with the Fish-
erbrand tape still attached) were transferred to a beaker with enzyme solution (S1 Table) to a
ratio of 10 ml solution to 1 g leaf tissue. Leaves were vacuum infiltrated for 10 min and then
shaken at 45 rpm for 1.5 h. After this initial incubation, the enzyme solution was poured off,
and fresh enzyme solution was added to the leaves. The incubation was then continued for an
additional 3 h at 45 rpm. After the incubation for both methods, the enzyme solution was poured into a 50 ml Falcon
tube, and an equivalent amount of W5 solution (S1 Table) was added to stop the digestion. The solution was poured through a 40 μM nylon mesh to remove large pieces of debris, then
centrifuged at 100 x g for 2 min at room temperature. The supernatant was removed, and each
tube was resuspended with an equivalent amount of W5 solution. Protoplasts were counted on a hemocytometer. The number of protoplasts in 4 squares of
the hemocytometer grid were counted to calculate protoplast concentration. The protoplast
density was then calculated as follows: protoplast number (per gram of tissue) = the average
count of protoplast per square × 104. PEG-mediated protoplast transfection Following the successful isolation of protoplasts, the scaled-up protoplast transfection method
was performed as adapted from the protocols of Wu and Hanzawa et al. 2018 and Li et al. 2011
[28, 30]. The 15 ml Falcon tubes were coated with 5% (w/v) fetal bovine serum, then spun
down at maximum speed for 1 min, and the excess serum was removed. Next, 60 μg of plasmid
DNA (pDNA) was added to each tube while keeping one tube as a negative control with no
pDNA added. A total of 400 μl of protoplasts with a concentration of 2.5 x 105 protoplasts/ml
were added to the tube and pipetted gently to mix with the pDNA. Subsequently, 460 μl of
freshly prepared 40% PEG-4000 solution (S1 Table) was added to each tube and pipetted
gently to mix. The tube was incubated at room temperature for 30 min. Finally, 3 ml of W5
solution was added to stop the transformation, and the tubes were centrifuged at 250 x g for 1
min at room temperature. The supernatant was removed without disturbing the protoplast
pellet. Protoplasts were then resuspended with 200 μl of W1 solution (S1 Table). Tubes were
covered in aluminum foil and kept at 4˚C for 72 h until DNA was extracted. Plasmid preparation and constructs Golden Gate Cloning was used to develop the VgPDS-targeting plasmid encoding the Cas9
enzyme and sgRNA cassette following the publication by Čerma´k, et al. 2017 [34]. For these,
three intermediate module plasmids A, B, and C were prepared for the construction of the
CRISPR-Cas9 vector. For module A, pMOD_A0101 (Addgene plasmid #90998) was used
where AtCas9 was expressed (Arabidopsis-codon optimized SpCas9) by a Cauliflower Mosaic
Virus 35S (CaMV 35S) promoter. The pMOD_B2303 vector was used for module B. The poly-
cistronic csy4 system [35] was used for the expression of multiple sgRNAs and the cassette
containing the four sgRNA sequences for VgPDS was synthesized (Macrogen) and placed in a
pUC57 intermediate vector (Genscript Biotech Ltd., Piscataway, NJ, USA), then was digested
in-house using restriction enzymes (PstI and XhoI) and placed in the pMOD_B2303 vector
using T4 Ligase (NEB) following the manufacturer’s recommendations. Expression of the
sgRNA cassette was driven by the CmYLCV (Cestrum Yellow Leaf Curling Virus) promoter. The pMOD_A0101, modified pMOD_B2303, and empty vector pMOD_C0000 (Addgene
#91081) were assembled into a non-binary vector, pTRANS_100 (Addgene #91198) and
binary vector pTRANS_210 (Addgene plasmid # 91108) by Golden Gate protocol using the
AarI enzyme. Two vectors were developed: a binary vector (pTRANS_210-VgPDS) to be used
for Agrobacterium transformations and a non-binary vector (pTRANS_100-VgPDS) to be
used for protoplast transformations. sgRNA design for VgPDS and in vitro ribonucleoprotein assay Single-guide RNAs (sgRNAs) from the outputs of both CRISPR-P 2.0 and CRISPRdirect
sgRNA design tools [31, 32] were selected to target the VgPDS gene. The DNA sequence of the
cowpea PDS gene was obtained from the Legume Information System (LIS) database (www. legumeinfo.org) and was used as the input in the programs for the sgRNA design. Four
sgRNAs (sgRNA1: CCGGCAATAACGACCTTCAACGG, sgRNA 2: CTTCAGTTCGTGCTTCT
AAGAGG, sgRNA 3: GAAGCTAGAGACGTTCTAGGTGG, and sgRNA 4: ATATGTGTCTGG
CGCCAAGCTGG) were designed from the outputs of the two programs and synthesized at
Synthego, Inc. Genomic DNA (gDNA) was extracted from young cowpea using the SDS method, follow-
ing a Cetyl Trimethyl Ammonium Bromide (CTAB) protocol based on the publications by
Doyle and Doyle 1987 [33]. The region of the PDS gene containing the sgRNA sequences was
genotyped (primers used listed in S2 Table) and amplicons were purified using the Qiagen
QIAquick Gel Extraction Kit (Hilden, Germany). TOPO-cloning followed by Sanger sequenc-
ing (Eurofins Genomics LLC, Louisville, KY) confirmed that the sgRNA target sequence in 3 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea our cultivar was identical to the one obtained from the LIS database. Additionally, purified
PCR fragments were used for an in vitro ribonucleoprotein assay to validate the designed
sgRNAs following the protocol “In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes
(M0386)” from New England Biolabs (Ipswich, MA, USA) with a few modifications. In this
case, a 27 μL reaction mixture containing 30 nM of synthesized sgRNA, 30 nM of Cas9 nucle-
ase, and 3 μL of 10× NEB buffer 3.1 were pre-incubated for 10 min at 25˚C. Afterward, 100 ng
substrate purified PCR product was added to make a total reaction volume of 30 μl and incu-
bated at 37˚C for 1 h. After adding 1 μl of proteinase K, the reaction mixture was kept for 10
min at 56˚C, and fragment analysis was then performed using a 2% (g/v) gel electrophoresis. Mutation analysis for the protoplast assay At four days post-transfection in dark conditions, the cowpea protoplasts were collected by
centrifugation at 13,000 rpm, and genomic DNA was then extracted with the CTAB protocol
[33]. The Cas9–sgRNAs target sites of DNA segments were amplified with Phusion polymerase
using pairs of allele-specific primers (S2 Table). Gradient PCR was performed with an initial
denaturation step of 98˚C for 30 s, followed by 32 cycles of 98˚C for 30 s, 55˚C– 58˚C for 30 s,
and 72˚C for 30 s, and a final extension of 72˚C for 7 min. The PCR product was then purified
by gel extraction and cloned into Topo vector (Thermofiser Scientific, Waltham, MA). Any
mutations in the VgPDS target site were characterized by sanger sequencing. Agroinfiltration assay Binary vectors (CmYLCVpromoter:GUS, 35spromoter:GUS vector and pTRANS_210-VgPDS)
were transferred into A. tumefaciens strains EHA105 and AGL-1 via the freeze-thaw transfor-
mation method [36] A single colony of Agrobacterium strains containing the desired plasmid
was inoculated into liquid LB media with specific antibiotics (Rifampicin was used for
EHA105, rifampicin and carbenicillin for AGL-1 agrobacterium strain selection, and kanamy-
cin was used for all vector selection). Cultures were put in a shaker incubator overnight at
28˚C at 250 rpm. Bacteria were pelleted by centrifugation (4,000 g for 30 min) and resuspended 4 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea to an OD600 = 1.0 in bacterial resuspension medium (1/2 MS, 3% sucrose, 10 mM MgCl2,
200 μM acetosyringone, pH 5.8). Bacterial suspensions were delivered into the underside of
leaves of 7- day-old plantlets using a blunt-tipped 1 ml plastic syringe by applying gentle pres-
sure. The infiltrated leaves were harvested three days later. GUS assay was performed in cow-
pea leaves that were infiltrated with A. tumefaciens strains EHA105 and AGL-1 containing
CmYLCVpromoter:GUS and 35spromoter:GUS vector [37]. Genomic DNA was isolated from the
leaves infiltrated with A. tumefaciens strain EHA105 containing binary vector (pTRANS_210-
VgPDS), and any mutations in the VgPDS target site were characterized by PCR and sanger
sequencing. High-efficiency protoplast isolation by the tape sandwich method Optimizing the exposure of mesophyll surface area is critical to obtain high numbers of proto-
plasts from young leaves. Our results showed that the tape method was more efficient than the
leaf-cutting method for preparing leaves for the enzyme solution (Table 1, Fig 1). The leaf-cut-
ting method isolated approximately 175,000 protoplasts per gram of starting tissue (proto-
plasts/g), with the total time to isolation taking approximately 7 h, while the tape sandwich
method isolated 200,000 protoplasts/g in 3 h (Table 1 and Fig 1). The time to isolation also
included the front-end work, such as making enzyme solution and preparing the leaves, as
well as post-enzyme incubation steps, such as counting protoplasts on the hemocytometer and
the 30 min incubation on ice. Different incubation conditions and different ages of plants were tested to optimize the tape
sandwich method. The incubation conditions of the Hibi et al. 1975 method [38] added an
extra 30 min preliminary incubation step and two additional hours relative to the unmodified
tape sandwich method (Table 1). Protoplast yield was significantly higher when using the lon-
ger, two-stage incubation, yielding approximately 1,675,000 protoplasts/g compared to
200,000 protoplasts/g from the shorter incubation. On the other hand, protoplasts isolated
from 12-day-old plants yielded 7,550,000 protoplasts/g, significantly higher than 6-7-day-old
plants. In addition, it was observed that the enzyme/protoplast solution from 12-day-old plants
had much less debris than that from 6-7-day-old plants (Table 1). Hence, the optimized tape
sandwich method was highly consistent, and each application of this methodology in subse-
quent trials produced large numbers of protoplasts, sufficient for dozens of transformations. g
We also tested the activity of the two constitutive promoters, 35S and CmYLCV, in cowpea
protoplasts. Transformation efficiency was calculated as follows: (the number of protoplasts
expressing GFP/total number of protoplasts) × 100. We found that protoplasts transformed
with 35S:GFP gave slightly higher transformation efficiency than CmYLCV:GFP based on the
number of GFP expressed protoplasts (Fig 2). Temperature, PEG and plasmid concentration
play a crucial role in protoplast transformation. In this study, we found the highest transforma-
tion efficiency at 40% PEG, 60 μg plasmid concentration and 13˚C for 24 h. Table 1. Comparison of protoplast isolation methods. High-efficiency protoplast isolation by the tape sandwich method Method Description
Tissue (days)
Time (h)
Average Yield (protoplasts/g)
Debris
Leaf cutting
6–7
7
~175,000
Medium
Tape sandwich
6–7
3
~200,000
Low
Optimized tape sandwich
6–7
5
~1,675,000
Low
Optimized tape sandwich
10–12
5
~7,550,000
Very Low
https://doi.org/10.1371/journal.pone.0283837.t001
OS O
|
//
/
/
/ Table 1. Comparison of protoplast isolation methods. Method Description
Tissue (days)
Time (h)
Average Yield (protoplasts/g)
Debris
Leaf cutting
6–7
7
~175,000
Medium
Tape sandwich
6–7
3
~200,000
Low
Optimized tape sandwich
6–7
5
~1,675,000
Low
Optimized tape sandwich
10–12
5
~7,550,000
Very Low
htt
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/10 1371/j
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0283837 t001 Table 1. Comparison of protoplast isolation methods. 5 / 14 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Fig 1. Comparison of protoplast isolation from leaf cutting method versus tape sandwich methods. (A)
Protoplasts isolated via the original leaf-cutting method. (B) Protoplasts isolated via the tape sandwich method from
7-day-old plants without optimization. (C) Protoplasts isolated via the tape-sandwich method from 7-day-old plants
with optimization. (D) Protoplasts isolated via the tape-sandwich method from 12-day-old plants with optimization. https://doi.org/10.1371/journal.pone.0283837.g001 Fig 1. Comparison of protoplast isolation from leaf cutting method versus tape sandwich methods. (A)
Protoplasts isolated via the original leaf-cutting method. (B) Protoplasts isolated via the tape sandwich method from
7-day-old plants without optimization. (C) Protoplasts isolated via the tape-sandwich method from 7-day-old plants
with optimization. (D) Protoplasts isolated via the tape-sandwich method from 12-day-old plants with optimization. h
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/10 13 1/j
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028383
001 In Vitro digestion of VgPDS targets with sgRNA and Cas9 In vitro ribonucleoprotein (RNP) assay for the four sgRNAs targeting a PCR amplicon flanking
the target site of the cowpea PDS gene was performed using the RNP complexes with purified
Cas9 (Invitrogen, Waltham, MA, USA) and synthetic gRNAs (Synthego, Redwood City, CA,
USA). The negative controls had uncut PCR products, while three bands were seen for the cut
amplicon with sgRNA1, sgRNA2, sgRNA3 and sgRNA4, indicating that all sgRNAs efficiently
cut their target nucleotide sequences in the VgPDS gene targets (Fig 3). We conducted agroinfiltration experiments using unifoliate leaves of greenhouse grown 7-
10-day-old cowpea plants under laboratory conditions. Our results showed that the transient
expression of the GUS reporter worked well under these conditions (Figs 4 and 5). We tested
the expression of a GUS reporter under the 35S and CmYLCV promoter separately. Two dif-
ferent Agrobacterium strains (EHA105 and AGL-1) were used in this study. Our results
showed that when using the 35S promoter, the EHA105 strain produced higher GUS expres-
sion than the AGL-1 strain (Fig 4). However, the CmYCLV promoter overall gave higher GUS
expression than the 35S promoter for both Agrobacterium strains (Fig 5). Editing of the VgPDS gene in cowpea protoplasts To test the gene-editing efficacy of the CRISPR-Cas9 vector for cowpea VgPDS, Cowpea proto-
plasts were transformed with CRISPR-Cas9_VgPDS_sgRNAs vector by our optimized PEG 6 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Fig 2. GFP expression under CmYLCV and 35S promoter in cowpea protoplasts. (A) CmYLCVpromoter:GFP and 35Spromoter:GFP
vectors used in this study. (B) Control protoplasts (no GFP plasmid). (C) Protoplasts with GFP expression under CmYLCV promoter. (D) protoplast with GFP expression under 35S promoter. (E) The transformation efficiency (TE) of protoplasts transformed with
CmYLCV:GFP and 35S:GFP plasmid. The protoplasts TE was evaluated after incubation in 40% PEG solution. Values represent
means ± SE (n = 7). Fig 2. GFP expression under CmYLCV and 35S promoter in cowpea protoplasts. (A) CmYLCVpromoter:GFP and 35Spromoter:GFP
vectors used in this study. (B) Control protoplasts (no GFP plasmid). (C) Protoplasts with GFP expression under CmYLCV promoter. (D) protoplast with GFP expression under 35S promoter. (E) The transformation efficiency (TE) of protoplasts transformed with
CmYLCV:GFP and 35S:GFP plasmid. The protoplasts TE was evaluated after incubation in 40% PEG solution. Values represent
means ± SE (n = 7). https://doi.org/10.1371/journal.pone.0283837.g002 https://doi.org/10.1371/journal.pone.0283837.g002 https://doi.org/10.1371/journal.pone.0283837.g002 mediated transformation protocol. Genomic DNA was extracted to amplify the DNA fragment
containing the target site. Sanger sequencing of targeted PCR products obtained from the iso-
lated genomic DNA of each protoplast pool was used to detect the editing efficiency and pat-
terns. The sequencing results revealed various large deletion mutations which covered
sgRNA2, sgRNA3 and sgRNA4. We identified 672 bp deletions in three samples (P1, P3 and
P4) and 681 bp deletions in two samples (P2 and P5) (Fig 6). However, we did not find any
mutation in the sgRNA1 region. Editing of the VgPDS Gene in cowpea leaves by agroinfiltration We also tested the editing efficiency of CRISPR-Cas9 vector for cowpea VgPDS by agroinfiltra-
tion. After seven days of agroinfiltration with Agrobacterium strain EHA105 containing
CRISPR-Cas9_VgPDSsgRNA, we isolated the DNA from the infiltrated cowpea leaves. Any
mutations of the target VgPDS sequencing were detected by sequencing. Similar to the proto-
plast editing results, we also identified large deletions in the VgPDS sgRNA regions. We found
669 bp deletions in three samples (S1, S2 and S5), 530 bp deletion in one sample (S3), and 530
bp deletion and 1 bp substitutions in one sample (S4) (Fig 7). Discussion The protoplast isolation protocol developed through this study combines simple procedures,
high yield, reproducible results, and a moderate experimental run time. Through this study,
we have demonstrated that the success of cowpea protoplast isolation mainly depends on the
method to expose the mesophyll area, the age of plant tissue, and the incubation conditions. Specifically, results indicate that the combination of complete removal of the lower epider-
mis,10-12-day-old plants and specific incubation times are crucial factors in establishing a 7 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Fig 3. Details of the cowpea phytoene desaturase (PDS) gene. (A) Schematic representation of cowpea VgPDS gene. (B) Target
region of PDS gene where four sgRNAs were designed. (C) Schematic diagram representation of cys4–sgRNAs cassette targeting the
VgPDS gene. (D) In vitro digestion of VgPDS. Lanes L1 and L6: 1 kb Plus ladder, L2: uncut VgPDS target region, L3: VgPDS target
region digested with Cas9 and sgRNA1 (expected bands of 753 bp and 218 bp), L4: uncut VgPDS target region, L5: VgPDS target
region digested with Cas9 and sgRNA2 (expected bands of 648 bp and 130 bp), L7: uncut VgPDS target region, L8: VgPDS target
region digested with Cas9 and sgRNA3 (expected bands of 741 bp and 230 bp), L9: uncut VgPDS target region, L10: VgPDS target
region digested with Cas9 and sgRNA4 (expected bands of 524 bp and 254 bp) (partially designed with BioRender.com, accessed on
10 December 2022). Original gel images are provided as S1 Raw images. https://doi.org/10.1371/journal.pone.0283837.g003 Fig 3. Details of the cowpea phytoene desaturase (PDS) gene. (A) Schematic representation of cowpea VgPDS gene. (B) Target
region of PDS gene where four sgRNAs were designed. (C) Schematic diagram representation of cys4–sgRNAs cassette targeting the
VgPDS gene. (D) In vitro digestion of VgPDS. Discussion Lanes L1 and L6: 1 kb Plus ladder, L2: uncut VgPDS target region, L3: VgPDS target
region digested with Cas9 and sgRNA1 (expected bands of 753 bp and 218 bp), L4: uncut VgPDS target region, L5: VgPDS target
region digested with Cas9 and sgRNA2 (expected bands of 648 bp and 130 bp), L7: uncut VgPDS target region, L8: VgPDS target
region digested with Cas9 and sgRNA3 (expected bands of 741 bp and 230 bp), L9: uncut VgPDS target region, L10: VgPDS target
region digested with Cas9 and sgRNA4 (expected bands of 524 bp and 254 bp) (partially designed with BioRender.com, accessed on
10 December 2022). Original gel images are provided as S1 Raw images. https://doi.org/10.1371/journal.pone.0283837.g003 method that is both high-yielding and performs consistently well. Likely the most crucial
parameter in the optimization process was testing how to remove the lower epidermis of the
primary leaves without causing damage. Maximizing the mesophyll surface area in contact
with the enzyme solution has been previously established to achieve both high yield and a
shorter incubation time in Arabidopsis [29]. Adapting the Arabidopsis-optimized tape method
for cowpea overcomes major hurdles to achieving isolation efficiency as protoplast yields
increase dramatically compared to the more commonly used leaf strip method [39]. The selection of plant tissue at the appropriate growth stage has also been previously
reported as an important factor in protoplast isolation in multiple species [23, 28, 38, 40]. Results testing different plant ages showed that 10-12-day-old cowpea leaves produce a greater,
more consistent yield and less debris than 6-7-day-old leaves. The 10-12-day-old leaves are
neither floppy nor stiff, and the lower epidermis peels very easily with one pass of tape, reduc-
ing the possibility of tissue damage. Very young leaves are commonly recommended for proto-
plast isolations when using the leaf-cutting method [23, 41]. However, trials in this study
found that leaves younger than six days old are not compatible with the use of the tape method. Younger leaves are too floppy when working with tape, and it is difficult to remove the lower
epidermis without damaging the leaf. As avoiding mesophyll damage is a specific priority of
the tape method, leaves younger than six days old are unsuitable for this method. PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 Discussion Furthermore,
previous publication in cowpea has reported that plants over 12 days old are not recom-
mended for use in protoplast work, as older protoplasts have low survival rates during 8 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Fig 4. Transient GUS expression via agroinfiltration with 35Spromoter:GUS construct in cowpea leaves. (A) 35Spromoter:
GUS construct used in this study. (B) Control leaves (infiltration with Agrobacterium strain EHA105 without vector). (C)
Cowpea leaves infiltration with Agrobacterium strain EHA105 containing 35Spromoter:GUS vector. (D) Cowpea leaves
infiltration with Agrobacterium strain AGL-1 containing 35Spromoter:GUS vector. Fig 4. Transient GUS expression via agroinfiltration with 35Spromoter:GUS construct in cowpea leaves. (A) 35Spromoter:
GUS construct used in this study. (B) Control leaves (infiltration with Agrobacterium strain EHA105 without vector). (C)
Cowpea leaves infiltration with Agrobacterium strain EHA105 containing 35Spromoter:GUS vector. (D) Cowpea leaves
infiltration with Agrobacterium strain AGL-1 containing 35Spromoter:GUS vector. https://doi.org/10.1371/journal.pone.0283837.g004 https://doi.org/10.1371/journal.pone.0283837.g004 Fig 5. Transient GUS expression via agroinfiltration with CmYLCVpromoter:GUS vector in cowpea leaves. (A)
CmYLCVpromoter:GUS construct used in this study. (B) Control leaves (infiltration with Agrobacterium strain AGL-1
without vector). (C) Cowpea leaves infiltration with Agrobacterium strain EHA105 containing CmYCLVpromoter:GUS
vector. (D) Cowpea leaves infiltration with Agrobacterium strain AGL-1 containing CmYLCVpromoter:GUS vector. Fig 5. Transient GUS expression via agroinfiltration with CmYLCVpromoter:GUS vector in cowpea leaves. (A)
CmYLCVpromoter:GUS construct used in this study. (B) Control leaves (infiltration with Agrobacterium strain AGL-1
without vector). (C) Cowpea leaves infiltration with Agrobacterium strain EHA105 containing CmYCLVpromoter:GUS
vector. (D) Cowpea leaves infiltration with Agrobacterium strain AGL-1 containing CmYLCVpromoter:GUS vector. https://doi.org/10.1371/journal.pone.0283837.g005 9 / 14 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 PLOS ONE Protoplast transformation and agroinfiltration in cowpea Fig 6. Sanger sequencing results of the VgPDS gene derived from cowpea protoplast after PEG mediated transformation. sgRNA 1, sgRNA 2, sgRNA 3
and sgRNA 4 positions are indicated. PAM sequences are highlighted in red letter. WT indicates wild type that was not edited. Dash lines (—) are the deleted
nucleotides. Fig 6. Sanger sequencing results of the VgPDS gene derived from cowpea protoplast after PEG mediated transformation. sgRNA 1, sgRNA 2, sgRNA 3
and sgRNA 4 positions are indicated. PAM sequences are highlighted in red letter. WT indicates wild type that was not edited. Dash lines (—) are the deleted
nucleotides. https://doi.org/10.1371/journal.pone.0283837.g006 https://doi.org/10.1371/journal.pone.0283837.g006 subsequent steps of the procedure [38]. Acknowledgments We thank Dr. B.B. Singh for providing the cowpea seeds used in this study. We thank Dr. B.B. Singh for providing the cowpea seeds used in this study. Author Contributions Conceptualization: Aya Bridgeland, Sudip Biswas, Nikolaos Tsakirpaloglou, Michael J. Thomson, Endang M. Septiningsih. Data curation: Aya Bridgeland, Sudip Biswas, Endang M. Septiningsih. Formal analysis: Aya Bridgeland, Sudip Biswas, Nikolaos Tsakirpaloglou, Endang M. Septiningsih. Funding acquisition: Michael J. Thomson, Endang M. Septiningsih. Investigation: Aya Bridgeland, Sudip Biswas. Methodology: Aya Bridgeland, Sudip Biswas, Nikolaos Tsakirpaloglou, Endang M. Septiningsih. Project administration: Endang M. Septiningsih. Project administration: Endang M. Septiningsih. Resources: Michael J. Thomson, Endang M. Septiningsih. Software: Aya Bridgeland, Sudip Biswas, Nikolaos Tsakirpaloglou. Supervision: Endang M. Septiningsih. Supervision: Endang M. Septiningsih. Validation: Aya Bridgeland, Sudip Biswas, Endang M. Septiningsih. Visualization: Aya Bridgeland, Sudip Biswas, Endang M. Septiningsih. Writing – original draft: Aya Bridgeland, Sudip Biswas. Writing – review & editing: Nikolaos Tsakirpaloglou, Michael J. Thomson, Endang M. Septiningsih. Discussion From this information, it can be concluded that there
is a limited window in which protoplast isolation conditions are optimal in a cowpea plant
when using the tape sandwich method. For the agroinfiltration experiments, the virulence of Agrobacterium strains varies from
plant to plant, which might be due to the ability of the Agrobacterium to attach to the plant
cells or T-DNA transfer mechanism [42, 43]. In this study, we found that EHA105-treated
cowpea leaves showed higher GUS expression than the AGL-1 strain with the 35S promoter,
while the CmYLCV promoter showed high levels of GUS expression with both Agrobacterium
strains. We chose the 35S promoter for expression of Cas9 and used Cestrum Yellow Leaf
Curling Virus promoter (CmYLCV) for gRNA expression to prevent the use of duplicate pro-
moters. The CmYLCV promoter drives comparable or higher levels of expression than the 35S
or maize (Zea mays) ubiquitin (ZmUbi) promoters in both dicots and monocots [44]. In this
study, we also used Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) for the expres-
sion of four gRNA simultaneously. It was reported that both Csy4 and tRNA expression sys-
tems are almost twice more effective than individual RNA polymerase III promoters systems
in gRNAs expression and creating mutation, although the Csy4 expression system performed
best regardless of the position of the gRNA in the array [34]. Finally, we tested our CRISPR-Cas9 vector containing designed sgRNAs in protoplasts by
PEG mediated transformation and in cowpea leaves through agroinfiltration. In both methods,
we found large deletions which covered all sgRNAs except sgRNA1. In summary, we have suc-
cessfully established efficient protoplast transformation and agroinfiltration in cowpea as a Fig 7. Sanger sequencing results of PDS gene derived from agroinfiltrated cowpea leaves. The sgRNA1, sgRNA2, sgRNA3 and sgRNA4 positions are
indicated. PAM sequences are highlighted in red letter. WT indicates wild type that was not edited. Dash lines (—) are the deleted nucleotides and one
substitution bp shows as a green letter in S4. https://doi.org/10.1371/journal.pone.0283837.g007 Fig 7. Sanger sequencing results of PDS gene derived from agroinfiltrated cowpea leaves. The sgRNA1, sgRNA2, sgRNA3 and sgRNA4 positions are
indicated. PAM sequences are highlighted in red letter. WT indicates wild type that was not edited. Dash lines (—) are the deleted nucleotides and one
substitution bp shows as a green letter in S4. Discussion https://doi.org/10.1371/journal.pone.0283837.g007 https://doi.org/10.1371/journal.pone.0283837.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0283837
April 5, 2023 10 / 14 PLOS ONE Protoplast transformation and agroinfiltration in cowpea testbed for optimizing genome editing using the CRISPR-Cas9 system by targeting the PDS
gene. Supporting information
S1 Fig. Schematic diagram of protoplast isolation for the leaf-cutting method (upper
panel) and the tape sandwich method (lower panel). (PDF)
S1 Table. Solutions used for protoplast isolation and transfection. (PDF)
S2 Table. Primers used to amplify the cowpea PDS gene. (PDF)
S1 Raw images. (PDF) testbed for optimizing genome editing using the CRISPR-Cas9 system by targeting the PDS
gene. testbed for optimizing genome editing using the CRISPR-Cas9 system by targeting the PDS
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https://openalex.org/W3133884082
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https://digital.csic.es/bitstream/10261/254546/1/Materials%20Science.pdf
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English
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Hyaluronic acid (HA)-coated naproxen-nanoparticles selectively target breast cancer stem cells through COX-independent pathways
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Materials science & engineering. C, Biomimetic materials, sensors and systems
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cc-by
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A B S T R A C T Keywords:
Breast cancer
Cancer stem cells
CD44
Hyaluronic acid
Nanoparticle
Naproxen Cytotoxic chemotherapy continues to be the main therapeutic option for patients with metastatic breast cancer. Several studies have reported a significant association between chronic inflammation, carcinogenesis and the
presence of cancer stem cells (CSC). We hypothesized that the use of non-steroidal anti-inflammatory drugs
targeted to the CSC population could help reducing tumor progression and dissemination in otherwise hard to
treat metastatic breast cancer. Within this study cationic naproxen (NAP)-bearing polymeric nanoparticles (NPs)
were obtained by self-assembly and they were coated with hyaluronic acid (HA) via electrostatic interaction. HA-
coated and uncoated NAP-bearing NPs with different sizes were produced by changing the ionic strength of the
aqueous preparation solutions (i.e. 300 and 350 nm or 100 and 130 nm in diameter, respectively). HA-NPs were
fully characterized in terms of physicochemical parameters and biological response in cancer cells, macrophages
and endothelial cells. Our results revealed that HA-coating of NPs provided a better control in NAP release and
improved their hemocompatibility, while ensuring a strong CSC-targeting in MCF-7 breast cancer cells. Furthermore, the best polymeric NPs formulation significantly (p < 0.001) reduced MCF-7 cells viability when
compared to free drug (i.e. 45 ± 6% for S-HA-NPs and 87 ± 10% for free NAP) by p53-dependent induction of
apoptosis; and the migration of these cell line was also significantly (p < 0.01) reduced by the nano-formulated
NAP (i.e. 76.4% of open wound for S-HA-NPs and 61.6% of open wound for NAP). This increased anti-cancer
activity of HA-NAP-NPs might be related to the induction of apoptosis through alterations of the GSK-3β-
related COX-independent pathway. Overall, these findings suggest that the HA-NAP-NPs have the potential to
improve the treatment of advanced breast cancer by increasing the anti-proliferative effect of NAP within the
CSC subpopulation. Eva Espinosa-Cano a,b, Miguel Huerta-Madro˜nal a,b, Patricia C´amara-S´anchez b,c,d,
Joaquin Seras-Franzoso d, Simo Schwartz Jr b,d, Ibane Abasolo b,c,d,*, Julio San Rom´an a,b,
Maria Rosa Aguilar a,b,* a Biomaterials Group, Institute of Polymer Science and Technology (ICTP-CSIC), 28006 Madrid, Spain
b Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain
c Functional Validation & Preclinical Research (FVPR), Universitat Aut`onoma de Barcelona (UAB), 08035 Barcelona, Spain
d Drug Delivery & Targeting, CIBBIM-Nanomedicine, Vall d’Hebron Institut de Recerca (VHIR), Universitat Aut`onoma de Barcelona Contents lists available at ScienceDirect Contents lists available at ScienceDirect * Corresponding authors at: Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain.
E-mail addresses: ibane.abasolo@vhir.org (I. Abasolo), mraguilar@ictp.csic.es (M.R. Aguilar). Available online 10 March 2021
0928-4931/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1016/j.msec.2021.112024
Received 23 November 2020; Received in revised form 23 February 2021; Accepted 5 March 2021 Materials Science & Engineering C 124 (2021) 112024 Materials Science & Engineering C 124 (2021) 112024 Available online 10 March 2021
0928-4931/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
* Corresponding authors at: Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain.
E-mail addresses: ibane.abasolo@vhir.org (I. Abasolo), mraguilar@ictp.csic.es (M.R. Aguilar).
https://doi.org/10.1016/j.msec.2021.112024
Received 23 November 2020; Received in revised form 23 February 2021; Accepted 5 March 2021 Hyaluronic acid (HA)-coated naproxen-nanoparticles selectively target
breast cancer stem cells through COX-independent pathways Eva Espinosa-Cano a,b, Miguel Huerta-Madro˜nal a,b, Patricia C´amara-S´anchez b,c,d,
Joaquin Seras-Franzoso d, Simo Schwartz Jr b,d, Ibane Abasolo b,c,d,*, Julio San Rom´an a,b,
Maria Rosa Aguilar a,b,* 2.1.1. Hyaluronic acid electrostatic coating of NAP-based NPs Self-assembled cationic NAP-bearing NPs were prepared via nano
precipitation method according to a recently described protocol [24]. Briefly, an organic solution of poly(HNAP-co-VI)(71:29) was added
dropwise to an aqueous solution (0.1 M acetic acid (AA, Sigma-Aldrich,
St. Louis, MO, USA)), 0.1 M or 0 M sodium chloride (NaCl, Panreac,
Spain) under continuous stirring. Then, the NPs suspension was kept in
dialysis for 2 days to eliminate the organic solvent. HA-coating was
performed by electrostatic interaction between cationic NAP-based NPs
and hyaluronic acid (HA, 200–300 KDa, Bioiberica, Spain). Briefly, 1 mL
of NAP NPs dispersion (0.1 M AA, 0.1 M NaCl at pH 4.0) was added
dropwise to a HA solution (2 mL, 0.1 M AA, 0.1 M NaCl at pH 4.0) under
continuous stirring. Different mass ratios (mg poly(HNAP-co-VI):mg HA
= 0.5, 0.4, 0.3, 0.2 and, 0.1) and, final concentration of nanoparticles
([NPs]F = 0.06 mg/mL, 0.25 mg/mL and 0.50 mg/mL) were tested. Finally, NPs were freeze-dried for 48 h and stored at 4 ◦C. The influence
of pH and ionic strength on hydrodynamic properties, as well as the
stability in suspension at pH 4.0 were assessed. NPs with larger and
smaller hydrodynamic diameter obtained when changing ionic strength
of the aqueous solutions were named L-NPs or S-NPs, respectively. Fluorescent NPs were prepared analogously following the same coating
protocol but using previously described coumarin 6 (c6, Sigma-Aldrich)-
loaded NAP-bearing NPs (c6-NPs) [24]. 2.1.2. Hydrodynamic properties: size, polydispersity and, surface charge
The particle size distribution and zeta potential (ξ) of the HA-coated
and uncoated NPs suspension was determined by dynamic light scat
tering (DLS) and laser Doppler electrophoresis (LDE), respectively, using
a Malvern Nanosizer NanoZS Instrument (Madrid, Spain) equipped with
a 4 mW He–Ne laser (λ = 633 nm) at a scattering angle of 173◦. Mea
surements were performed according to manufacturer’s instructions. The particle size distribution (% intensity) was obtained using ZetaSizer
Software 7.10 version, as well as the mean hydrodynamic diameter
(size) and the particle dispersion index (PdI) based on the Sto
kes−Einstein equation, assuming spherical NPs morphology. The sta
tistical average and standard deviation of samples data were calculated
from 3 measurements of 11 runs each one, in case of size and PdI, and
from 3 measurements of 20 runs each one, in case of ξ. In a previous work, our group described cationic NAP-bearing
polymeric NPs (NPs) with strong anti-inflammatory capacity [24]. 1. Introduction combination of surgery, chemotherapy, radiation, and targeted therapy. However, cytotoxic chemotherapy continues to be the main, if not the
only, therapeutic option for patients with metastatic breast cancer. Paradoxically, the increased use of chemotherapy at early stages of the
disease often derives in the acquisition of drug resistances and the
development of early, aggressive metastatic relapses [2]. This
chemotherapy-resistance as well as the metastatic dissemination seem to
be sustained by the presence of cancer stem cells (CSC), a small cell
subpopulation within the tumors with high tumorigenic potential and
the ability to self-renew. In breast cancer, CD44+/CD24−phenotype and Breast cancer is the most frequently diagnosed cancer among
women, accounting for 24.5% of all new cancer cases in 2020 world
wide. Importantly, for the first time, female breast cancer has become
the most common globally diagnosed cancer (11.7%), surpassing lung
cancer (11.4%). In addition, breast cancer is the leading cause of cancer
death in women worldwide, accounting for 15% of all female cancer
deaths [1], highlighting the need of more effective therapies. Treatment
of early breast cancer involves a multimodal strategy with a Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. high aldehyde deshydrogenase 1 (ALDH1) expression are the major CSC
markers described [3]. Moreover, several studies have reported an
aberrant activation of different signaling pathways in CSC, including
Hedgehog, Wnt/β-catenin, PI3K/PTEN and nuclear factor-κB, essential
to maintain their CSC-like properties [4,5]. However, these pathways
have also been proved decisive in inflammatory processes [6,7]. l may translate into a better controlled, stimuli-dependent, release of the
drug. Results show that S-HA-NPs have the potential to improve the
treatment of advanced breast cancer by increasing the anti-proliferative
effect of NAP within the CSC subpopulation. 2.1.1. Hyaluronic acid electrostatic coating of NAP-based NPs Un
fortunately, their positive surface charge precluded further in vivo
testing [25]. Remarkably, the negative charge and hydrophilic nature of
hyaluronic acid (HA) makes HA-coating an appropriate approach to
improve NAP-based NPs stability, reduce protein adsorption and in
crease their hemocompatibility [26,27]. Moreover, HA is the most
common natural ligand for CD44, a widely accepted marker of CSC [28]
and immune cells like macrophages [29,30]. Hence, enhanced cellular
uptake and selectivity towards immune cells and CSC can be achieved by
directly targeting this receptor. In fact, previous studies have already
demonstrated the potential use of HA-based NPs as tumor site-specific
drug delivery of chemotherapeutic drugs in breast cancer models [31]
and as anti-inflammatory agents in inflammatory diseases [32], towing
to its high binding affinity for the CD44 receptor. For example, HA-
doxorubicin nanoparticles [33] and HA-Lipid-PTX nanoparticles [34]
have been examined in ovarian and breast cancer preclinical models,
respectively. Both NPs leaded to significant reduction of tumor growth
and increased the lifespan of mice. 2.1. Preparation and characterization of hyaluronic acid-coated poly
(HNAP-co-vinyl imidazole(VI))-based NPs 2.1. Preparation and characterization of hyaluronic acid-coated poly
(HNAP-co-vinyl imidazole(VI))-based NPs 2.1.1. Hyaluronic acid electrostatic coating of NAP-based NPs 2.1.3. Morphology: cryoTEM The images of Cryo-TEM of the HA-NPs were taken with a Jeol 1230
electron microscope (Bergen, Norway) operated at 100 kV and equipped
with a Gatan liquid nitrogen specimen holder (Warrendale, PA, United
States) and a CMOS Tvips TemCam-F416 camera (Gauting, Germany), at
40,000 nominal magnifications under low dose conditions. Samples
were applied to holey carbon grids (Quantifoil, Großl¨obichau, Germany)
after glow-discharge and immediately blotted and vitrified using a FEI
Vitrobot cryo-plunger (Thermo Scientific, Madrid, Spain). 2.1.4. Quantification of HA-complexed: CTAB turbidimetric assay i 2. Materials and methods l
Chronic inflammation has long been linked to carcinogenesis [8]. Previous studies have reported that inflammatory responses play deci
sive roles at different stages of tumor development, including initiation,
promotion, malignant conversion, invasion, and metastasis [9]. As such,
anti-inflammatory drugs have been explored as therapeutic candidates
for cancer treatment and prevention. In the past few decades, research
has shown that non-steroidal anti-inflammatory drugs (NSAIDs)
decrease the risk to develop certain types of cancer and reduce tumor
progression, recurrence and mortality of colorectal and breast cancer
[10,11]. Moreover, improved outcomes had been reported when
combining NSAIDs with anti-tumor drugs in preclinical studies [12]. In
particular, naproxen (NAP), a well-known NSAID, and its derivatives
have been proven effective in the treatment of colorectal cancer [13],
urinary bladder cancer [14] and breast cancer [15]. NAP anti-cancer
properties are mainly attributed to the reduction of prostaglandin E(2)
(PGE2) levels, the major product of the activation cascade of both
cyclooxygenase (COX) isoforms (COX1/COX2) [15]. However, there is
increasing evidence of COX-independent mechanisms playing a role in
the anti-tumorigenic activity of NSAIDs [16,17]. It was recently shown
that NAP can inhibit GSK-3β pathway causing an increase in p53 cyto
solic accumulation that compromises MCF-7 breast cancer cells migra
tion and survival [18]. Of note, both pathways have been linked to CSC
maintenance [19] suggesting a potential use of NAP as an adjuvant in
chemotherapy. In fact, the potential of NAP and its derivatives to
enhance breast cancer treatment has been demonstrated recently
[15,18]. Unfortunately, repeated-administration and efficacy of free
forms of NAP are hampered by the drug’s high hydrophobicity and by
dose and time-dependent gastrointestinal side-effects [20]. In this sce
nario, nanomedicine offers the possibility of increasing the potential of
the free drugs while reducing their side effects [21]. Because of that, in
the last years, several works have focused on the preparation of lipid or
polymeric nanoformulations encapsulating NAP or NAP derivatives for
the treatment of breast cancer [22,23]. However, to the best of our
knowledge, there are no actively targeted formulations incorporating
covalently link naproxen. 2.4.1. Non-phagocytic MCF7 breast cancer cells p
g
y
The same coumarin-6 loaded nanoparticulated systems were used to
study the differential uptake of NPs by CSC and non-CSC populations. For this assay, an in vitro CSC fluorescent model of MCF-7 cell line, in
which CSC can be identified by the expression of tdTomato fluoro
chrome was used [40]. The stemness nature of tdTomato-expressing
cells has been already fully confirmed in vitro by increased expression
of stemness markers, mammosphere formation and in vivo tumorigenic
capacity [40]. In brief, 200,000 cells of MCF-7.Fluc2-ALDH1-tdTomato
cell line, containing 1–2% of CSC-tdTomato+ stabilized cell subpopu
lation, were seeded in complete DMEM medium in 6-well plates and
allowed to attach for 24 h. The medium was replaced with the corre
sponding NP suspension at 0.02 mg/mL and incubated for different time
points: 2, 5, 10, 15, 20, 30 and 120 min at 37 ◦C. Then, cells were washed
with PBS 1× (Cytiva Hyclone, Thermo Fisher Scientific, CA, USA) to
discard c6-loaded NPs that were not internalized, harvested with 0.25%
trypsin-EDTA (Sigma-Aldrich) and resuspended in PBS 1× supple
mented with 10% FBS and DAPI (10 μg/mL, Sigma-Aldrich) for vital
staining. Cell suspensions were examined by flow cytometry (BD LSR
Fortessa™ Cytometer, Becton Dickinson Bioscience, USA). Data were
analyzed with FCS Express 5 Flow Research Edition software (De Novo
Software, Glendale, CA, USA). For each sample, at least 50,000 indi
vidual cells were collected and the mean fluorescence intensity was
evaluated. %NAP released = mass of NAP released
initial mass of NAP × 100
(3) %NAP released = mass of NAP released
initial mass of NAP × 100
(3) (3) 2.3. Hemocompatibility of HA-NPs. Hemolysis and plasma aggregation
assays As developed nanomaterials were aimed for intravenous adminis
tration, their safety upon contact with blood was studied. In detail, two
types of assays were performed to test the hemocompatibility of the
NAP-bearing NPs. On the one hand, their effect on the integrity of red
blood cells was measured using a hemolysis test through the determi
nation of hemoglobin by a quantitative colorimetric determination using
the Drabkin’s reagent (Sigma-Aldrich, Madrid, Spain). On the other
hand, their potential interference with blood coagulation was studied by
analyzing plasma coagulation times. The concentrations tested were the
maximum permitted in both assays. Hemolysis assay was based on
ASTM E2524-08 guideline [36]. 2.4.1. Non-phagocytic MCF7 breast cancer cells In detail, for the hemolysis test, red
blood cells (RBC) were isolated from volunteer donors, resuspended in
2% (v/v) of PBS, and exposed to different concentrations of test NPs
during 1 h at 37 ◦C in duplicates. The amount of released hemoglobin
was measured in a spectrophotometer at 405 nm (microplate reader
ELx800, BioTek, Germany) after centrifugation (1000 g, 10 min). Absorbance values were referred to a positive control of 100% hemolysis
obtained after incubating RBC with 1% of Triton-X (Sigma-Aldrich,
Madrid, Spain). Samples with <5% are considered non hemolytic
[36,37]. The effect of the NPs in plasma coagulation was tested using
Start4 equipment (Stago, France) and following the manufacturer’s
protocol according to NCL method ITA-12 (2020) [38]. Activated partial
thromboplastin time (aPTT), prothrombin time (PT) and thrombin time
(TT) were determined as a measure of the extrinsic and intrinsic coag
ulation pathways and to assess fibrin formation from fibrinogen, 2.3.1. Cell culture 2.3.1. Cell culture
The RAW264.7 (murine macrophages, Sigma-Aldrich), MCF-7
(human mammary adenocarcinoma cells, European Collection of Cell
Cultures, ECACC), HepG2 (human hepatocarcinoma cells, American
Type Culture Collection, ATCC) and HUVEC (human umbilical vascular
endothelial cells, Sigma-Aldrich) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM, Sigma-Aldrich), supplemented with 10% fetal
bovine serum (FBS, Sigma-Aldrich), 2% L-glutamine, 1% penicillin/
streptomycin (P/S, Sigma-Aldrich). HUVEC culture medium was addi
tionally supplemented with 0.4% Glutamax (Sigma-Aldrich), 0.04%
heparin (Sigma-Aldrich) and, 0.04% Endothelial Cell Growth Supple
ment (ECGS, Sigma-Aldrich). The MCF-7-ALDH1A1/tdTomato CSC
model (which accounted for 1–2% of CSC-tdTomato+ cells) [40] was
cultured in complete medium supplemented with 1 μg/mL Blasticidin
and 1 μg/mL Puromycin as selection antibiotics. All cell lines were
incubated under permissive conditions (37 ◦C and 5% CO2). Briefly, right after preparation, HA-NPs were centrifuged at 12,000
rpm for 30 min at 4 ◦C. Then, 750 μL of HA standard solution
(0.625–0.15 mg/mL) or supernatant sample was added to 750 μL of
Hexadecyltrimethylammonium bromide (CTAB, Sigma-Aldrich) solu
tion (10 mM in 0.1 M AA, 0.1 M NaCl at pH 4.0). The absorbance of the
precipitated complex was read at 570 nm four subsequent times within
1 min using NanoDrop One spectrophotomer (Thermo Scientific). All
measurements were performed in triplicates. 2.4. NP uptake studies to evaluate CD44-mediated active targeting non-
phagocytic cells with differential CD44 expression 2.4. NP uptake studies to evaluate CD44-mediated active targeting non-
phagocytic cells with differential CD44 expression 2.4.1. Non-phagocytic MCF7 breast cancer cells 2.2. Evaluation of the influence of HA-coating and esterase on naproxen
release kinetics in PBS 5 mL of S-HA-NPs resuspended in PBS after freeze-drying or S-NPs
diluted 1:5 (v/v) in phosphate buffer saline (PBS, Sigma-Aldrich) solu
tion containing 10% fetal bovine serum (FBS, Sigma-Aldrich), in the
presence (+) or absence (−) of with 15 u/mL of esterase from porcine
liver (Sigma-Aldrich) were dialyzed against 15 mL of PBS solution
containing 0.5% (v/v) of Tween-80 (Sigma-Aldrich). The release was
performed at 37 ◦C using a 3.5–5 kDa MWCO membrane (Spectrum
Laboratories, Piscataway, NJ, USA) and esterase content was refreshed
every 48 h. After different time-points up to 14 days, 1 mL of the dia
lyzing medium was withdrawn and the same volume (1 mL) of PBS
solutions was replenished. Concentration of NAP released was calcu
lated by measuring the absorbance of the aliquots at λ = 262 nm using
NanoDrop One spectrophotomer (Thermo Scientific) and extrapolating
data to a calibration curve of NAP in PBS, 0.5% (v/v) Tween-80 solution
(i.e. Abs (λ = 262 nm) = 0,0231 [NAP] + 0,0773; R2 = 0.9994). The
experiment was carried out in duplicates and the percentage of initial
NAP released was computed according to Eq. (3). Quantification of HA-complexed: CTAB turbidimetric assay Thus, this work is aimed at developing HA-coated NAP-based NPs
(HA-NPs) with improved CD44-mediated active-targeting capacity and
superior anti-cancer properties than free administered NAP. This
nanoplatform, unlike previously described NAP-based systems, might
reduce adverse effects by avoiding off target effect and, particularly, GI
risk by chemically modifying the –COOH group on NAP structure and i
HA complexed with nanoparticles was quantified by a reported in
direct method using a CTAB turbidimetric assay [35]. The amount of
complexed HA was calculated by subtracting the amount of HA in the
supernatant determined by CTAB turbidimetric analysis to the initial
amount of HA (Eq. (1)), and the % of adsorbed HA was calculated
relative to the initial amount of HA in the suspension (Eq. (2)). 2 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. E. Espinosa-Cano et al. [HA]complexed = [HA]initial −[HA]measured (1) respectively. Normal coagulation time for aPTT is ≤34.1 s, for PT is
≤13.4 s and for TT ≤21 s, as reported in the literature [39]. Samples
were tested in duplicate in all three in vitro tests. %HAadsorbed = [HA]complexed
[HA]initial
× 100
(2) l %HAadsorbed = [HA]complexed
[HA]initial
× 100l (2) 2.3.1. Cell culture 3.1. Preparation and characterization of hyaluronic acid (HA)-decorated
poly(HNAP-co-VI)-based NPs HA-NPs were spherical and the size increase after HA adsorp
tion was observed when comparing to previously reported cryoTEM
images of the uncoated NPs [43]. NPs showed an irregular surface due to
the folding of HA chains as they adsorbed onto NAP-NPs surface. A low
density (i.e. light grey), highly hydrated HA shell could be distinguished
as well as regions of higher density (i.e. darker grey) at the core of the
NPs where the HA and NPs co-exist. The HA-NPs were not aggregated,
and particle size was approximately 250–400 nm ratifying DLS results. 2.8. Wound healing assay to evaluate S-HA-NPs capacity to inhibit MCF-
7 migration Hydrodynamic properties as a function of the pH were studied due to
the electrostatic nature of the coating and the previously reported lack of
stability of uncoated NPs at pH values higher than 4.5 [24]. In order to
avoid the influence of [NPs]Final, HA-NPs were freeze-dried and resus
pended in the same volume of 0.1 M AA, 0.1 M NaCl aqueous solution at
different pH values (4.5, 5.5, 6.5 or, 7.5). Dispersion after freeze-drying
of the system was achieved by simple manual agitation and 5 min of bath
sonication. Fig. 3a shows the evolution of the size distribution (% in
tensity) and the surface charge determined by DLS and LDE, respec
tively. The size distribution was wider and the intensity of the peak in
the microscale increased with pH while the surface charge remained
unaltered. The larger sizes could be explained by the higher hydration of
the HA shell and the weaker electrostatic interaction between VI imid
azole groups and HA carboxylic groups that occurred due to ionization
of –COOH or –NH as pH values get between their pKa and pKb, respec
tively. Stability at physiological pH was achieved after HA coating as
demonstrated in Fig. 3b. Uncoated NPs precipitated at pH above 5.5 Whole cell population of MCF-7 cells (800,000 cells/mL) were
seeded in a 6 well-plate. After 48 h, cells were exposed to either C.M. (positive control), different concentrations of S-HA-NPs (50, 25 and,
12.5 μg/mL) in suspension or free NAP equivalent concentration (30, 15
and 7.5 μg/mL). Once the cells reached 90% confluence, a “wound” was
made by manually scraping the middle of cell monolayers with a stan
dard 200 μL pipette tip. 3.1. Preparation and characterization of hyaluronic acid (HA)-decorated
poly(HNAP-co-VI)-based NPs NAP-NPs were obtained by nanoprecipitation method as previously
described by our group [24]. Then, HA was physically adsorbed onto the
surface of the cationic NPs at pH 4.0 in order to favor electrostatic
interaction between the imidazole groups of poly(VI-co-HNAP) and the
carboxylic groups of HA (pKa ~ 3.0–4.0) [42] and do not reach the
aggregation pH of NAP-NPs (pH ~ 4.5–5.0) [24]. Different mass ratios
(mg NPs:mg HA) were tested in order to achieve an efficient electrostatic
coating. The size distribution (by % intensity) obtained by DLS and zeta
potential (ξ) values obtained by LDE are displayed in Fig. 1b and c. At
mass ratios below 0.2 or above 0.4, precipitation of the HA and NAP-NPs
mixture occurred. However, at mass ratios within this range, it was
observed an inversion in sign of surface charge and a significant increase
in NPs diameter that confirmed polyanion (HA) physical adsorption. In
particular, 0.2 was chosen as the optimal mass ratio as the size increase
was minimized, the size distribution was narrower and, there were no
presence of large aggregates. Then, at that mass ratio, the maximum
final concentration of NPs ([NPs]Final) that can be achieved without
precipitation was 0.5 mg/mL. It was observed an increase in NPs size as
the [NPs]Final increases with no significant changes in surface charge
(Fig. 1c). The optimized HA-coating protocol of NAP-bearing NPs is
schematically presented in Fig. 1a. i 2.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells 2.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells As breast cancer tumors are mainly constituted by bulk non-CSC, we
tested the effect of S-HA-NPs using the whole cell population of the MCF-
7 cell line, which exhibits a stable CSC-population (1–2%). Accordingly,
a total of 6000 cells/well of MCF-7 were seeded in a 96 well-plate. After
24 h, cells were exposed to either C.M. (positive control) or different
concentrations of S-HA-NPs (50, 25 and, 12.5 μg/mL) in suspension or
free NAP at equivalent concentrations (30, 15 and 7.5 μg/mL). Every 24
h, cell media was collected and replaced by fresh C.M. PGE2 and VEGF
released after 72 h of treatment was measured by human PGE2 and
VEGF ELISA kit (Invitrogen) following manufacturer’s protocol. 2.7. Apoptosis in NAP-treated MCF-7 cells. Quantification of p53 levels
by ELISA Whole cell population of MCF-7 cells (750,000 cells/mL), were
seeded in a 6 well-plate under permissive conditions. 3.1. Preparation and characterization of hyaluronic acid (HA)-decorated
poly(HNAP-co-VI)-based NPs After 24 h, cells
were exposed to either C.M. (positive control), different concentrations
of S-HA-NPs (50, 25 and, 12.5 μg/mL) in suspension or free NAP at the
concentration that was present on each NPs sample (30, 15 and 7.5 μg/
mL). Every 24 h, extracts were replaced by fresh C.M. and, after 48 h,
cells were collected, lysed and p53 levels in MCF-7 cells lysates were
measured by human p53 ELISA kit (Abcam, Cambridge, United
Kingdom) according to manufacturer’s protocol. Size and morphology of HA-NPs were confirmed by cryoTEM
(Fig. 2). HA-NPs were spherical and the size increase after HA adsorp
tion was observed when comparing to previously reported cryoTEM
images of the uncoated NPs [43]. NPs showed an irregular surface due to
the folding of HA chains as they adsorbed onto NAP-NPs surface. A low
density (i.e. light grey), highly hydrated HA shell could be distinguished
as well as regions of higher density (i.e. darker grey) at the core of the
NPs where the HA and NPs co-exist. The HA-NPs were not aggregated,
and particle size was approximately 250–400 nm ratifying DLS results. Hydrodynamic properties as a function of the pH were studied due to
the electrostatic nature of the coating and the previously reported lack of
stability of uncoated NPs at pH values higher than 4.5 [24]. In order to
avoid the influence of [NPs]Final, HA-NPs were freeze-dried and resus
pended in the same volume of 0.1 M AA, 0.1 M NaCl aqueous solution at
different pH values (4.5, 5.5, 6.5 or, 7.5). Dispersion after freeze-drying
of the system was achieved by simple manual agitation and 5 min of bath
sonication. Fig. 3a shows the evolution of the size distribution (% in
tensity) and the surface charge determined by DLS and LDE, respec
tively. The size distribution was wider and the intensity of the peak in
the microscale increased with pH while the surface charge remained
unaltered. The larger sizes could be explained by the higher hydration of
the HA shell and the weaker electrostatic interaction between VI imid
azole groups and HA carboxylic groups that occurred due to ionization
of –COOH or –NH as pH values get between their pKa and pKb, respec
tively. Stability at physiological pH was achieved after HA coating as
demonstrated in Fig. 3b. Uncoated NPs precipitated at pH above 5.5 Size and morphology of HA-NPs were confirmed by cryoTEM
(Fig. 2). %open wound = wound area after 24 h
Initial wound area
× 100 %open wound = wound area after 24 h
Initial wound area
× 100 (4) 2.5.2. Evaluation of S-HA-NPs cytotoxicity in cells from the mammary
tumor microenvironment: RAW264.7, HUVEC and MCF-7 All results are expressed as the mean ± standard deviation (SD) of
several experimental replicates, unless otherwise specified. ANOVA
tests, student’s t-tests or equivalent non-parametric tests were used to
investigate the differences between NPs and controls, using Prism 6.02
software (GraphPad Software, Inc., CA, USA). Statistical differences
were accepted as significant when p < 0.01 (*) or p < 0.05 (**) and as
highly significant when p < 0.001 (***). 10,000, 2000 and 6000 cells/well of HUVEC, RAW264.7 and MCF-7
lines were seeded in a 96-well plate. After 24 h, all cell lines were
exposed to either culture media (C.M., positive control) or different
concentrations of S-HA-NPs (50, 25 and, 12.5 μg/mL) in suspension
whereas MCF-7 cells were also treated with free NAP (0.1% DMSO
(Sigma-Aldrich) in culture media) at the concentration that was present
on each NPs sample (30, 15 and 7.5 μg/mL). Then, cell viability was
determined after 72 h of incubation. Briefly, cells were incubated for 3 h
with a solution 10% (v/v) AlamarBlue (Invitrogen, Waltham, MA, USA)
in phenol red-free DMEM at 37 ◦C in a humidified incubator with 5%
CO2. Then, absorbance at 570 nm was measured by a Multi-Detection
Microplate Reader Synergy HT (BioTek). The treatments were done in
replicates (n = 8). Results of the experiments were expressed as per
centage of relative cell viability (% respect to the control). 2.9. Statistical analysis 2.5.2. Evaluation of S-HA-NPs cytotoxicity in cells from the mammary
tumor microenvironment: RAW264.7, HUVEC and MCF-7 2.5.2. Evaluation of S-HA-NPs cytotoxicity in cells from the mammary
tumor microenvironment: RAW264.7, HUVEC and MCF-7 2.5. Cell viability studies to demonstrate specific cytotoxicity against
CD44-expressing cancer cells 2.5.1. Evaluation of the cytotoxicity of coated and uncoated NPs in cancer
cells with distinct CD44 expression: MCF-7 and HepG2 2.5.1. Evaluation of the cytotoxicity of coated and uncoated NPs in cancer
cells with distinct CD44 expression: MCF-7 and HepG2 As CSC only represented 1–2% of the total cell population within a
tumor [40], whole cell population was used in order to better mimic the
tumor situation. Therefore, MCF-7 cells and HepG2 cells were seeded on
96-well plates at a density of 10,000 and 15,000 cells per well, respec
tively, and left to attach for 24 h. Then, cells were incubated with serial
dilutions of both sizes (large and small) of coated and uncoated NPs for
72 h. Complete medium was used as negative control and 10% DMSO
(Sigma-Aldrich) as positive control of toxicity. Subsequently, 5 mg/mL
of MTT were added, cells were incubated for 4 h and formazan pre
cipitates were resuspended in DMSO before measuring absorbance at
590 nm (microplate reader ELx800, BioTek). Cell viability was calcu
lated and normalized to non-treated cells (100% viability) and positive 3 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. controls (10% DMSO, 0% viability). Dose-response curves were plotted
and half maximal inhibitory concentration (IC50) values were deter
mined using Prism 6 Software (GraphPad Software, Inc., CA, USA). All
experiments were run in triplicate. 3.1. Preparation and characterization of hyaluronic acid (HA)-decorated
poly(HNAP-co-VI)-based NPs Scratches were washed three times with PBS and
photographed using an inverted microscope (20-fold magnification,
Nikon Eclipse TE 2000-S). Subsequently, the cells were incubated in
fresh C.M. for additional 24 h and photographed again. The area of the
open wound was computed using Image J software [41] and the % of
open wound after 24 h was calculated using Eq. (4). Experiments were
performed independently twice, evaluating 3 scratches in each
experiment. 4 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. Fig. 1. Synthesis and characterization of NAP-NPs. a) Schematic presentation of the optimized protocol of HA physical adsorption onto NPs surface. Size distribution
(by intensity) obtained by DLS, and z-potential (ξ, in brackets) obtained by LDE of NAP-NPs (red) and HA-NPs (black) b) at different mass ratios (mg poly(HNAP-co-
VI):mg HA) and, c) at different final concentration of NPs in mg/mL (mass ratio fixed at 0.2); (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.) Fig. 1. Synthesis and characterization of NAP-NPs. a) Schematic presentation of the optimized protocol of HA physical adsorption onto NPs surface. Size distribution
(by intensity) obtained by DLS, and z-potential (ξ, in brackets) obtained by LDE of NAP-NPs (red) and HA-NPs (black) b) at different mass ratios (mg poly(HNAP-co-
VI):mg HA) and, c) at different final concentration of NPs in mg/mL (mass ratio fixed at 0.2); (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.) Fig. 2. Morphology of HA-coated and uncoated NAP-NP. Cryogenic transmission electron microscopy (cryoTEM) micrograph of the NPs. a) Uncoated NPs, without
HA covering b) HA-NPs at low magnification, c) HA-NPs at higher magnification. Fig. 2. Morphology of HA-coated and uncoated NAP-NP. Cryogenic transmission electron microscopy (cryoTEM) micrograph of the NPs. a) Uncoated NPs, without
HA covering b) HA-NPs at low magnification, c) HA-NPs at higher magnification. complexed onto the surface of the NPs when compared to those prepared
at 0.1 M ionic strength as confirmed by CTAB turbidimetric analysis of
the samples (Table 1). However, the reported higher HA adsorption did
not lead to a higher size increment suggesting that higher charge also
increased system compactness. 3.1. Preparation and characterization of hyaluronic acid (HA)-decorated
poly(HNAP-co-VI)-based NPs From now on, nanoparticles will be
labelled as ‘large’ (L) and ‘small’ (S) depending on their size: L-NPs or S-
NPs (uncoated NAP NPs) and L-HA-NPs or S-HA-NPs (HA-coated NAP
NPs). because of a decrease in electrostatic repulsive forces as imidazole
groups of VI deprotonate, whereas the HA-coating kept the surface
charge constant avoiding precipitation of the system over the whole
range of pH under study. However, the improvement in stability was
accompanied by a significant increase in mean size of the NPs, from
131.5 ± 3.3 nm (NAP NPs, pH 4.5) to 351.5 ± 22 nm (HA-NAP NPs, pH
4.5) and to almost 620.4 ± 4.2 nm (HA-NAP NPs, pH 7.5). CD44 is the
hyaluronic acid receptor and therefore HA-NPs actively target CD44-
receptor. However, their large size might hamper this interaction
[44,45]. Changes in the ionic strength of aqueous media affect surface
charge, size, and compactness of NPs with ionic groups on its structure
[46,47]. The modulation of hydrodynamic properties of NAP- NPs and
HA-NPs by ionic strength was studied at pH 4.0 (Table 1) [48]. An in
crease in the ionic strength caused a decrease in the absolute value of z
potential, from an initial value of +40.7 ± 0.9 mV or −36.9 ± 2.7 mV in
the absence of salt to +28.8 ± 0.97 mV or −18.2 ± 0.9 mV at an ionic
strength of 0.1 M NaCl for uncoated NPs or HA-coated NPs, respectively. Moreover, there was a reduction of about 30 nm on the size of the un
coated NPs and of about 50 nm on the size of HA-NPs as the ionic
strength decreases from 0.1 M to 0 M. The higher positive surface charge
of NAP- NPs at 0 M ionic strength translated into almost twice HA To further characterize the obtained NPs, their stability was studied
in detail. Regarding stability, as it occurred with L-NPs [24], newly
described S-NPs, L-HA-NPs and, S-HA-NPs systems were stable at
preparation pH (i.e. pH 4.0) up to one month. A slight swelling was
observed in the first 2 days after preparation of S-HA-NPs with no sig
nificant changes in hydrodynamic properties from that time point up to
one month (Fig. 4). Table 1 Hydrodynamic properties of NPs right after preparation. Hydrodynamic prop
erties and Zeta potential of uncoated nanoparticles (NPs) and HA-coated NPs
(HA- NPs) at a concentration of 500 μg/mL, and, percentage of initial HA
adsorbed onto the NAP-bearing NPs surface obtained by CTAB turbidimetric
analysis at pH 4.0 and 0.1 M NaCl (large, L) and 0 M NaCl (small, S) ionic
strengths. Results are presented as mean ± SD of three independent samples (n
= 3) of each system. Moreover, at any time point, the HA-coated system presented lower
percentage of NAP released than its uncoated homologous with 94 ± 9%
and 65 ± 8% of the drug released after 14 days by S-NPs and S-HA-NPs,
respectively (Fig. 5, grey lines). These data also confirmed that the HA-
coating acted as a barrier layer providing a better control in NAP release
as it occurred with other drugs or proteins encapsulated in HA-coated
systems described in the literature [50,51]. System
[NaCl]
(M)
Sizea
(nm)
PdIb
ξc
(mV)
%
HAadsorbed
d
L-NPs
0.1
131.5 ± 3
0.115 ±
0.016
+28.8 ±
0.7
–
S-NPs
0
99.1 ± 10
0.106 ±
0.020
+40.7 ±
0.9
–
L-HA-
NPs
0.1
351.5 ±
22
0.124 ±
0.054
−18.2 ±
0.9
24
S-HA-
NPs
0
297.0 ± 6
0.113 ±
0.017
−36.9 ±
2.7
46 System
[NaCl]
(M)
Sizea
(nm)
PdIb
ξc
(mV)
%
HAadsorbed
d
L-NPs
0.1
131.5 ± 3
0.115 ±
0.016
+28.8 ±
0.7
–
S-NPs
0
99.1 ± 10
0.106 ±
0.020
+40.7 ±
0.9
–
L-HA-
NPs
0.1
351.5 ±
22
0.124 ±
0.054
−18.2 ±
0.9
24
S-HA-
NPs
0
297.0 ± 6
0.113 ±
0.017
−36.9 ±
2.7
46
a Mean hydrodynamic diameter (% intensity). b Polydispersity of the size distribution. c Surface charge and. d Percentage of the initial HA adsorbed to the NPs surface. 3.2. Evaluation of the influence of HA-coating and esterases on naproxen
release kinetics in PBS Naproxen is linked to the polymeric backbone via an ester bond
involving the –COOH group [24] to reduce adverse effects associated to 5 Materials Science & Engineering C 124 (2021) 112024 Materials Science & Engineering C 124 (2021) 112024
Fig. 3. Size distribution and Zeta potential of NAP-NPs upon different pHs. a) Evolution of the size distribution (% intensity) and the surface charge (in brackets) of
HA-NPs at different pHs as determined by DLS and LDE, respectively. b) Hydrodynamic diameter (Size), polydispersity index (PdI) and zeta potential of uncoated
NAP- NPs (red) and HA-coated NAP-NPs (black) as a function of the pH. *Samples too polydisperse for accurate measurement. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
E. Espinosa-Cano et al. E. Espinosa-Cano et al. Materials Science & Engineering C 124 (2021) 11202
pinosa-Cano et al. Fig. 3. Size distribution and Zeta potential of NAP-NPs upon different pHs. a) Evolution of the size distribution (% intensity) and the surface charge (in brackets) of
HA-NPs at different pHs as determined by DLS and LDE, respectively. b) Hydrodynamic diameter (Size), polydispersity index (PdI) and zeta potential of uncoated
NAP- NPs (red) and HA-coated NAP-NPs (black) as a function of the pH. *Samples too polydisperse for accurate measurement. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.) amphiphilic copolymer (poly(VI-co-HNAP) in a more hydrophilic
terpolymer (poly(VI-co-HNAP-co-HEMA), as NAP residues change into
HEMA residues after NAP release. This fact might induce a change in the
core of the NPs that was well-organized and of difficult access at the
beginning of the experiment. The increasing hydrophilicity of the
formed copolymer might accelerate the release of NAP after 144 h, as
shown in Fig. 5 (plain lines). Table 2 The same trend was observed for small
(S) systems but the hemolytic behavior appeared at lower concentra
tions than for the L systems. S-HA-NPs were above the hemolytic limit
(5%) at the highest concentration (250 μg/mL), showing intermediate
hemolytic properties at 125 μg/mL and no hemolytic activity at any
other concentration tested. Its uncoated homologous, S-NPs, was highly 3.3. Hemocompatibility of HA-NPs. Hemolysis and plasma aggregation
assays a Mean hydrodynamic diameter (% intensity). Since NPs were envisioned for intravenous administration, to test the
hemocompatibility of NPs, the prothrombin time (PT), activated partial
thromboplastin time (aPTT), and thrombin time (TT) values were
determined after blood samples were challenged with the HA- coated
and uncoated systems. Data obtained from these tests are presented in
Table 2. PT, aPTT and TT values were within normal ranges for all
coated and uncoated systems. There were no significant changes when
compared to the negative control (C-) or complete culture medium (C. M.) but for the aPTT after treatment with S-NPs. However, for all sam
ples, PT, aPTT or TT were within the normal clinical limits established
by the NCL ITA-12 method [38]. Therefore, none of the NPs under study
compromised any of the main coagulation pathways (intrinsic (APTT),
extrinsic (PT) and common (TT)). b Polydispersity of the size distribution. g
d Percentage of the initial HA adsorbed to the NPs surface. the carboxylic group [13,49]. Therefore, the drug can be released either
chemically, by hydrolysis of this bond, or enzymatically, in the presence
of esterase. The hydrolytic stability, influence of HA-coating and
esterase-mediated in vitro release of NAP was studied by a dialysis
diffusion method. Fig. 5 shows the in vitro NAP release profile at 37 ◦C
during 14 days in PBS. A burst release of 16 ± 1% and 20 ± 0.1% of NAP
was observed over the first 72 h for the uncoated systems in the absence
and presence of esterase, respectively, while between 0 and 4 ± 0.1% of
NAP was released from the HA-coated systems in the absence or pres
ence of esterase, correspondingly. After this point, there were no further
drug release unless esterase were added (plain lines) confirming an
esterase-dependent release of the drug as well as the hydrolytic stability
of the systems. The continuous exposure of the NPs to esterase induce
the progressive release of NAP and the transformation of the To further assess the hemocompatibility of the systems, the per
centage of hemolysis (i.e. red blood cells (RBC) lysis) was determined
after 1 h of incubation with up to 250 μg/mL of L-NPs, S-NPs, L-HA-NPs
or S-HA-NPs (Fig. 6). Table 2 Comparison of the coagulation properties of HA-coated and uncoated NPs. Prothrombin time (PT), activated partial thromboplastin time (aPTT) and
thrombin time (TT) values in seconds (s) after incubating blood samples with
PBS 1× (negative control, C-), complete culture medium (C.M.) and with 50 μg/
mL of the HA-uncoated (S-NPs and L-NPs) and coated systems (L-HA-NPs and S-
HA-NPs). Data is shown as mean ± SD of two replicates. cytotoxic against RBC at concentrations of 30 μg/mL and above. Ac
cording to previous studies on positively charged NPs, the cationic
surface charge of NAP NPs might be the reason for RBC cytotoxicity due
to charge interaction with the plasma membrane and/or with
negatively-charged cell components [25]. Moreover, L-systems showed
lower hemolytic activity than their homologous S-systems. Although
surface charge might be the main factor contributing to the higher he
molysis rate, there are also previous reports in which size plays a role in
haemocompatibility, i.e. small size NPs caused higher thrombocyte and
granulocyte activation, and hemolysis when compared to larger NPs
with the same chemical structure [53]. However, at concentrations
below 125 μg/mL none of HA-coated systems were above the hemolytic
limit (5%). Therefore, concentrations equal or lower than 125 μg/mL
were selected for further studies. As a conclusion, these data confirmed
the already described HA capacity to improve hemocompatibility of
biomaterials [54]. In particular, here it was demonstrated a decrease
hemolysis of RBC after electrostatic HA coating of cationic NAP NPs. p
PT (s)
aPTT (s)
TT (s)
PBS (C-)
12.80 ± 0.14
34.40 ± 0.01
15.85 ± 0.07
C.M. 13.30 ± 0.01
33.00 ± 0.71
18.10 ± 0.28
L-NPs
13.30 ± 0.14
29.00 ± 0.85
17.25 ± 0.21
S-NPs
13.20 ± 0.01
40.25 ± 0.07
18.10 ± 0.28
L-HA-NPs
12.70 ± 0.01
27.95 ± 0.78
18.20 ± 0.14
S-HA-NPs
12.70 ± 0.01
34.70 ± 0.01
17.80 ± 0.28
Normal coagulation times: PT ≤13.4 s, aPTT ≤34.1 s and TT ≤21 s. Pathological coagulation times: PT ≥20 s, aPTT ≥61 s and TT ≥42 s. hemolytic activity, whereas values below 2% imply no significant he
molytic activity [52]. According to that, the L-HA-NPs showed slight
RBC cytotoxicity at the highest concentration (250 μg/mL) and no
cytotoxicity at any other concentration tested. Its uncoated homologous,
L-NPs showed percentage of hemolysis above those of HA-coated system
for the whole range of concentration and almost 100% of hemolysis at
concentrations above 60 μg/mL. 3.3. Hemocompatibility of HA-NPs. Hemolysis and plasma aggregation
assays (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.) Fig. 6. Hemolysis of NAP NPs. Percentage of hemolysis after 1 h of incubation Fig. 6. Hemolysis of NAP NPs. Percentage of hemolysis after 1 h of incubation
of blood samples with different concentrations of NPs. Data is shown as mean ±
SD of two replicates. Fig. 5. Naproxen release kinetics from HA-coated or uncoated NPs. In vitro
release profile of NAP from S-HA-NPs (grey) and S-NPs (red) in the presence
(+Est, continuous line) or absence (-Est, dashed line) of esterase, in PBS at
37 ◦C, over 14 days (336 h). Esterase were refreshed every 48 h. Data are shown
as mean ± SD of two replicates. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.) Fig. 6. Hemolysis of NAP NPs. Percentage of hemolysis after 1 h of incubation
of blood samples with different concentrations of NPs. Data is shown as mean ±
SD of two replicates. 3.3. Hemocompatibility of HA-NPs. Hemolysis and plasma aggregation
assays According to the criterion in the ASTM E2524-08
standard [36], a hemolytic behavior is characterized by percentage of
hemolysis above 5%, values between 2 and 5% denote a slightly 6 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. Materials Science & Engineering C 124 (2021) 112024
Fig. 4. Stability of HA-coated or uncoated NPs over time. Mean hydrodynamic diameter (size, nm) and polydispersity (PdI) obtained by DLS, and z-potential (ξ, mV)
obtained by LDE of S-NPs (red), L-HA-NPs (black) and, S-HA-NPs (grey) at pH 4.0 after 2, 7, 14, 21 and 28 days of storage at 4 ◦C. Data represent mean ± SD of three
replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
E. Espinosa Cano et al. Fig. 4. Stability of HA-coated or uncoated NPs over time. Mean hydrodynamic diameter (size, nm) and polydispersity (PdI) obtained by DLS, and z-potential (ξ, mV)
obtained by LDE of S-NPs (red), L-HA-NPs (black) and, S-HA-NPs (grey) at pH 4.0 after 2, 7, 14, 21 and 28 days of storage at 4 ◦C. Data represent mean ± SD of three
replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 5. Naproxen release kinetics from HA-coated or uncoated NPs. In vitro
release profile of NAP from S-HA-NPs (grey) and S-NPs (red) in the presence
(+Est, continuous line) or absence (-Est, dashed line) of esterase, in PBS at
37 ◦C, over 14 days (336 h). Esterase were refreshed every 48 h. Data are shown
as mean ± SD of two replicates. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Hemolysis of NAP NPs. Percentage of hemolysis after 1 h of incubation
f bl
d
l
ith diff
t
t
ti
f NP
D t i
h
± Fig. 5. Naproxen release kinetics from HA-coated or uncoated NPs. In vitro
release profile of NAP from S-HA-NPs (grey) and S-NPs (red) in the presence
(+Est, continuous line) or absence (-Est, dashed line) of esterase, in PBS at
37 ◦C, over 14 days (336 h). Esterase were refreshed every 48 h. Data are shown
as mean ± SD of two replicates. 3.4. Uptake studies in phagocytic and non-phagocytic cells with
differential CD44 expression Faster internalization of S-HA-NPs when compared to their large
homologous (L-HA-NPs) as well as their uncoated homologous (S-NPs)
by either phagocytic M1-polarized macrophages (i.e. treated with LPS,
lipopolysaccharide from Escherichia; +LPS) or none-polarized (un
treated, -LPS) RAW264.7 macrophages was demonstrated (Fig. S1). This 7 Materials Science & Engineering C 124 (2021) 112024 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. Fig. 7. Uptake of large and small HA-coated NPs in CSC and non-CSC in MCF
cells. Time dependent internalization of smaller HA-coated in non-CSC (light
grey) and CSC (light red) or larger HA-coated NPs in non-CSC (dark grey) and
CSC (dark red) represented as a) the percentage of positive cells for c6 and b)
the mean fluorescence intensity per cell. Data show the mean ± SD of three
replicates. Statistical significance is shown with black asterisks for S-HA-NPs
and L-HA-NPs when comparing CSC with non-CSC (t-student, *p < 0.05, **p <
0.01, ***p < 0.001). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.) data suggested that both size and surface coating play a key role in NPs
internalization by phagocytic cells. In particular, M1-polarized macro
phages overexpress CD44-receptor while M0 non-polarized macro
phages do not [29]. However, their phagocytic activity also increased
with M1 polarization. This might explain why the internalization
increased in a similar manner for coated and uncoated NPs after LPS-
treatment. Therefore, these results were not conclusive in terms of
CD44-mediated active targeting as large anionic particles were reported
to be preferentially passively internalized by macrophages over smaller
cationic ones [37]. Therefore, to confirm that the increased internali
zation was occurring because of the HA-CD44 interaction, the rate of
internalization of HA-coated NPs (L or S) by cancer cells showing dif
ferential CD44 expression was investigated (Supporting Material,
Fig. S2). In particular, an in vitro CSC fluorescent model of MCF-7 cell
line was used. In this model, CSC can be sorted by the expression of
tdTomato fluorochrome (tdTomato+ cells) obtaining a CSC-enriched
population with increased expression of stemness markers, including
CD44 (Supporting Material, Fig. S3) [40]. Using these MCF-7.Fluc2-
ALDH1-tdTomato cells, the CSC specific uptake of NPs with CD44-
mediated active targeting was demonstrated by flow cytometry. 3.4. Uptake studies in phagocytic and non-phagocytic cells with
differential CD44 expression l
When comparing L-HA-NPs and S-HA-NPs, it is clear that the rate of
internalization of both NPs in CSC and non-CSC subpopulations is very
different (Fig. 7). When analyzing the CSC, after 2 min of incubation
16.3 ± 1.89% of CSC were positive in c6 for L-HA-NPs, whereas almost
all CSC (97.7 ± 1.56%) were positive on c6 for S-HA-NPs. This higher
internalization of S-HA-NPs in CSC was especially significant at short
incubation times, but was kept even after 120 min of incubation. Apart
from the number of positive cells, the mean fluorescence intensity (MFI)
in each cell was also analyzed. In CSC, more than twice the MFI was
observed after incubation for 10 min with S-HA-NPs when compared to
L-HA-NPs. Such differences between S-HA-NPs and L-HA-NPs were also
observed in non-CSC, but internalization rates and the MFI were
significantly lower at almost all times in non-CSC compared to CSC
(Fig. 7 and Supporting Material, Fig. S4). Therefore, smaller NPs were
shown to internalize faster than the larger ones in MCF-7 cancer cells,
regardless their CD44-expression, as it occurred for macrophages. For
both types of NPs, large or small, the faster and more intense internal
ization of HA-NPs in CD44+ CSC [40,55], seems to indicate that CD44-
HA interaction plays a key role in internalization of NAP NPs. Further
more, these results strongly suggest that CSC have a significantly higher
endocytosis rate of coated-NPs than the differentiated breast cancer
cells, which is consistent with previous studies [56,57]. Altogether, our
results suggested that CD44-HA interaction could be considered a good
tool for drug delivery approaches to increase the anti-CSC efficiency of
anti-cancer drugs. Fig. 7. Uptake of large and small HA-coated NPs in CSC and non-CSC in MCF
cells. Time dependent internalization of smaller HA-coated in non-CSC (light
grey) and CSC (light red) or larger HA-coated NPs in non-CSC (dark grey) and
CSC (dark red) represented as a) the percentage of positive cells for c6 and b)
the mean fluorescence intensity per cell. Data show the mean ± SD of three
replicates. Statistical significance is shown with black asterisks for S-HA-NPs
and L-HA-NPs when comparing CSC with non-CSC (t-student, *p < 0.05, **p <
0.01, ***p < 0.001). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.) 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 a) Percentage of cell
viability of MCF-7 cells (high expression of CD44)
after 72 h of treatment with different concentrations
of S-NPs (red), L-NPs (dark red), S-HA-NPs (grey) or
L-HA-NPs (black). b) Cell viability experiments in
HepG2 cells (low expression of CD44) using the same
NPs. Data is represented as mean ± SD of three rep
licates. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.) Fig. 8. In vitro cytotoxicity of HA-NPs in cells with
differential expression of CD44. a) Percentage of cell
viability of MCF-7 cells (high expression of CD44)
after 72 h of treatment with different concentrations
of S-NPs (red), L-NPs (dark red), S-HA-NPs (grey) or
L-HA-NPs (black). b) Cell viability experiments in
HepG2 cells (low expression of CD44) using the same
NPs. Data is represented as mean ± SD of three rep
licates. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.) 7.5, 15 and 30 μg/mL) in order to demonstrate the improved anti-cancer
effect of S-HA-NPs over free NAP. Fig. 9a presents the cell viability of
MCF-7 cells after 72 h of treatment with S-HA-NPs or free NAP relative
to a positive control (i.e. cells treated with culture medium, C.M.). Although there was a direct relationship between S-HA-NPs concentra
tion and cytotoxicity, no significant differences were observed in MCF-7
viability after treatment with NPs at the three concentrations tested. However, cytotoxicity when compared to the positive control (C.M.) was
evident with a percentage of cell viability below 50% in all cases. In case
of free NAP, again the decreasing trend with the increase in concen
tration was observed with no statistical differences in cytotoxicity at the
three concentrations tested. However, the cell viability was 80% or
higher in all cases. Therefore, the superiority of the NAP-bearing NPs
over freely administered drug in terms of MCF-7 cytotoxicity was
demonstrated at all concentrations tested. i would be advantageous [15]. In contrast, S-HA-NPs affected the cell
viability of HUVEC cells and a direct relationship between NPs con
centration and HUVEC cell viability was observed. However, cell
viability values in HUVEC cells reached the 66 ± 6% for 50 μg/mL of NP
dose, compared to the 45 ± 6% of cell viability in MCF-7 cells. 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 The in
hibition of the neoangiogenic process and by extension the inhibition of
the proliferative capacity of endothelial cells is highly desirable in an
anti-tumoral treatment. In this regard, NSAID inhibition of HUVEC
proliferation is extensively described [59,60] as well as its benefits in
terms of reduction of angiogenesis and tumor growth. The advantage in
this case is that, similarly to what happened with cancer cells, S-HA-NPs
was superior to free NAP in terms of cytotoxicity against HUVEC
(Fig. S6) after 72 h of treatment. 3.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 NAP into the HA-coated delivery systems increases the specificity and
sensitivity towards CD44 expressing cells. Altogether, these results
demonstrate that CD44-mediated active targeting using HA-coated NAP
NPs translated into reduction of the NAP dose needed to achieve anti-
cancer activity in MCF-7 cells. Cell viability of MCF-7 cells (high CD44 expression, 92.62 ± 4.35%)
was evaluated after 72 h of treatment with different concentrations of S-
NPs, L-NPs, S-HA-NPs and L-HA-NPs, and compared to the viability of
HepG2 cells (low CD44 expression, 0.85 ± 0.18%), in order to assess the
effect of CD44-expression in the anti-cancer activity of NAP-bearing NPs
(Fig. 8 and Fig. S2). Results showed that the uncoated systems did not
reduce MCF-7 or HepG2 cells viability below 90% at any of the tested
concentrations with no significant differences between S-NPs and L-NPs. However, their HA-coated analogs were cytotoxic at concentrations of
polymer above 0.1 μg/mL for MCF-7 or above 1 μg/mL for HepG2, that
corresponded to 0.06 and 0.6 μg/mL of NAP, respectively. Therefore,
HA-NPs is 10 times less effective in cells with low CD44 expression
(HepG2) when compared to CD44-overexpressing cells (MCF-7). Previ
ous studies in the literature have shown that MCF-7 and HepG2 have
similar sensitivity to free NAP [18], indicating that the incorporation of The IC50 of HA-NPs against MCF-7 cells after 72 h of treatment was
approximately 10 μg/mL and, therefore, concentrations above IC50 were
chosen for further experiments. Furthermore, S-HA-NPs were used for
further studies as they are internalized significantly faster than L-HA-
NPs whereas no significant differences were observed in terms of cyto
toxicity or hemocompatibility within this concentration range. 3.5.2. Cytotoxicity of S-HA-NPs in cell models from the mammary tumor
microenvironment: RAW264.7, HUVEC, and MCF-7 cells Cytotoxicity of S-HA-NPs at 12.5, 25 and 50 μg/mL was compared to
that of free NAP at the concentration that it was present in the NPs (NAP: 8 8 Materials Science & Engineering C 124 (2021) 112024
7.5, 15 and 30 μg/mL) in order to demonstrate the improved anti-cancer
effect of S-HA-NPs over free NAP. Fig. 9a presents the cell viability of
MCF-7 cells after 72 h of treatment with S-HA-NPs or free NAP relative
to a positive control (i.e. 3.5.1. Cytotoxicity of coated and uncoated NPs against cancer cells with
different CD44 expression: MCF-7 and HepG2 cells treated with culture medium, C.M.). Although there was a direct relationship between S-HA-NPs concentra
tion and cytotoxicity, no significant differences were observed in MCF-7
viability after treatment with NPs at the three concentrations tested. However, cytotoxicity when compared to the positive control (C.M.) was
evident with a percentage of cell viability below 50% in all cases. In case
of free NAP, again the decreasing trend with the increase in concen
tration was observed with no statistical differences in cytotoxicity at the
three concentrations tested. However, the cell viability was 80% or
higher in all cases. Therefore, the superiority of the NAP-bearing NPs
over freely administered drug in terms of MCF-7 cytotoxicity was
demonstrated at all concentrations tested. would be advantageous [15]. In contrast, S-HA-NPs affected the cell
viability of HUVEC cells and a direct relationship between NPs con
centration and HUVEC cell viability was observed. However, cell
viability values in HUVEC cells reached the 66 ± 6% for 50 μg/mL of NP
dose, compared to the 45 ± 6% of cell viability in MCF-7 cells. The in
hibition of the neoangiogenic process and by extension the inhibition of
the proliferative capacity of endothelial cells is highly desirable in an
anti-tumoral treatment. In this regard, NSAID inhibition of HUVEC
proliferation is extensively described [59,60] as well as its benefits in
terms of reduction of angiogenesis and tumor growth. The advantage in
this case is that, similarly to what happened with cancer cells, S-HA-NPs
was superior to free NAP in terms of cytotoxicity against HUVEC
(Fig. S6) after 72 h of treatment. Fig. 8. In vitro cytotoxicity of HA-NPs in cells with
differential expression of CD44. a) Percentage of cell
viability of MCF-7 cells (high expression of CD44)
after 72 h of treatment with different concentrations
of S-NPs (red), L-NPs (dark red), S-HA-NPs (grey) or
L-HA-NPs (black). b) Cell viability experiments in
HepG2 cells (low expression of CD44) using the same
NPs. Data is represented as mean ± SD of three rep
licates. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.)
E. Espinosa-Cano et al. E. Espinosa-Cano et al. Materials Science & Engineering C 124 (2021) 112024 Materials Science & Engineering C 124 (2021) 112024
Fig. 8. In vitro cytotoxicity of HA-NPs in cells with
differential expression of CD44. 3.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells The specificity of S-HA-NPs against cancer cells was demonstrated by
comparing their cytotoxicity against three cell types co-existing in the
mammary tumor microenvironment: malignant MCF-7 cells and non-
malignant RAW264.7 and HUVEC cell lines. Fig. 9b shows that, in
contrast to what happens with cancer MCF-7 cells (grey bar, data
duplicated in both figures to facilitate results’ interpretation), the cell
viability of RAW264.7 cells is not compromised with S-HA-NPs at doses
below 50 μg/mL. In the case of macrophages, it was also important to
discard the pro-inflammatory activity of low molecular weight HA
which has been reported in the literature [58]. To further confirm this
extent, internalization of NPs in M1-polarized macrophages (i.e. LPS-
activated, Fig. S1) and the release of nitric oxide (NO) (Fig. S5b) were
evaluated. Results showed that after 24 h of treatment with S-HA-NPs a
clear reduction in NO in macrophages treated with S-HA-NPs, while cells
incubated with free HA kept high NO levels. Of note, macrophages serve
as the first line of defense during tumor establishment, meaning that a
drug delivery system with little to no toxic effect against macrophages According to literature, the main anti-cancer mechanism of NSAIDs
is COX-dependent, especially in PGE2 overexpressing cells [61]. How
ever, some studies concluded that COX-independent mechanisms also
play a role in anti-tumorigenic activity of NSAIDs [16,17]. Here, in order
to better understand the mechanism of action of S-HA-NPs and its su
periority over free drug, S-HA-NPs and free NAP effect on PGE2 released
by MCF-7 cells was evaluated. ELISA assay results (Fig. 10a) revealed
that the levels of PGE2 released by MCF-7 cells were not significant. Moreover, the treatment with either S-HA-NPs or free NAP had no effect
on this variable. These data correlated with previous works reporting
that MCF-7 cells are not PGE2 expressing cells [62] and supported the
idea of COX-independent anti-cancer mechanisms. S-HA-NPs have already demonstrated anti-angiogenic properties as
they affect the proliferative capacity of endothelial cells (Fig. 9b). Pre
vious studies revealed that NAP might affect the expression of VEGF Fig. 9. Cell viability assays by Alamar Blue in MCF-7,
RAW264.7 and HUVEC cells treated with S-HA-NPs. a) Percentage of viable MCF-7 cells relative to control
(culture media of MCF-7 cells) after 72 h of treatment
with different concentrations of S-HA-NPs (dark grey)
or free NAP (orange, at equivalent doses to S-HA-
NPs). 3.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells b) Percentage of MCF-7 (dark grey), RAW264.7
(blue) and HUVEC (pink) cell viability in respect to
controls (cells treated with culture media) after 72 h
of treatment with different concentrations of S-HA-
NPs. The graphs include the mean ± SD (n = 8), and
the significance of the statistical comparison non-
treated cells (asterisks on top of bars) and between
the different systems (asterisks on brackets). Statis
tical analysis was performed by one-way ANOVA test
with *p < 0.01, **p < 0.05 and *** p < 0.001. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.) Fig. 9. Cell viability assays by Alamar Blue in MCF-7,
RAW264.7 and HUVEC cells treated with S-HA-NPs. a) Percentage of viable MCF-7 cells relative to control
(culture media of MCF-7 cells) after 72 h of treatment
with different concentrations of S-HA-NPs (dark grey)
or free NAP (orange, at equivalent doses to S-HA-
NPs). b) Percentage of MCF-7 (dark grey), RAW264.7
(blue) and HUVEC (pink) cell viability in respect to
controls (cells treated with culture media) after 72 h
of treatment with different concentrations of S-HA-
NPs. The graphs include the mean ± SD (n = 8), and
the significance of the statistical comparison non-
treated cells (asterisks on top of bars) and between
the different systems (asterisks on brackets). Statis
tical analysis was performed by one-way ANOVA test
with *p < 0.01, **p < 0.05 and *** p < 0.001. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.) Fig. 9. Cell viability assays by Alamar Blue in MCF-7,
RAW264.7 and HUVEC cells treated with S-HA-NPs. a) Percentage of viable MCF-7 cells relative to control
(culture media of MCF-7 cells) after 72 h of treatment
with different concentrations of S-HA-NPs (dark grey)
or free NAP (orange, at equivalent doses to S-HA-
NPs). b) Percentage of MCF-7 (dark grey), RAW264.7
(blue) and HUVEC (pink) cell viability in respect to
controls (cells treated with culture media) after 72 h
of treatment with different concentrations of S-HA-
NPs. The graphs include the mean ± SD (n = 8), and
the significance of the statistical comparison non-
treated cells (asterisks on top of bars) and between
the different systems (asterisks on brackets). 3.7. Apoptosis in NAP-treated MCF-7 cells. Quantification of p53 levels
by ELISA 3.7. Apoptosis in NAP-treated MCF-7 cells. Quantification of p53 levels
by ELISA Fig. 11. Intracellular p53 in S-HA-NPs or free NAP treated MCF-7 cells. ELISA
quantification of intracellular concentration of p53 in MCF-7 cells after 48 h of
treatment with S-HA-NPs (dark grey) or free NAP (orange, at equivalent doses
in S-HA-NPs). The graph includes the mean, ± SD (n = 2), and the statistical
comparison with non-treated cells (asterisks on top of bars) and between the
different systems (asterisks on brackets). Statistical analysis was performed by
one-way ANOVA test with *p < 0.01 and **p < 0.05. Among COX-independent mechanisms, NAP has demonstrated to
reduce MCF7 viability and proliferation via the inhibition of GSK-3β
[18]. GSK-3β is one of the main components of the complex responsible
for β-catenin proteolytic degradation in the Wnt/β-catenin signaling
pathway overexpressed in CSCs. In fact, pharmacologic inhibition of
GSK3 activity can lead to disruption of this complex and β-catenin sta
bilization and activation of β-catenin and TCF-dependent gene tran
scription [71]. It has been demonstrated that interferences in β-catenin
proteolytic degradation, like that caused by GSK3β inhibition, might
results in accumulation of p53 [72]. It has been previously observed that
treatment of MCF-7 cells with free NAP diminishes p53 mitochondrial
translocation promoting apoptosis of this cell line via mitochondrial
pathways [19]. In order to evaluate the apoptosis in NAP-treated MCF-7
cells, we measured the cytosolic accumulation of p53 by ELISA (Fig. 11). A significant dose-dependent accumulation of p53 was observed after
MCF-7 incubation with S-HA-NPs at 12.5 and 25 μg/mL. However, this
effect was not appreciated at the highest concentration of NPs. An in
crease in the concentration was associated with an increase in the hy
drodynamic properties of the NPs that could reduce in the
internalization efficiency of the NPs at high doses. this study, the percentage of open wound was graphically and numeri
cally presented in Fig. 12 and Table S1, respectively. S-HA-NPs at 12.5
and 25 μg/mL demonstrated a significant, concentration-dependent,
inhibition of cells migration. Moreover, the nanoparticle system had
significantly superior properties when compared to free drug at these
concentrations. S-HA-NPs at the highest concentration (50 μg/mL) did
not inhibit migration in a significant manner but had no effect as
compared with the control, as observed in the p53 experiment. 3.7. Apoptosis in NAP-treated MCF-7 cells. Quantification of p53 levels
by ELISA Previous works had demonstrated that COX-dependent pathways
does not affect migration capacity of MCF-7 cells [61] and that through
GSK-3β inhibition, NAP can compromise MCF-7 migration and survival
without affecting VEGF expression [19]. Therefore, the migration study,
together with all previous data, supported the idea of S-HA-NPs acting
through GSK-3β-related COX-independent pathway. 3.8. Wound healing assay to evaluate S-HA-NPs capacity to inhibit MCF-
7 migration 3.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells Statis
tical analysis was performed by one-way ANOVA test
with *p < 0.01, **p < 0.05 and *** p < 0.001. (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web
version of this article.) Materials Science & Engineering C 124 (2021) 112024
Fig. 10. ELISA quantification of extracellular PGE2 and VEGF in S-HA-NPs or free NAP treated MCF-7 cells. ELISA quantification of a) PGE2 and b) VEGF released by
MCF-7 cells after 72 h of treatment with either S-HA-NPs (dark grey) and free NAP (orange). The graphs include the mean ± SD (n = 2), and the statistical sig
nificance of the comparison with basal levels (dotted line). Statistical analysis was performed by one-way ANOVA test with **p < 0.05. E. Espinosa-Cano et al. Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. Fig. 10. ELISA quantification of extracellular PGE2 and VEGF in S-HA-NPs or free NAP treated MCF-7 cells. ELISA quantification of a) PGE2 and b) VEGF released by
MCF-7 cells after 72 h of treatment with either S-HA-NPs (dark grey) and free NAP (orange). The graphs include the mean ± SD (n = 2), and the statistical sig
nificance of the comparison with basal levels (dotted line). Statistical analysis was performed by one-way ANOVA test with **p < 0.05. [63], a growth factor that plays a key role in angiogenesis [64]. NSAIDs’
inhibitory effect on angiogenic factors are cell-, dose- and, time-
dependent. Depending on these variables, VEGF expression can be
reduced [65–67], increased [68], or non-significantly changed [69] by
NSAIDs. Moreover, according to literature, reduction in VEGF levels is
usually related to inhibition of PGE2 [66,70]. Therefore, to further
assess anti-angiogenic properties and mechanism of action of S-HA-NPs,
a study on VEGF production by MCF-7 cells after treatment with these
NAP-bearing NPs or free NAP was carried out (Fig. 10b). Basal levels of
VEGF were not reduced by S-HA-NPs nor free NSAID at any of the
concentrations tested. However, a tendency of lower VEGF levels after
treatment with S-HA-NPs when compared to free drug was appreciated. In fact, at the higher concentration of free drug, there was a significant
increase when compared to basal levels of VEGF, while no significant
changes were observed for the NP system. 3.6. COX-dependent markers (PGE2 and VEGF) in S-HA-NP-treated
MCF-7 cells Hence, these data correlated
with previous results on PGE2 supporting that anti-cancer activity of S-
HA-NPs occurs through COX-independent mechanisms. [63], a growth factor that plays a key role in angiogenesis [64]. NSAIDs’
inhibitory effect on angiogenic factors are cell-, dose- and, time-
dependent. Depending on these variables, VEGF expression can be
reduced [65–67], increased [68], or non-significantly changed [69] by
NSAIDs. Moreover, according to literature, reduction in VEGF levels is
usually related to inhibition of PGE2 [66,70]. Therefore, to further
assess anti-angiogenic properties and mechanism of action of S-HA-NPs,
a study on VEGF production by MCF-7 cells after treatment with these
NAP-bearing NPs or free NAP was carried out (Fig. 10b). Basal levels of
VEGF were not reduced by S-HA-NPs nor free NSAID at any of the
concentrations tested. However, a tendency of lower VEGF levels after
treatment with S-HA-NPs when compared to free drug was appreciated. In fact, at the higher concentration of free drug, there was a significant
increase when compared to basal levels of VEGF, while no significant
changes were observed for the NP system. Hence, these data correlated
with previous results on PGE2 supporting that anti-cancer activity of S-
HA-NPs occurs through COX-independent mechanisms. 0
10
20
30
40
50
[p53] (ng/mL) per million cells
[S-HA-NPs] ( g/mL)
0
50
25
12.5
CONTROL
S-HA-NPs
Free NAP
*
**
*
*
Fig. 11. Intracellular p53 in S-HA-NPs or free NAP treated MCF-7 cells. ELISA
quantification of intracellular concentration of p53 in MCF-7 cells after 48 h of
treatment with S-HA-NPs (dark grey) or free NAP (orange, at equivalent doses
in S-HA-NPs). The graph includes the mean, ± SD (n = 2), and the statistical
comparison with non-treated cells (asterisks on top of bars) and between the
different systems (asterisks on brackets). Statistical analysis was performed by
one-way ANOVA test with *p < 0.01 and **p < 0.05. 0
10
20
30
40
50
[p53] (ng/mL) per million cells
[S HA NP ] (
/ L)
0
50
25
12.5
CONTROL
S-HA-NPs
Free NAP
*
**
*
* CONTROL
S-HA-NPs
Free NAP [S-HA-NPs] ( g/mL) 3.8. Wound healing assay to evaluate S-HA-NPs capacity to inhibit MCF-
7 migration 4. Conclusion Hyaluronic acid electrostatic coating of previously described poly
(HNAP-co-VI)-based NPs was successfully carried out and the resultant
system was extensively characterized and tested in terms of GSK-3β-dependent anti-tumor activity of S-HA-NPs was further
investigated by evaluating migration of MCF-7 cells, a key step in the
metastasis sequence, which is also affected by GSK-3β inhibition [19]. In 10 Materials Science & Engineering C 124 (2021) 112024 E. Espinosa-Cano et al. Fig. 12. Wound healing assay in S-HA-NPs or free NAP treated MCF-7 cells. Effect of different concentrations of S-HA-NPs or free NAP on MCF-7 migration in vitro: a)
Inverted microscope images (20-fold magnification) of the wound at the beginning of the assay (0 h) and 24 h post-scratching and; b) Percentage of open wound after
24 h of treatment when compared to the original wound size. The graph includes the mean ± SD (n = 2) with non-treated cells (asterisks on top of bars) and between
the different systems (asterisks on brackets). Statistical analysis was performed by one-way ANOVA with *p < 0.01 and **p < 0.05. Fig. 12. Wound healing assay in S-HA-NPs or free NAP treated MCF-7 cells. Effect of different concentrations of S-HA-NPs or free NAP on MCF-7 migration in vitro: a)
Inverted microscope images (20-fold magnification) of the wound at the beginning of the assay (0 h) and 24 h post-scratching and; b) Percentage of open wound after
24 h of treatment when compared to the original wound size. The graph includes the mean ± SD (n = 2) with non-treated cells (asterisks on top of bars) and between
the different systems (asterisks on brackets). Statistical analysis was performed by one-way ANOVA with *p < 0.01 and **p < 0.05. honoraria; educational grants; participation in speakers’ bureaus;
membership, employment, consultancies, stock ownership, or other
equity interest; and expert testimony or patent-licensing arrangements),
or non-financial interest (such as personal or professional relationships,
affiliations, knowledge or beliefs) in the subject matter or materials
discussed in this manuscript. internalization, safety and therapeutic activity in different models. HA-
coating enhanced pH stability of the NP, provided a better control in the
esterase-dependent release of NAP and improved NP’s hemocompati
bility. Furthermore, HA-coated NPs are better and faster internalized in
CSC than in conventional non-stem cancer cells as a direct consequence
of the CD44 targeting. [1] H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray,
Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality
worldwide for 36 cancers in 185 countries, CA Cancer J Clin. (2021 Feb 4), https://
doi.org/10.3322/caac.21660. Epub ahead of print. PMID: 33538338. 4. Conclusion This feature coupled to the anti-inflammatory
activity from NAP makes HA-coating of poly(HNAP-co-VI)-based NPs
a potent strategy for targeting CSC subpopulation within breast tumors
by either directly attacking CSC or preventing their occurrence derived
in response to a pro-inflammatory state. The system also allowed to
reduce the dose of NAP needed to achieve pro-apoptotic and anti-
migratory activity against luminal breast cancer cells. Moreover, re
sults suggest that the anti-cancer activity of NPs could be related to the
induction of apoptosis through alterations of GSK-3β-related COX-
independent pathways. In summary, the developed NPs are a good
platform for the development of new anti-CSC therapies for metastatic
breast cancer treatment. Acknowledgement Authors would like to thank the Spanish Ministry of Science, Inno
vation and Universities (MAT2017-84277-R) and CIBER-BBN for the
financial support of this project. CIBER-BBN is financed by the Instituto
de Salud Carlos III (ISCIII) with assistance from the European Regional
Development Fund (ERDF).The work was also partially funded by ISCIII
(PI18_00871 co-founded by ERDF), and CIBER-BBN (EXPLORE) granted
to I.A. ICTS “NANBIOSIS” has participated in this work, more specif
ically the U20/FVPR in the hemocompatibility assays (http://www. nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). E. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.msec.2021.112024. Supplementary data to this article can be found online at https://doi. org/10.1016/j.msec.2021.112024. [2] R.M. Lu, M.S. Chen, D.K. Chang, C.Y. Chiu, W.C. Lin, S.L. Yan, Y.P. Wang, Y.S. Kuo,
C.Y. Yeh, A. Lo, H.C. Wu, Targeted drug delivery systems mediated by a novel
peptide in breast cancer therapy and imaging, PLoS One 8 (6) (2013), e66128. CRediT authorship contribution statement Espinosa-Cano would like to thank the training program for Academic
Staff (FPU15/06109) of the Spanish Ministry of Education Culture and
Sport. The kind support by Alvaro Gonz´alez-G´omez, Rosana Ramírez
from the Biomaterials Group (ICTP-CSIC) and Rafael Nu˜nez from the
Center for Biological Research (CIB-CSIC), in the synthesis, cell culture
and cryoTEM experiments, respectively, is greatly appreciated. Eva Espinosa-Cano: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Writing - original draft, Visualization. Miguel Huerta-Madro˜nal: Methodology, Validation, Investigation;
Patricia C´amara-S´anchez: Conceptualization, Methodology, Valida
tion, Formal analysis, Investigation, Writing - original draft, Visualiza
tion. Joaquin Seras-Franzoso: Methodology, Validation, Writing -
review & editing. Simo Schwartz Jr.: Methodology, Validation, Writing
- review & editing; Ibane Abasolo: Conceptualization, Resources,
Writing - review & editing, Supervision, Project administration, Funding
acquisition. Julio San Rom´an: Conceptualization, Resources, Writing -
review & editing, Supervision, Project administration, Funding acqui
sition; Maria Rosa Aguilar: Conceptualization, Resources, Writing -
review & editing, Supervision, Project administration, Funding
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https://openalex.org/W2279442433
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English
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PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET
|
Studies and Scientific Researches. Economics Edition
| 2,014
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cc-by
| 4,118
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Studies and Scientific Researches. Economics Edition, No 20, 2014 Studies and Scientific Researches. Economics Edition, No 20, 2014 http://sceco.ub.ro JEL Classification
M31 JEL Classification
M31 PARTICULARITIES OF CONSUMER BEHAVIOR IN THE
COSMETICS MARKET Eugenia Harja
“Vasile Alecsandri” University of Bacau
eugenia.harja@ub.ro
Laura Cătălina Ţimiraş
“Vasile Alecsandri” University of Bacau
timiras.laura@ub.ro Abstract Based on some results of a research organized in the county of Bacau on consumers of
cosmetics, using the questionnaire, this article analyses a number of issues with regard to
consumer behavior, namely: cosmetics brand most commonly purchased by consumers,
cosmetics category to which are allocated the largest sums of money, the amounts of money
that consumers are willing to spend per month to purchase these types of products, the
importance of some of the main criteria considered when buying cosmetic products and
differences manifested in categories of respondents by a number of variables such as age, sex,
marital status, income and living environment. The research was conducted using a sample of
500 respondents non-randomly selected, so that the results presented refer only to the studied
sample, being a guide to community from which it was extracted. Keywords
i Keywords
cosmetics; consumer behavior; brand; product selection criteria; expenses for cosmetics Research results Referring to cosmetics brand most frequently purchased by investigated persons, of
the 500 respondents, 28 did not provide information on this topic (indicated response
option "do not know / no answer" in the questionnaire). The results presented below
refer only to the 472 respondents who said what brand of cosmetics purchase most
often. Thus, from this point of view, Avon ranks first with a share of over 60% of
those who said they most often purchase this brand. The second place, but at a
considerable distance (15% of total respondents) stands Oriflame, a brand comparable
to first one in terms of specific distribution system (through direct sales), followed by
Nivea, L'Oreal, Adidas and Farmec. 3.6% of respondents stated that acquires most
common "other brand". For the category "other brand" respondents indicated: Puma
(0.6% of consumers) and Faberlic, Yves Rocher, Dolce & Gabana, Amway, Chanel,
Christian Dior, Fa, Gerocossen, Gucci, No.7., Versace, each of these being indicated
by less than 0.5% of consumers. For the categories of persons by various socio-economic and demographic criteria, we
found a number of differences concerning cosmetic brand most often purchased as
follows: •
By age groups, Avon is preferred to a greater extent by people over 20 years
(and especially over 30 years), while Nivea and Adidas are preferred to a
greater extent by the very young (15-20 years) compared with other age
groups. L'Oreal and Oriflame brands were only given by the category over
20 years and Farmec by those over 30; •
By gender, Avon products are preferred to a greater extent by women than
men, while a higher proportion of men (compared to women) use most often:
Oriflame, Nivea, L'Oreal, Adidas; •
Depending on civil status, people living together prefer in greater extent
Avon to those who live alone, while Nivea is preferred to a greater extent by
those who live alone compared to those who live together; •
In terms of income, there is not an obvious influence on preferences, i.e. there is no guidance showing greater or lesser extent in buying certain brands
with revenue growth; •
By residence, consumers show greater orientation towards Avon, Oriflame
and Adidas in urban areas, while L'Oreal and Nivea are preferred by those in
rural areas (compared to people in urban areas). Introduction During March-April 2014, in the county of Bacau, we organized a direct research,
using the questionnaire, in order to know the particularities of consumer behavior for
cosmetics. The research was exploratory, the obtained results being a guide for
reference in Bacău market, given that the determination of the investigated sample
(size and selection process) did not respect the principles of the theory of the survey. Some of the objectives pursued through this research and to which we referred in the
article were: knowledge of the cosmetics brand acquired by most of shoppers,
identifying the categories of cosmetics for which the consumers allocate the largest
sums of money, knowledge of the amount of money that a consumer is willing to
spend per month to purchase these products, determining the importance of some of
the main criteria for assessing the products in purchasing decision-making process,
and the extent to which variables such as age, sex, marital status, income and
environment influence the issues outlined above. During March-April 2014, in the county of Bacau, we organized a direct research,
using the questionnaire, in order to know the particularities of consumer behavior for
cosmetics. The research was exploratory, the obtained results being a guide for
reference in Bacău market, given that the determination of the investigated sample
(size and selection process) did not respect the principles of the theory of the survey. (size and selection process) did not respect the principles of the theory of the survey. Some of the objectives pursued through this research and to which we referred in the
article were: knowledge of the cosmetics brand acquired by most of shoppers,
identifying the categories of cosmetics for which the consumers allocate the largest
sums of money, knowledge of the amount of money that a consumer is willing to
spend per month to purchase these products, determining the importance of some of
the main criteria for assessing the products in purchasing decision-making process,
and the extent to which variables such as age, sex, marital status, income and
environment influence the issues outlined above. The community studied was the population over 15 years. We used the snowball
sampling, the questionnaires being administrated by interviewers. Investigated sample
of 500 people, represented the different categories of persons in terms of age (except
those under 15, these are not undertaken under study), gender, living environment,
income or marital status. 188 Research results Oriflame, 15.0%
Avon, 60.8%
Nivea, 7.8%
L'Oreal, 3.2%
Adidas, 2.8%
Farmec, 2.1%
London, 1.1%
Maybelline, 1.9%
Other brand, 3.6%
Zara, 0.8%
Metropolitan, 0.8%
Figure 1 Structure of respondents* depending on the brand of cosmetics most
frequently purchased
* were exempted respondents who did not answer London, 1.1% Farmec, 2.1% Avon, 60.8% Figure 1 Structure of respondents* depending on the brand of cosmetics most
frequently purchased
* were exempted respondents who did not answer q
y p
* were exempted respondents who did not answer 189 Harja, Țimiraș Harja, Țimiraș Table 1 Structure of respondents * depending on the brand of cosmetics most
frequently purchased by age, sex, marital status, income and living environment
(%)
Brand
Age
Gender
Marital status
15-20
years old
21-30
years old
31-40
years old
41-50
years old
Over 51
years old
Females
Males
Persons
living
alone
People
living
together
Avon
27
41
67
63
55
81
46
51
64
Oriflame
0
22
17
14
15
6
22
14
15
Nivea
36
7
5
8
9
5
10
11
7
L'Oreal
0
7
3
2
6
2
4
4
3
Adidas
9
4
2
2
3
0
5
4
2
Farmec
0
0
2
2
2
2
2
2
2
Other brand
27
19
3
8
11
5
11
15
6
Total
100
100
100
100
100
100
100
100
100
Table 1 (continuation)
Brand
The average monthly income of the household
(average per person)
Residence
under 500
lei
between
500 and
1000 lei
between
1000 and
1500 lei
between
1500 and
2000 lei
between
2000 and
2500 lei
over 2500
lei
Urban
Rural
Avon
61
56
54
52
54
68
67
59
Oriflame
14
21
14
9
15
15
17
13
Nivea
11
10
4
4
8
8
6
8
L'Oreal
2
4
11
13
6
0
1
3
Adidas
2
4
7
0
4
2
3
1
Farmec
5
0
0
0
2
2
1
2
Other brand
7
4
11
22
11
5
5
14
Total
100
100
100
100
100
100
100
100
* were exempted respondents who did not answer Table 1 Structure of respondents * depending on the brand of cosmetics most
frequently purchased by age, sex, marital status, income and living environment
(%) Table 1 Structure of respondents * depending on the brand of cosmetics most
frequently purchased by age, sex, marital status, income and living environment
(%) Table 1 Structure of respondents * depending on the brand of cosmetics most
frequently purchased by age, sex, marital status, income and living environment
(%) Cosmetics for which investigated persons have allocated the most money were body
care products (soaps, shower gels, deodorants, lotions, etc.), followed by perfumes
and makeup. PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET •
By gender, more women (compared to men) allocate the largest amount of
money to perfumes, followed by makeup products, while more men (than
women) allocate the largest sums of money for skin, hair and body care; •
Depending on civil status, most people who live alone allocates the largest
amount of money to perfumes, and most of the people living along allocate
large sums to body care products; •
By income categories, it is noted that for perfume are allocated large sums of
money especially by high income categories, while most people with modest
incomes allocate the largest amount of money for body care products. There
is an orientation following the increasing of incomes, more and more people
investing in skin care products and perfumes (the largest sums of money are
allocated to these categories), to the detriment of products for hair and body; •
Most of the people in urban areas allocate money for perfumes, while in
rural areas, body care products are preferred. •
Most of the people in urban areas allocate money for perfumes, while in
rural areas, body care products are preferred. Research results The smallest amount of money was allocated for skin care products
(creams, cleansers, masks, shaving products, etc.) and hair care products (shampoo,
conditioner, treatment, etc.). Information presented refer exclusively to the 434
respondents who provided information on this issue. By socio-economic and demographic criteria, there is real differences between
respondents in terms of product categories for which they are allocated the largest
amount of money, namely: By socio-economic and demographic criteria, there is real differences between
respondents in terms of product categories for which they are allocated the largest
amount of money, namely: •
By age groups, it is observed that most young allocate large sums of money
especially for body or hair care products, while people over 50 years for
perfumes. With age there is a shift in needs so that more people allocate the
largest sums of money for skin care products and fragrances at the expense
of hair and body care. To makeup products are allocated the largest sums of
money by people of 31-40 years; 190 PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET Body care
products (soaps,
shower gels,
deodorants,
lotions etc.),
33.4%
Skin-care
products (creams,
cleansers, masks,
shaving products
etc.), 10.4%
Make-up, 15.7%
Hair care products
(shampoo,
conditioner,
treatment etc.),
11.1%
Perfumes, 29.5%
Figure 2 Structure of respondents* depending on the products to which are
allocated the largest sums of money
* were exempted respondents who did not answer Body care
products (soaps,
shower gels,
deodorants,
lotions etc.),
33.4% Hair care products
(shampoo,
conditioner,
treatment etc.),
11.1% Figure 2 Structure of respondents* depending on the products to which are
allocated the largest sums of money
* were exempted respondents who did not answer Table 2 Structure of respondents * depending on the products to which are
allocated the largest sums of money by age, sex, marital status, income and living
environment (%)
Product category
Age
Gender
Marital
status
15-20 years
old
21-30 years
old
31-40 years
old
41-50 years
old
Over 51
years old
Females
Males
Persons
living alone
People living
together
Skin-care products (creams,
cleansers, masks, shaving products
etc.)
3
4
13
10
16
5
17
9
11
Make-up
3
6
35
11
13
29
0
27
10
Perfumes
0
4
31
31
56
32
27
39
25
Hair care products (shampoo,
conditioner, treatment etc.)
29
40
10
3
6
5
18
9
12
Body care products (soaps, shower
gels, deodorants, lotions etc.)
65
46
13
46
9
29
38
16
41
Total respondents
100
100
100
100
100
100
100
100
100 Table 2 Structure of respondents * depending on the products to which are Table 2 Structure of respondents * depending on the products to which are
located the largest sums of money by age, sex, marital status, income and living
environment (%) 191 Harja, Țimiraș Table 2 (continuation)
Product category
The average monthly income of the household
(average per person)
Residence
under 500
lei
between
500 and
1000 lei
between
1000 and
1500 lei
between
1500 and
2000 lei
between
2000 and
2500 lei
over 2500
lei
Urban
Rural
Skin-care products (creams,
cleansers, masks, shaving
products, etc.)
3
6
17
16
14
15
16
4
Make-up
22
6
13
31
5
10
19
12
Perfumes
7
34
23
31
74
65
44
15
Hair care products (shampoo,
conditioner, treatment, etc.)
21
17
8
1
0
0
5
17
Body care products (soaps,
shower gels, deodorants,
lotions, etc.)
48
38
38
19
7
10
15
52
Total respondents
100
100
100
100
100
100
100
100
* were exempted respondents who did not answer Table 2 (continuation) Regarding the amount of money consumers are willing to spend monthly for the
purchase of cosmetics, of the 498 respondents who provided information on this
subject, most of them - 47% - indicated a value between 50 and 100 lei. PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET between 100
and 150 lei,
13.2%
between 50 and
100 lei, 47.0%
less than 50 lei,
27.8%
between 150
and 200 lei,
5.0%
between 200
and 300 lei,
2.8%
over 300 lei,
3.8%
Figure 3 Structure of respondents* according to the amount of money they are
willing to spend monthly for the purchase of cosmetics
* were exempted respondents who did not answer between 100
and 150 lei,
13.2%
between 50 and
100 lei, 47.0%
less than 50 lei,
27.8%
between 150
and 200 lei,
5.0%
between 200
and 300 lei,
2.8%
over 300 lei,
3.8% between 200
and 300 lei,
2.8% over 300 lei,
3.8% Figure 3 Structure of respondents* according to the amount of money they are
willing to spend monthly for the purchase of cosmetics
* were exempted respondents who did not answer 192 PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET Table 3 Structure of respondents* according to the amount of money they are
willing to spend per month to purchase cosmetics by age, gender, marital status,
income and living environment (%)
Monthly allowance
Age
Gender
Marital
status
15-20 years
old
21-30 years
old
31-40 years
old
41-50 years
old
Over 51
years old
Females
Males
Persons
living alone
People living
together
less than 50 lei
51
55
50
11
14
25
31
30
24
between 50 and 100 lei
34
33
37
57
49
47
48
34
54
between 100 and 150 lei
14
8
7
17
17
16
10
17
11
between 150 and 200 lei
0
4
3
7
4
5
5
7
4
between 200 and 300 lei
0
0
1
1
13
2
4
5
2
over 300 lei
0
0
2
6
4
5
2
7
2
Total respondents
100
100
100
100
100
100
100
100
100
Table 3 (continuation)
Monthly allowance
The average monthly income of the
household (average per person)
Residence
under 500 lei
between 500
and 1000 lei
between 1000
and 1500 lei
between 1500
and 2000 lei
between 2000
and 2500 lei
over 2500 lei
Urban
Rural
Less than 50 lei
42
33
23
13
16
8
22
35
between 50 and 100 lei
30
36
69
76
33
48
53
42
between 100 and 150 lei
16
10
4
4
36
36
12
14
between 150 and 200 lei
5
7
4
3
2
8
5
5
between 200 and 300 lei
3
7
1
0
2
0
3
3
over 300 lei
4
6
0
3
11
0
6
1
Total respondents
100
100
100
100
100
100
100
100
* were exempted respondents who did not answer Table 3 Structure of respondents* according to the amount of money they are
willing to spend per month to purchase cosmetics by age, gender, marital status,
income and living environment (%) Table 3 Structure of respondents* according to the amount of money they are
willing to spend per month to purchase cosmetics by age, gender, marital status,
income and living environment (%) Table 3 (continuation)
Monthly allowance
The average monthly income of the
household (average per person)
Residence
under 500 lei
between 500
and 1000 lei
between 1000
and 1500 lei
between 1500
and 2000 lei
between 2000
and 2500 lei
over 2500 lei
Urban
Rural
Less than 50 lei
42
33
23
13
16
8
22
35
between 50 and 100 lei
30
36
69
76
33
48
53
42
between 100 and 150 lei
16
10
4
4
36
36
12
14
between 150 and 200 lei
5
7
4
3
2
8
5
5
between 200 and 300 lei
3
7
1
0
2
0
3
3
over 300 lei
4
6
0
3
11
0
6
1
Total respondents
100
100
100
100
100
100
100
100
* were exempted respondents who did not answer Referring to the importance of some of the main criteria for selecting cosmetics
(shown in table 4), we note that, firstly, consumers appreciate the best quality-price
options, followed by convenience in purchasing products, the nature of the ingredients
(natural) and finally, brand awareness. PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET About 28%
are willing to spend less than 50 RON and 13% between 100 and 150 lei. The differences are observed by categories of respondents, as follows: •
With age consumers are willing to spend more money to cosmetics, most
until 40 years (50%) indicating the range of "less than 50 lei", while this
range is indicated by under 15 % of those over 40 years; •
By gender, we found slight differences, women being decided in a greater
extent, to allocate more money to cosmetics; •
Depending on civil status, most people living together (54%) are willing to
spend between 50 and 100 lei per month for the purchase of cosmetics. The
category of those living alone show a greater heterogeneity in terms of
amounts that they are willing to allocate to cosmetic products during a
month; ;
•
As expected, higher income consumers are willing to spend more money to
cosmetics, compared to those with low incomes; •
People in urban areas are also willing to allocate more money to cosmetics
compared to those in rural areas. PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET Note that all 500 respondents expressed their
views on this issue. Significant differences are recorded in the categories of persons by considered socio-
economic and demographic criteria, namely: Significant differences are recorded in the categories of persons by considered socio-
economic and demographic criteria, namely: •
By age groups, the importance of all 4 criteria in the purchase of cosmetics is
higher for older age categories. The biggest differences between young and
old is registered in terms of the importance attributed to convenience in
acquiring products; •
By gender, differences are found in terms of the importance attributed to the
acquisition comfort and used ingredients. Thus, if more men appreciate the
convenience of buying process, women are more interested in natural
ingredients; 193 Harja, Țimiraș Harja, Țimiraș •
Depending on civil status, the importance attributed to the acquisition
comfort is different, being more appreciated by those who live together. A
similar situation is seen for the use of natural ingredients criterion, but
differences between categories of respondents being smaller; •
In relation to household income, the biggest difference in terms of the
importance given to the process of buying cosmetic products is registered for
the use of natural ingredients and brand awareness, followed by convenience
in purchasing, all being considered important by those with higher incomes; p
g
g
p
y
g
•
By residence, a greater importance in the purchase decision is given in urban
areas compared to rural area to brand awareness and use of natural
ingredients. On the other hand, compared to those in urban areas, rural
respondents indicated that the most important are best quality-price option
and convenience in the purchase. Table 4 Distribution of respondents according to the importance given to some of
the main selection criteria for cosmetic products on a scale from 5 (very
important) to 1 (unimportant) and calculated average score for each criterion PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET PARTICULARITIES OF CONSUMER BEHAVIOR IN THE COSMETICS MARKET Table 5 (continuation)
Criteria
The average monthly income of the
household (average per person)
Residence
under 500 lei
between 500
and 1000 lei
between 1000
and 1500 lei
between 1500
and 2000 lei
between 2000
and 2500 lei
over 2500 lei
Urban
Rural
Best quality-price
option
4.5
4.1
4.9
4.8
4.7
4.5
4.3
4.9
Brand awareness
2.7
2.5
3.4
3.9
4.7
4.6
3.6
2.8
Convenience in the
purchase
3.8
3.4
3.8
4.1
4.5
4.5
3.7
4.1
Use of natural
ingredients
2.8
3.3
3.8
4.7
4.7
4.8
4.1
3.3 Table 5 (continuation) Conclusions In conclusion, based on the results of exploratory research undertaken using a sample
of 500 individuals, it was revealed that the most commonly purchased brand of
cosmetics is Avon (over 60% of persons who provided information on this subject),
followed by Oriflame (15%). Cosmetics for which investigated persons allocated the
most money are body care products (soaps, shower gels, deodorants, lotions, etc.),
followed by perfumes and makeup. Nearly half of respondents (47%) are willing to
monthly spend for cosmetics an amount between 50 and 100 lei, around 28% under
50 lei and 13% between 100 and 150 lei. The criteria rated as the most important in
the process of buying cosmetic products are: best quality-price ratio, followed by
convenience in purchasing products. Ţimiraş, C. Laura (2007), Tendinţe în evoluţia marketingului agroalimentar în contextul
extinderii Uniunii Europene, Editura EduSoft, Bacău. Table 4 Distribution of respondents according to the importance given to some of
the main selection criteria for cosmetic products on a scale from 5 (very
important) to 1 (unimportant) and calculated average score for each criterion Criteria
Awarded score
Average
score
5
4
3
2
1
Best quality-price option
325
168
7
-
-
4.6
Brand awareness
114
98
114
105
69
3.2
Convenience in the
purchase
228
105
66
72
29
3.9
Use of natural ingredients
158
159
74
84
25
3.7 Table 5 Average scores calculated on a scale from 5 (very important) to 1
(unimportant) of some of the main criteria for selecting cosmetics, depending on
the focus of consumer in the process of buying cosmetic products by age, sex,
marital status, income and living environment Criteria
Age
Gender
Marital status
15-20 years
old
21-30 years
old
31-40 years
old
41-50 years
old
Over 51
years old
Females
Males
Persons
living alone
People living
together
Best quality-price
option
3.9
4.5
4.7
4.6
4.8
4.8
4.4
4.6
4.6
Brand awareness
2.1
1.9
2.7
3.9
3.5
3.1
3.3
3.1
3.3
Convenience in the
purchase
2.7
2.9
4.4
4
4.3
3.5
4.3
3.3
4.2
Use of natural
ingredients
2.8
2.7
3.3
4.2
4.1
4
3.3
3.4
3.9 194 Harja, Eugenia, Laura C. Ţimiraş. (2010), Metode statistice utilizate în cercetarea de
marketing, Editura Alma Mater, Bacău. Aaker, David A., V. Kumar, George S. Day (1998), Marketing research, 6th Edition, John
Wiley & Sons, Inc., New York, ş.a.
Cătoiu, Iacob, Nicolae Teodorescu (2003), Comportamentul consumatorului, Editura Uranus,
Bucureşti. References:
Aaker, David A., V. Kumar, George S. Day (1998), Marketing research, 6th Edition, John
Wiley & Sons, Inc., New York, ş.a.
Cătoiu, Iacob, Nicolae Teodorescu (2003), Comportamentul consumatorului, Editura Uranus,
Bucureşti.
Cătoiu, Iacob (coordonator) (2002), Cercetări de marketing, Editura Uranus, Bucureşti.
Harja, Eugenia, Laura C. Ţimiraş. (2010), Metode statistice utilizate în cercetarea de
marketing, Editura Alma Mater, Bacău.
Ţimiraş, C. Laura (2007), Tendinţe în evoluţia marketingului agroalimentar în contextul
extinderii Uniunii Europene, Editura EduSoft, Bacău. References: Aaker, David A., V. Kumar, George S. Day (1998), Marketing research, 6th Edition, John
Wiley & Sons, Inc., New York, ş.a. y
ş
Cătoiu, Iacob, Nicolae Teodorescu (2003), Comportamentul consumatorului, Editura Uranus,
Bucureşti. Cătoiu, Iacob (coordonator) (2002), Cercetări de marketing, Editura Uranus, Bucureşti. Harja, Eugenia, Laura C. Ţimiraş. (2010), Metode statistice utilizate în cercetarea de
marketing, Editura Alma Mater, Bacău. Ţimiraş, C. Laura (2007), Tendinţe în evoluţia marketingului agroalimentar în contextul
extinderii Uniunii Europene, Editura EduSoft, Bacău. 195
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Oral Health: The Need for Both Conventional Microbial and Molecular Characterization
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Oral Health: The Need for Both Conventional Microbial and
Oral Health: The Need for Both Conventional Microbial and
Molecular Characterization
Molecular Characterization Elisheva Friedman
Touro College of Pharmacy Follow this and additional works at: https://touroscholar.touro.edu/tcopny_pubs Part of the Oral Biology and Oral Pathology Commons Part of the Oral Biology and Oral Pathology Commons Touro Scholar
Touro Scholar Touro Scholar
Touro Scholar Touro College of Pharmacy (New York)
Publications and Research Touro College of Pharmacy (New York) Elisheva Friedman 1, Negin Alizadeh 1 and Zvi Loewy 1,2,* 1
Department of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy, New York, NY 10027,
USA; eswartz@student.touro.edu (E.F.); nalizade@student.touro.edu (N.A.)
2
Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA
*
Correspondence: zvi.loewy@touro.edu; Tel.: +1-646-981-4718
Academic Editor: Massimo Negrini
Received: 10 July 2017; Accepted: 27 July 2017; Published: 1 August 2017 1
Department of Pharmaceutical and Biomedical Sciences, Touro College of Pharmacy, New York, NY 10027,
USA; eswartz@student.touro.edu (E.F.); nalizade@student.touro.edu (N.A.)
2
Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA
*
Correspondence: zvi.loewy@touro.edu; Tel.: +1-646-981-4718 Academic Editor: Massimo Negrini
Received: 10 July 2017; Accepted: 27 July 2017; Published: 1 August 2017 Received: 10 July 2017; Accepted: 27 July 2017; Published: 1 August 2017 Abstract: This study aims to consider the microbial distribution in oral disease, as well as gene
analysis and expression, in elucidating: 1, the fundamental underpinnings of oral disease, and
2, the potential relationship between oral diseases and systemic health. A key focus is identifying
the microbiota associated with oral disease manifestations characterized by both conventional
microbiological and molecular methods. Variations in the observed microbial populations
characterized by conventional and molecular approaches have been identified for caries, periodontitis,
peri-implantitis, and stomatitis. The discovery of therapeutic approaches for oral disease will require
comprehensive microbial and genomic analysis. This study evaluated the current state of the relevant
microbial and genomic information for several prevalent oral diseases. Keywords: oral disease; systemic health; oral microbiota; microarrays Oral Health: The Need for Both Conventional
Microbial and Molecular Characterization Elisheva Friedman 1, Negin Alizadeh 1 and Zvi Loewy 1,2,* Recommended Citation
Recommended Citation Friedman, E., Alizadeh, N., & Loewy, Z. G. (2017). Oral health: The need for both conventional microbial
and molecular characterization. High-Throughput, 6(3) [Article 2]. This Article is brought to you for free and open access by the Touro College of Pharmacy (New York) at Touro
Scholar. It has been accepted for inclusion in Touro College of Pharmacy (New York) Publications and Research by
an authorized administrator of Touro Scholar. For more information, please contact touro.scholar@touro.edu. 2. Caries Dental caries is one of the most widespread chronic diseases [4]. Current epidemiological studies
indicate a marked increase in the prevalence of dental decay among all age groups [5]. Dental caries
is a polymicrobial infection that results from an imbalance of the dynamic metabolic process in the
dental biofilm [6]. The ultimate harm to the teeth is not apparent until the mineralization balance in
the supragingival biofilm is disturbed, affecting homeostasis in the biofilm [7]. Classical experimental
techniques have provided significant information on the microorganisms associated with dental caries. Tooth decay is initially activated by early colonizers such as Streptococcus oralis, followed by adherence
of Streptococcus sobrinus and Stretococcus mutans [8]. More recently, the Human Oral Microbe Identification Microarray (HOMIM) has been used
to provide a more comprehensive description of the biofilm composition associated with the oral
cavity. It can detect pathogens regardless of whether they can be cultivated or not [6,8]. This new
metagenomic approach may also be useful to assess the dynamic process of the metabolic activity of
biofilms. Microarrays hold high promise for advancements in oral biology. They are specifically useful
for the diagnosis, prevention, and monitoring of microorganisms in the oral cavity, which should lead
to better management of patients’ oral health [9]. One of the areas in clinical dentistry where microarrays have proved to be very effective is
the analysis of the oral microbiota in pediatric patients between the age of three months and three
years [10]. Using gene chip analysis, the relationship between the microorganisms and the presence
or absence of caries can be assessed. Microarray analysis has shown that many of the bacteria that
colonize the oral cavity at three months of age continue to be present at three years of age. A few of the
early bacteria cease to exist as the baby matures, and correspondingly, new pathogens start colonizing
at age three for more than 50% of children [10]. Although the microbiota composition at three months
of age is unrelated to caries development at a later age, several pathogens present in the oral biofilms
of three-year-olds can be linked to caries [10]. DNA sequencing has also been used to examine the bacterial community associated with caries. The 454 sequencing technology (454 Life Sciences, Branford, CT, USA) was used to evaluate microbial
diversity influenced by the pH of cavitated lesions [11]. 2. Caries pH within a dentinal cavitated lesion was
found to significantly affect the microbial population of 42% of the caries-associated bacteria. Interestingly, bacterial culture methodologies continue to provide information on the bacterial
composition associated with caries. An anaerobic culture of bacteria associated with caries has been very
valuable in elucidating the bacterial population. Using rich non-selective media and anaerobic incubation
has resulted in the improved detection of Actinobacteria as compared to PCR and cloning/sequencing
analysis [12]. Table 1 summarizes the representative bacteria associated with dental caries. Table 1. Microbiota associated with caries. Conventional Methods
Molecular Methods
References
Streptococcus
Lactobacillus
Actinomyces
Streptococcus
Lactobacillus
Actinomyces
Bifidobacterium
Propionibacterium
Veillonella
Selenomonas
Atopobium
[11,12]
ostics Table 1. Microbiota associated with caries. Conventional Methods
Molecular Methods
References
Streptococcus
Lactobacillus
Actinomyces
Streptococcus
Lactobacillus
Actinomyces
Bifidobacterium
Propionibacterium
Veillonella
Selenomonas
Atopobium
[11,12]
stics Table 1. Microbiota associated with caries. Table 1. Microbiota associated with caries. 1. Introduction Over the past 30 years, research has begun to elucidate the etiology of oral diseases. A key contributor to oral disease is the complex oral microbiome. The oral cavity is home to
about 700 microbial species, many of which interact with host factors. Oral disease resulting from
microbe–host interaction can cause systemic diseases, some of which may be lethal. The composition of the oral microbiota in dentate individuals differs significantly from that of
edentulous individuals [1]. The oral microbiome of dentate individuals contains greater proportions of
anaerobes and spirochetes, while the prosthetic dentures of edentate patients harbor a larger percentage
of aerobic organisms, as well as yeast and lactobacilli [2,3]. This dissimilarity must be considered
when examining a potential oral and systemic connection. Figure S1 summarizes the similarities and
differences of microbial communities present in dentate and edentulous populations [1]. The study of genomes, both human and microbial, can provide insight into the etiology of
disease, as well as help elucidate potential approaches for therapeutic intervention. DNA microarrays,
also referred to as gene chips, allow for massively parallel, rapid screening of thousands of genes. The methodology allows for the identification of genes that are expressed differentially. Gene chips
show the modulation of mRNA expression levels, suppression of gene expression, and activation of
gene expression. This technology allows for large-scale genomic analysis between patients with and
without a given disease. The objectives of this paper include: (i) to provide a perspective for the role of microbes in
several oral diseases; (ii) to identify differences between microbes detected by classical microbiological
approaches as compared to molecular analyses; and (iii) to provide a genomic perspective on the
relationship between oral and systemic health. 2017, 6, 2; doi:10.3390/ht6030002 www.mdpi.com/journal/high-throughput 2017, 6, 2 2017, 6, 2 2 of 9 3. Salivary Diagnostics Saliva is a biological fluid secreted by the salivary glands. Saliva contains bacteria originating
from the surfaces of various intraoral surfaces, including teeth, gingival crevices, tongue and buccal
mucosa [13]. Bacterial species prevalent in saliva are summarized in Table 2. Saliva is also a rich source of proteins, mRNA, miRNA (non-coding RNA), and antibodies [14–19]. It therefore has immense diagnostic potential, both for identifying individuals with a given disease 3 of 9 2017, 6, 2 2017, 6, 2 and for tracking patients’ disease progression and treatment response. An obvious advantage of this
strategy is that the collection of saliva is a non-invasive procedure. and for tracking patients’ disease progression and treatment response. An obvious advantage of this
strategy is that the collection of saliva is a non-invasive procedure. Table 2. Microbiota prevalent in saliva. Bacteria
References
Streptococcus
[13]
Granulicatella
Neisseria
Rothia
Prevotella Bacteria
References
Streptococcus
[13]
Granulicatella
Neisseria
Rothia
Prevotella Microarrays have proven to be excellent experimental platforms to analyze saliva [14,17]. The results provided by microarrays are rapid and remarkably precise, two factors that are key
in diagnostics. Salivary analysis by microarrays has been used to identify and track numerous systemic
diseases. For example, fiber microarrays have been used to demonstrate the altered salivary protein
profile of patients with asthma and cystic fibrosis [20], whereas a microsphere-based array could not
identify a significant alteration. Investigators have even developed an effective discriminatory salivary
test, in which microarray detection of the downregulation of five salivary mRNA biomarkers reliably
indicates the presence of ovarian cancer [21]. Many other diseases have been shown to impact the saliva’s contents. While microarrays have
not yet been used as a diagnostic tool for these clinical states, there is certainly potential for the
development of such procedures. There are numerous diseases whose impact has been illustrated:
19 genes are differentially expressed in the saliva of Sjogren’s syndrome patients [22], and autoantigens
in the saliva differ according to the subtype of systemic lupus erythematous [23]. The blood-borne
pathogens, human immunodeficiency virus [24] and hepatitis C virus antibodies can be found in
salivary concentrations that correlate to the systemic viral load [25]. With respect to metabolic diseases,
65 proteins are differentially expressed in the saliva of patients with type II diabetes mellitus [26]. 4. Gingivitis and Periodontitis The composition of the oral microbiota has been investigated for well over half a century. With the
advent of molecular diagnostic assays including DNA probes and PCR, as well as immunoassays
designed to characterize the association between the subgingival microbiota and the levels of
biomarkers released by tissues and cells measured in gingival crevicular fluid (GCF), significant
progress has been made in elucidating the composition of the subgingival microbiota. The role
of five main microbial complexes in the gingival biofilm was characterized and described using
a checkerboard DNA–DNA hybridization [32]. Using cloning and Sanger sequencing, as well as
next-generation sequencing techniques, suggested that cultivatable as well as not-yet-cultivatable
microbial species are involved in the etiology of periodontitis [33,34]. Based upon current information,
it appears that periodontal disease is the result of infection, with a relatively small number of interacting
species. Periodontal microbiota identified by classical microbiology methods as well as molecular
approaches are summarized in Table 4. A systemic review was reported that showed the association
of 17 species/phylotypes from the Bacteria domain, the Candidatus Saccharibacteria phylum, and the
Archaea domain with the etiology of periodontitis [35]. Table 4. Microbiota associated with periodontitis. Conventional Methods
Molecular Methods
References
Porphyromonas
Bacteroides
[35,36]
Prevotella
Firmicutes
Tannerella
Proteobacteria
Treponema
Spirochaeta
Fusobacterium
Candida
Campylobacter Table 4. Microbiota associated with periodontitis. In clinical practice, microarrays can be used to detect and quantify the specific pathogens
responsible for periodontitis [37]. They can also be utilized to identify whether the pathogens are
the ones more likely to be associated with refractory periodontitis, and to assess the efficacy of
periodontal therapy [38]. Kinney et al. [39] demonstrated that by examining the salivary concentrations
of pathogens, matrix metalloproteinase-8 and -9 (MMP-8, MMP-9), calprotectin, osteoprotegerin
(OPG), tumor necrosis factor (TNFα), interferon (IFN), and numerous interleukins (ILs), one can
examine the progression or non-progression of periodontal disease. Furthermore, a number of these
salivary solutes can be used predictively: high concentrations of pathogens Fusobacterium nucleatum,
Campylobacter rectus and Prevotella intermedia predict disease progression, while low levels of MMP-8,
MMP-9, OPG and IL-1β predict stability [39]. From a research perspective, microarrays are valuable tools for gaining further understanding
of the pathophysiology of periodontitis. 3. Salivary Diagnostics There is potential for advancement in cancer salivary diagnostics as well; proteins, mRNA and miRNA
have been shown to have distinct patterns in patients with breast cancer [27], head and neck squamous
cell carcinoma [28], lung cancer [29], oral squamous cell carcinoma [30], and resectable pancreatic
cancer [31] (Table 3). Table 3. Cancer salivary biomarkers. Table 3. Cancer salivary biomarkers. Cancer
Up-Regulation
Down-Regulation
Reference
Breast Cancer
Vascular Endothelial Growth Factor (VEGF)
Epidermal Growth Factor (EGF)
Carcinoembryonic Antigen (CEA)
[27]
Head and Neck
Squamous Cell
Carcinoma
miRNA-9
miRNA-191
miRNA-134
[28]
Lung Cancer
CCNI
FGF19
FRS2
GREB1
EGFR
[29]
Oral Squamous Cell
Carcinoma
miRNA-24
miRNA-27b
miRNA-136
miRNA-147
miRNA-1250
miRNA-148a
miRNA-632
miRNA-646
miRNA668
miRNA-877
miRNA-503
miRNA-220a
miRNA-323-5p
[30]
Resectable Pancreatic
Cancer
miRNA-940
miRNA-3679-5p
[31]
CCNI: Cyclin I; FGF19: Fibroblast Growth Factor 19; FRS2: Fibroblast Growth Factor Receptor Substrate 2; GREB1:
Growth Regulation by Estrogen in Breast Cancer 1. 2017, 6, 2 4 of 9 4. Gingivitis and Periodontitis For example, they have been used to demonstrate the
involvement of long non-coding RNAs (IncRNAs) in the pathogenesis, with the upregulation of
4313 and downregulation of 4612 lncRNAs in chronic periodontitis tissue [40], and to establish
that both chronic and aggressive periodontitis have similar gene expression profiles, with limited
differences in the gingival transcription patterns [41]. Interestingly, Schaefer [42] used microarray
analysis to reveal that certain genetic variations thought to bear connection to periodontitis do not,
in fact, predispose patients to development of this disease [42]. Microarrays have even been used to
identify an apoptotic pathway as a potential anti-periodontitis pharmacological target [43]. Microarrays are often used in comparative studies to determine altered levels of bacterial
pathogens, or the expression of genes in patients with and without periodontitis. Belstrom et al. demonstrated that certain bacterial taxa are upregulated only in the saliva of periodontitis patients,
rather than the saliva of patients with good oral health [44]. MicroRNA (miRNA) has been a large focus
in this subfield; Xie discovered that in comparison to healthy gingival tissue, inflamed gingival tissue
caused the upregulation of 91 miRNAs and downregulation of 34 miRNAs, all over two-fold [45]. 5 of 9 2017, 6, 2 Similarly, Lee showed that six miRNAs are upregulated in periodontitis [46]. It has been suggested
that salivary miRNAs will comprise the next generation of diagnostic periodontitis tests, but it will be
necessary to first develop standardized tests and protocols [47]. 5. Peri-Implantitis Reconstructive dentistry today is largely facilitated by dental implants, as compared to fixed or
removable partial dentures. During the past decade, the rate of growth for osseointegrated dental
implants in Americans has been estimated at approximately 500,000 per year [48]. The increase in the
number of implants has also given rise to a corresponding increase in clinical problems associated with
the implants. Two diseases, peri-implant mucositis and peri-implantitis, have emerged as diseases
associated with dental implants. Peri-implant mucositis is similar to gingivitis; it is characterized by
inflammation of the mucosa without a corresponding bone loss. Peri-implantitis is characterized as a
more severe inflammatory lesion, with a loss of supporting bone around an implant. Like gingivitis
and periodontitis, peri-mucositis and peri-implantitis are also initiated with microbial infections. Peri-implantitis can result in a loss of the implant as well as infection of other implants or the remaining
natural teeth. Peri-implantitis is asymptomatic, since pain is infrequent. As a consequence, patients do
not recognize that a problem exists until the onset of implant mobility. Of all implant recipients, the
prevalence of peri-implantitis has been estimated at 28–56% [48]. Peri-implantitis is a polymicrobial infection [48]. There are relatively few studies that have used
molecular approaches to characterize the peri-mucositis and peri-implantitis microbiota [49]. The use
of 16S-based sequencing suggests that the peri-implant microbiome may be distinct from that of
the periodontal microbiome. To date, no deep metagenomic sequencing analyses of peri-implantitis
samples have been reported. Table 5 summarizes the microbiota associated with peri-implantitis, as
identified by both conventional as well as molecular methodologies [50,51]. Table 5. Microbiota associated with peri-implantitis. Table 5. Microbiota associated with peri-implantitis. Table 5. Microbiota associated with peri-implantitis. Conventional Methods
Molecular Methods
References
Bacillus
Aggregatibacter
Candida
Staphylococcus
Enterococcus
Streptococcus
Porphyromonas
Fusobacterium
Prevotella
Bacillus
Neisseria
Kingella
Veillonella
Capnocytophaga
Paracoccus
Leptotrichia
Tannerella
Treponema
[49–51] Bacillus
Aggregatibacter
Candida
Staphylococcus Limited studies focused on the genomic and gene expression profiles for peri-implantitis have
been reported [52]. Peri-implant healing has been associated with the differential expression of several
genes, including cytokines, growth factors, transcription factors and secretory products. 6. Stomatitis In denture stomatitis (DS), the denture is a major reservoir of many microbes, especially
Candida albicans, a chronic source of infection. The denture surface provides a matrix that allows
for the development of a pathogenic yeast biofilm. The surface under a denture is more acidic and
less open to the oral mucosal saliva. This provides for an ideal environment for C. albicans enzymatic 2017, 6, 2 6 of 9 activity, which leads to C. albicans biofilm colonization and resistance. C. albicans biofilms are the
reservoirs for infection; they are enclosed within their own extracellular matrix (ECM) and attached to
the surface. The biofilm composition and structure protects the fungi from the environment, physical and
chemical stress agents, and provides resistance to antifungal agents as well. Indeed, the biofilm is up
to 1000-fold more resistant to antifungal agents than planktonic free-floating cells [53]. Planktonic cells
are thin, and have to undergo irreversible genetic changes to provide resistance. However, biofilms are
200–300 nm thick, and are able to persist due to their physical presence in a phase-specific manner,
regardless of genetic alteration. The biofilm resistance is correlated with efflux pumps that develop
during the intermediate phases of biofilm formation and extracellular matrix (ECM) production. Table 6 lists the microbes associated with stomatitis that have been identified by conventional
microbial methodologies. The microbes related to stomatitis that have been identified by molecular
analytical approaches are catalogued in Figure S1. The contribution of molecular biology to the
elucidation of stomatitis-related microbes is readily apparent upon comparing the microbial population
identified by conventional methods with those characterized by molecular approaches. Table 6. Microbiota associated with stomatitis. To better understand the molecular underpinnings of DS, microarray technologies have been
used to assess gene expression variability, from an early stage of biofilm growth to the maturity
associated with DS. The genomic composition of the mucosa of healthy denture wearers has been
compared to the mucosa of denture wearers with DS, and it was found that more than 3000 genes
are subjected to transcriptional expression changes in the diseased state as compared to the healthy
state [55]. Among those genes differentially expressed, 71 genes were downregulated. These genes
code for neutrophil, lymphocyte, monocyte, keratins, and epithelial adhesion molecules, all of which
mediate an innate response and the release of inflammatory mediators in DS. 6. Stomatitis In contrast, 235 genes
were upregulated in response to hyphae that were inserted by Candida albicans biofilm into underlying
epithelial layers. All of the upregulated genes increase the ability of C. albicans to bind and penetrate the oral
epithelial mucosa, and so lead to the increase in inflammatory response. Knowing what genes are
expressed differently, and whether they are upregulated or downregulated, and also knowing at what
phase of biofilm formation these changes of gene expression are developed, provides an understanding
as to why biofilms are mostly antifungal resistant. References Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental
Disease. PLoS ONE 2015, 10, e0128657. [CrossRef] [PubMed] 9. Gupta, S.J.; Bains, V.K.; Madan, R.; Gupta, V.; Rizvi, I. Microarray: An Emerging Diagnostic Tool in Dentistry. Asian J. Oral Health Allied Sci. 2012, 2, 78–83. 10. Lif Holgerson, P.; Öhman, C.; Rönnlund, A.; Johansson, I. Maturation of oral microbiota in children with or
without dental caries. PLoS ONE 2015, 10, e0128534. [CrossRef] [PubMed] 11. Kianoush, N.; Adler, C.J.; Nguyen, K.-A.T.; Browne, G.V.; Simonian, M.; Hunter, N. Bacterial Profile of
Dentine Caries and the Impact of pH on Bacterial Population Diversity. PLOS ONE 2014, 9, e92940. [CrossRef]
[PubMed] 12. Tanner, A.C.R. Anaerobic culture to detect periodontal and caries pathogens. J. Oral Biosci. 2015, 57, 18–26. [CrossRef] [PubMed] 13. Takeshita, T.; Kageyama, S.; Furuta, M.; Tsuboi, H.; Takeuchi, K.; Shibata, Y.; Shimazaki, Y.; Akifusa, S.;
Ninomiya, T.; Kiyohara, Y.; et al. Bacterial diversity in saliva and oral health-related conditions:
The Hisayama Study. Sci. Rep. 2016, 6, 22164. [CrossRef] [PubMed] Kaczor-Urbanowicz, K.E.; Martin Carreras-Presas, C.; Aro, K.; Tu, M.; Garcia-Godoy, F.; Wong, D.T
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Current
iews and directions Exp Biol Med (Maywood) 2016 29 [CrossRef] [PubMed 14. Kaczor-Urbanowicz, K.E.; Martin Carreras-Presas, C.; Aro, K.; Tu, M.; Garcia-Godoy, F.; Won
Saliva Diagnostics - Current views and directions. Exp. Biol. Med. (Maywood) 2016, 29. [CrossRef] [Pu 14. Kaczor-Urbanowicz, K.E.; Martin Carreras-Presas, C.; Aro, K.; Tu, M.; Garcia-Godoy, F.; Wong, D.T. Saliva Diagnostics - Current views and directions. Exp. Biol. Med. (Maywood) 2016, 29. [CrossRef] [PubMed]
15. Li, Y.; Zhou, X.; St John, M.A.; Wong, D.T. RNA profiling of cell-free saliva using microarray technology. J. Dent. Res. 2004, 83, 199–203. [CrossRef] [PubMed] Kaczor-Urbanowicz, K.E.; Martin Carreras-Presas, C.; Aro, K.; Tu, M.; Garcia-Godoy, F.; Wong, D.T
Saliva Diagnostics - Current views and directions. Exp. Biol. Med. (Maywood) 2016, 29. [CrossRef] [PubMed 15. Li, Y.; Zhou, X.; St John, M.A.; Wong, D.T. RNA profiling of cell-free saliva using microarray technology. J. Dent. Res. 2004, 83, 199–203. [CrossRef] [PubMed] 16. Lin, X.; Lo, H.; Wong, D.T.; Xiao, X. Noncoding RNAs in human saliva as potential disease biomarkers. Front. Genet. 2015, 6, 175. [CrossRef] [PubMed] 17. Majem, B.; Rigau, M.; Reventos, J.; Wong, D.T. Non-coding RNAs in saliva: Emerging biomarkers for
molecular diagnostics. Int. J. Mol. Sci. 2015, 16, 8676–8698. [CrossRef] [PubMed] 18. Moriyama, S.; Tanaka, H.; Uwataki, M.; Muguruma, M.; Ohta, K. References 1. Campos, M.S.; Marchini, L.; Bernardes, L.A.S.; Paulino, L.C.; Nobrega, F.G. Biofilm microbial communities
of denture stomatitis. Oral Microbiol. Immunol. 2008, 23, 419–424. [CrossRef] [PubMed] 1. Campos, M.S.; Marchini, L.; Bernardes, L.A.S.; Paulino, L.C.; Nobrega, F.G. Biofilm microbial communities
of denture stomatitis. Oral Microbiol. Immunol. 2008, 23, 419–424. [CrossRef] [PubMed] 2. Loesche, W.J.; Abrams, J.; Terpenning, M.S.; Bretz, W.A.; Dominguez, B.L.; Grossman, N.S.; Hildebrandt, G.H.;
Langmore, S.E.; Lopatin, D.E. Dental findings in geriatric populations with diverse medical backgrounds. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 1995, 80, 43–53. [CrossRef] 2. Loesche, W.J.; Abrams, J.; Terpenning, M.S.; Bretz, W.A.; Dominguez, B.L.; Grossman, N.S.; Hildebrandt, G.H.;
Langmore, S.E.; Lopatin, D.E. Dental findings in geriatric populations with diverse medical backgrounds. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 1995, 80, 43–53. [CrossRef] 3. Terpenning, M.; Bretz, W.; Lopatin, D.; Langmore, S.; Dominguez, B.; Loesche, W. Bacterial colonization of
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research of World Health Assembly 2007, World Health Organization. Community Dent. Oral Epidemiol. 2009,
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perspective. Caries Res. 2013, 47, 89–102. [CrossRef] [PubMed] 6. Nyvad, B.; Crielaard, W.; Mira, A.; Takahashi, N.; Beighton, D. Dental caries from a molecular microbiological
perspective. Caries Res. 2013, 47, 89–102. [CrossRef] [PubMed] 7. Edwina, A.; Fejerskov, O. Essentials of Dental Caries, 4th ed.; Oxford University Press: London, UK, 7. Edwina, A.; Fejerskov, O. Essentials of Dental Caries, 4th ed.; Oxfo 8. Terai, T.; Okumura, T.; Imai, S.; Nakao, M.; Yamaji, K.; Ito, M.; Nagata, T.; Kaneko, K.; Miyazaki, K.;
Okada, A.; et al. 7. Conclusions A systems approach has been presented to catalogue the microbes involved in several oral diseases. Both conventional microbiology as well as new molecular analytical methodologies are needed to
comprehensively define the representative microbial populations in oral disease. Discovery of the
right therapeutic interventions will require microbiology classification, DNA information, clinical
information (medical records) and lifestyle information. The hope is that analyzing the microbiota, the
microbial genomes, and the host human genome, alongside performing functional genomic analysis,
will reveal critical pathways associated with the initiation and progression of oral disease, and provide
candidate targets for drug therapies. 7 of 9 2017, 6, 2 Supplementary Materials: The following are available online at www.mdpi.com/2571-5135/6/1/2/s1, Figure S1:
Microbial flora present in dentate and edentulous populations. Supplementary Materials: The following are available online at www.mdpi.com/2571-5135/6/1/2/s1, Figure S1:
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molecular and metagenomic analyses. Virulence 2015, 6, 183–187. [CrossRef] [PubMed] 50. Valente, N.A.; Andreana, S. Peri-implant disease: What we know and what we need to know. J. Periodontal
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in Patients with and without Clinical Signs of Inflammation. Dent. J. 2015, 3. [CrossRef] 52. Shanbhag, S.; Shanbhag, V.; Stavropoulos, A. Genomic analyses of early peri-implant bone healin
A systematic review. Int. J. Implant Dent. 2015, 1. [CrossRef] [PubMed] 53. Ramage, G.; Rajendran, R.; Sherry, L.; Williams, C. Fungal Biofilm Resistance. Int. J. Microbiol. 2012, 2012. [CrossRef] [PubMed] 54. References Pereira, C.A.; Toledo, B.C.; Santos, C.T.; Pereira Costa, A.C.B.; Back-Brito, G.N.; Kaminagakura, E.;
Jorge, A.O.C. Opportunistic microorganisms in individuals with lesions of denture stomatitis. Diagn. Microbiol. Infect. Dis. 2013, 76, 419–424. [CrossRef] [PubMed] 55. Offenbacher, S.; Barros, S.P.; Bencharit, S.; Yu, N.; Preisser, J.; Moss, K.; Loewy, Z.G. Differential mucosal
gene expression patterns in candidiasis-associated chronic oral denture stomatitis. In preparation. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Tendência temporal de atividade física em adolescentes brasileiros: análise da Pesquisa Nacional de Saúde do Escolar de 2009 a 2019
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Tendência temporal de atividade física em
adolescentes brasileiros: análise da Pesquisa
Nacional de Saúde do Escolar de 2009 a 2019 Temporal trend of physical activity in Brazilian
adolescents: analysis of the Brazilian National
Survey of School Health from 2009 to 2019 Tendencia temporal de la actividad física en
adolescentes brasileños: análisis de la Encuesta
Nacional de Salud del Escolar de 2009 a 2019 Carlos Alex Martins Soares 1,2
Otávio Amaral de Andrade Leão 3
Matheus Pintanel Freitas 2
Pedro Curi Hallal 3
Mário Bernardes Wagner 1 doi: 10.1590/0102-311XPT063423 doi: 10.1590/0102-311XPT063423 Tendência temporal de atividade física em
adolescentes brasileiros: análise da Pesquisa
Nacional de Saúde do Escolar de 2009 a 2019 Este é um artigo publicado em acesso aberto (Open Access) sob a licença
Creative Commons Attribution, que permite uso, distribuição e reprodu
ção em qualquer meio, sem restrições, desde que o trabalho original seja
corretamente citado. Cad. Saúde Pública 2023; 39(10):e00063423 1 Programa de Pós-graduação em Saúde da Criança e do
Adolescente, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brasil.
2 Programa de Pós-graduação em Educação Física, Universidade
Federal de Pelotas, Pelotas, Brasil.
3 Programa de Pós-graduação em Epidemiologia, Universidade
Federal de Pelotas, Pelotas, Brasil. Introdução Observa-se que estudos semelhantes em outros países são realizados há mais de quatro déca
das, como o transnacional Health Behaviour School-based Children (HBSC; Estudo Comportamental de
Saúde de Escolares) em mais de 40 países europeus, o Global School-based Student Health Survey (GSSH;
Pesquisa Global de Saúde dos Escolares) da OMS e o Youth Risk Behavior Surveillance System (YRBSS;
Sistema de Vigilância de Comportamentos de Risco Juvenil) do Centro de Controle e Prevenção de
Doenças dos Estados Unidos (CDC). Há um alerta nesses estudos em relação à atividade física dos
adolescentes, pois a grande maioria dos jovens não está atingindo as recomendações para atividade
física semanal, como explicitam o HBSC/2016 (82%) 12 e a PeNSE/2019 (81,9%) 10. Em nível mundial, as aferições da atividade física em adolescentes têm como base o ponto de corte
para atividade física recomendado pela OMS 5, ou seja, atividade física de intensidade moderada à
vigorosa, frequência diária e duração mínima de 60 minutos por sessão 7,13. Essas recomendações
foram reafirmadas e atualizadas em propostas posteriores, tanto pela OMS 13 quanto pelo Departa
mento de Saúde e Serviços Humanos dos Estados Unidos (DHHS) 14 – e, inclusive, pelo Ministério
da Saúde brasileiro 15. Assim, o acompanhamento do nível de atividade física dos adolescentes insere-se na vigilância em
saúde do Ministério da Saúde, ou seja, na necessidade de identificar e acompanhar os principais indi
cadores de saúde e a probabilidade de o adulto desenvolver alguma DCNT, pois a prática de atividade
física nessa faixa etária é preditora do comportamento fisicamente ativo ou inativo na idade adulta 16. Se a aquisição e incorporação da atividade física como hábito saudável ocorre pela influência dos
pais, dos pares e do ambiente escolar e deve ser desenvolvida a longo prazo 17, consequentemente,
a vigilância do nível de atividade física dos adolescentes contribui para um planejamento em saúde
abrangente, elaborado e direcionado à promoção de ações que visem mitigar os efeitos negativos da
inatividade física. Nesse cenário, a relevância e a análise dos dados da PeNSE permitirão compreender a tendência
da atividade física entre adolescentes na última década, projetar o futuro, elencar ações necessárias
para reduzir o desenvolvimento precoce dos fatores de risco e o subsequente surgimento de DCNT. Introdução As doenças crônicas não transmissíveis (DCNT) se desenvolvem silenciosamente ao longo da vida,
têm desfechos precoces (entre 30 e 69 anos) e são consequências de uma associação de fatores genéti
cos, fisiológicos, ambientais e comportamentais. Cerca de um quarto dos brasileiros tem pelo menos
uma DCNT 1,2. Destacam-se doenças cardiovasculares, cânceres, doenças respiratórias crônicas e dia
betes tipo 2, que foram responsáveis por 71% dos óbitos e por 85% das mortes prematuras no mundo
em 2016 3. No Brasil, causaram 54,7% dos óbitos em 2019 e, dessas mortes, 41,8% foram prematuras,
sendo que em 2000 havia ocorrido 47,4% de óbitos precoces 4. A prevenção para esse tipo de doenças
ocorre a partir do monitoramento e da vigilância, seguidos pelo desenvolvimento de políticas públicas
que contribuam para alcançar as metas da Organização Mundial da Saúde (OMS) de reduzir a inati
vidade física em 10% até 2025 e em 15% até 2030 5,6. Em 2016, 28% dos adultos (18+ anos) não atenderam às recomendações da OMS para atividade
física, ou seja, não cumpriram de 150 a 300 minutos por semana de atividade física com intensidade
moderada à vigorosa. A prevalência de inatividade física é mais do que o dobro em países de alta renda
em relação aos de média e baixa rendas, as mulheres são menos ativas na maioria dos países membro
da OMS e, nos últimos 15 anos, os níveis de inatividade física não diminuíram 7. Além disso, o nível de
atividade física costuma declinar durante a adolescência 8. Relatórios da Pesquisa Nacional de Saúde do
Escolar (PeNSE) 9,10 mostram que o percentual de adolescentes que não atingiram as recomendações
para atividade física cresceu de 56,9% na PeNSE/2009 para 71,8% da PeNSE/2012. Certamente, entre
as justificativas para não atender às diferentes recomendações, está a tendência mundial de baixos
níveis de atividade física e altos níveis de comportamento sedentário na infância e na adolescência 11. Enquanto isso, o Brasil caminha lentamente no monitoramento e vigilância da saúde dos adoles
centes. Resumo Correspondência
C. A. M. Soares
Rua Uruguai 1785, Pelotas, RS 96010-630, Brasil. carlos.alex@ufrgs.br O objetivo do estudo foi analisar a tendência de atividade física dos escolares
brasileiros e as associações com variáveis demográficas, socioeconômicas e
comportamentais, por meio da Pesquisa Nacional de Saúde do Escolar
(PeNSE) em suas quatro edições – 2009, 2012, 2015 e 2019. Foram usados
dados dos escolares (13-17 anos) participantes das quatro edições da PeNSE
(n = 392.922). Descrevemos o percentual de ativos, a média e valores
percentuais da atividade física de intensidade moderada à vigorosa em
minutos/semana. A regressão de Poisson foi ajustada para sexo, idade,
cor da pele, escore de bens e comportamento sedentário (≥ 2 horas/dia para
assistir TV e ≥ 3 horas/dia de tempo sentado). Como limitação, a amostra da
PeNSE/2009 refere-se apenas às capitais brasileiras. O percentual de ativos
reduziu de 43,1% em 2009 para 18,2% em 2019. A média em atividade física
de intensidade moderada à vigorosa da PeNSE/2009 (média = 318,4 minutos/
semana; IC95%: 313,4-323,4) reduziu 50% em 2019. Na educação física, a
média semanal em atividade física de intensidade moderada à vigorosa das
meninas foi menor que 50 minutos, ao passo que a dos meninos foi maior
que 60 minutos, nas quatro edições da PeNSE. Ainda, 22,7% das meninas
relataram (PeNSE/2019) não ter tido aulas de educação física, enquanto o
mesmo é relatado por 19,7% dos meninos. O comportamento sedentário sofreu
redução no hábito de assistir TV, porém o tempo sentado aumentou de 50,1%
(IC95%: 48,9-51,3) para 54% (IC95%: 53,1-54,9) entre a PeNSE/2009 e a
PeNSE/2019. Como consequência da queda nos níveis de atividade física,
são necessárias políticas públicas que promovam a atividade física, como
aumentar as aulas de educação física na escola para, no mínimo, três vezes
por semana. 1 Programa de Pós-graduação em Saúde da Criança e do
Adolescente, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brasil. 2 Programa de Pós-graduação em Educação Física, Universidade
Federal de Pelotas, Pelotas, Brasil. 3 Programa de Pós-graduação em Epidemiologia, Universidade
Federal de Pelotas, Pelotas, Brasil. 1 Programa de Pós-graduação em Saúde da Criança e do
Adolescente, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brasil. 2 Programa de Pós-graduação em Educação Física, Universidade
Federal de Pelotas, Pelotas, Brasil. 3 Programa de Pós-graduação em Epidemiologia, Universidade
Federal de Pelotas, Pelotas, Brasil. Adolescente; Atividade Física; Fatores de Risco; Estudos de Séries Temporais Soares CAM et al. 2 População-alvo A população-alvo da PeNSE são adolescentes, de ambos os sexos, matriculados e frequentes nos Ensi
nos Fundamental e Médio de escolas públicas e privadas situadas nas zonas urbanas e rurais de todo o
território brasileiro. A série e a idade foram definidas por estarem relacionadas, respectivamente, com
o mínimo de escolarização e a fase em que os indivíduos já têm autonomia necessária, preconizada
pela OMS, para responder a um questionário autoaplicável. Na primeira edição da PeNSE (2009), a coleta de dados foi restrita às 27 capitais brasileiras, com
N = 618.553 e uma amostra de 61.434 escolares. Em 2012, foi mantida a coleta de dados nas capitais
e foram incluídas cidades do interior, agrupadas em cada uma das cinco grandes regiões político
-administrativas do país (Norte, Nordeste, Centro-oeste, Sudeste e Sul), elevando a população-alvo
(N = 3.153.314) e obtendo amostra de 106.480 escolares. Em 2015, o plano amostral sofreu adaptações e foram desenvolvidas duas amostras. Neste estudo,
utilizamos a amostra 1, composta pelas 27 capitais e pelos municípios do interior, agrupados por
Unidades da Federação e abrangendo todo o território brasileiro. Assim, foram criados 53 estratos
geográficos, com a população-alvo estimada em 2.630.835 de escolares e a amostra final com 100.110
escolares. Finalmente, em 2019, a partir da população-alvo, estimada em 11.851.941 de escolares na faixa
etária de 13 a 17 anos, matriculados do 7o ao 9o do Ensino Fundamental e do 1o ao 3o ano do Ensino
Médio, a amostra totalizou 124.898 escolares. Delineamento Esta é uma pesquisa epidemiológica, transversal, de base escolar e composta por análises ecológicas 18,
pois utiliza dados governamentais coletados pelo Instituto Brasileiro de Geografia e Estatística (IBGE)
em quatro edições da PeNSE (2009, 2012, 2015 e 2019), a partir da proposição de vigilância em saúde
do adolescente realizada pela Secretaria de Vigilância em Saúde do Ministério da Saúde. Amostra A amostra deste estudo é o conjunto das quatro edições da PeNSE, cujas amostras foram aleatórias,
probabilísticas, estratificadas e dimensionadas para estimar parâmetros populacionais (proporções
ou prevalências), representativas para terem validade interna, externa e significância estatística. A
amostra foi a estimativa da proporção populacional, calculada para fornecer estimativas de propor
ções de algumas características de interesse, em cada um dos estratos geográficos, utilizando-se erro
amostral máximo de 3%, nível de 95% de confiança, prevalência de 50%, pois para proporções desse
valor a variância dos estimadores amostrais é máxima. Assim, este estudo é baseado em dados públicos e os projetos originais foram submetidos e
aprovados pela Comissão Nacional de Ética em Pesquisa (CONEP). Nossa amostra é composta por
392.922 escolares, de ambos os sexos, da faixa etária de 13 a 17 anos e oriundos da PeNSE/2009
(n = 61.434), PeNSE/2012 (n = 106.480), PeNSE/2015 (n = 100.110) e PeNSE/2019 (n = 124.898). Desde 2009, o Ministério da Saúde, sob a liderança da Secretaria de Vigilância em Saúde, tem rea
lizado a PeNSE, em parceria com o IBGE e o Instituto Nacional de Estudos e Pesquisas Educacionais
Anísio Teixeira (INEP) do Ministério da Educação, além do suporte das Secretarias Estaduais e Muni
cipais de Saúde e de Educação dos estados e municípios brasileiros. Nesse período, por meio de estu
dos epidemiológicos transversais, de base escolar, quatro edições da PeNSE (2009, 2012, 2015 e 2019)
coletaram dados de adolescentes dos Ensinos Fundamental e Médio de escolas públicas e privadas. Introdução Portanto, nosso objetivo é analisar a tendência de atividade física dos escolares brasileiros e as associa
ções com variáveis demográficas, socioeconômicas e comportamentais por meio da PeNSE em suas
quatro edições – 2009, 2012, 2015 e 2019. Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
3 Variáveis do estudo A variável dependente foi a atividade física acumulada. Esse desfecho foi obtido por autorrelato,
utilizando informações de três domínios: deslocamentos casa-escola-casa, atividade física nas aulas Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 4 de educação física e atividade física de lazer – consideram-se as atividades realizadas na escola, mas
distintas da aula regular de educação física e atividades físicas diversas realizadas em outros espaços
(clubes, associações, praças etc.). Com essas informações, foi realizada a multiplicação da frequência
(dias) pela duração (tempo de prática diária) da atividade física em cada domínio, seguida pela soma
dos produtos de cada domínio, resultando na atividade física acumulada. Posteriormente, a atividade física acumulada foi dicotomizada em atingir (≥ 300 minutos/semana)
e não atingir (< 300 minutos/semana) as recomendações para atividade física. A categoria “atingir”
foi denominada “percentual de ativos fisicamente” e teve seu intervalo de 95% de confiança (IC95%)
reportado para cada edição da PeNSE, distribuição geográfica (Brasil, capitais e interior) e estratifi
cado por sexo, idade e escore de bens (Tabela 1). Destaca-se que o tempo mínimo recomendado de
atividade física para jovens é de 60 minutos por dia de atividade física de intensidade moderada à
vigorosa 19,20, frequência diária 7,13,14, sendo que em três dias devem ser desenvolvidas atividade de
fortalecimento osteomuscular 14. As variáveis independentes são demográficas (etnia, sexo, idade), socioeconômicas (escore de posse
de bens e serviços) e comportamentais (comportamento sedentário relacionado ao tempo assistindo
televisão e relacionado ao tempo sentado). Com relação ao tempo assistindo televisão, foi considerado
excessivo o tempo ≥ 2 horas por dia; e quanto ao tempo sentado, ≥ 3 horas por dia. A idade incluída foi
limitada à faixa etária de 13 a 17 anos e o sexo restringiu-se à determinação biológica, masculino ou
feminino. Como de praxe, a determinação da etnia/cor da pele foi autodeclarada e seguiu as categorias
propostas pelo IBGE (branca, parda, preta, amarela e indígena). Partiu-se das questões sobre bens e serviços, incluídas na PeNSE, para gerar um escore de bens
e serviços, conforme relatório do IBGE. Para o escore, foram consideradas as variáveis de posse de
celular (0 = não; 1 = sim), computador ou notebook (0 = não; 1 = sim), acesso à Internet em casa (0 =
não; 1 = sim) e ter banheiro completo na residência (0 = não; 1 = sim). Variáveis do estudo O escore foi contabilizado
como a soma dos itens, sendo “0” um indivíduo que não tem nenhum dos itens e “4” o indivíduo que
tem todos os itens. Análise estatística A análise dos dados foi realizada primeiramente mediante a descrição da frequência relativa (Mate
rial Suplementar: https://cadernos.ensp.fiocruz.br/static//arquivo/suppl-e00063423_6796.pdf) das
variáveis utilizadas nas quatro edições da PeNSE (2009, 2012, 2015 e 2019). A atividade física foi
utilizada para verificar a diferença entre os inquéritos e descrita por meio de: (i) percentual de ativos
fisicamente e seus respectivos IC95%, conforme a distribuição geográfica (Brasil, capitais e interior),
considerando sexo, idade e escore de bens e serviços (Tabela 1); (ii) médias da atividade física e seus
respectivos IC95%, conforme a distribuição geográfica (Brasil, capitais e interior), considerando os
domínios da atividade física e o sexo (Tabela 2); e (iii) média geral (ambos os sexos) e dos percentis
da atividade física, conforme a distribuição geográfica (Brasil, capitais e interior), considerando os
domínios da atividade física e a atividade física acumulada (Tabela 3). Realizamos a regressão de Poisson (Tabela 4) para compreendermos a associação entre o percen
tual de ativos fisicamente e as variáveis demográficas (sexo, idade e cor da pele), socioeconômicas
(escore de bens e serviços) e comportamentais (tempo assistindo televisão e tempo sentado). Como consequência da seleção da amostra em conglomerado, incluímos no software estatístico
Stata, versão 16 (https://www.stata.com), o efeito do delineamento amostral, utilizado pelo IBGE,
para a obtenção de todas as estatísticas descritivas e de associação, considerando IC95% e valor de
p ≤ 0,05 como resultados estatisticamente significantes 21. Aspectos éticos Em cada edição da PeNSE, houve aprovação da CONEP para a realização dos inquéritos:
PeNSE/2009 (registro no 11.537); PeNSE/2012 (registro no 16.805); PeNSE/2015 (registro no
1.006.467); e PeNSE/2019 (parecer no 3.249.268). Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
5 5 Tabela 1 Percentual de ativos fisicamente segundo distribuição geográfica, sexo, idade e escore de bens e serviços da Pesquisa Nacional de Saúde do Escolar
(PeNSE) de 2009, 2012, 2015 e 2019. mente segundo distribuição geográfica, sexo, idade e escore de bens e serviços da Pesquisa Nacional de Saúde do Escolar Percentual de ativos fisicamente segundo distribuição geográfica, sexo, idade e escore de bens e serviços da Pesquisa Nacional de Saúde do Escolar
(PeNSE) de 2009, 2012, 2015 e 2019. * Criado a partir da posse de celular, computador, acesso à Internet e banheiro em casa. IC95%: intervalo de 95% de confiança. Aspectos éticos Variáveis
PeNSE
2009 (n = 61.434)
2012 (n = 106.480)
2015 (n = 100.110)
2019 (n = 124.898)
%
IC95%
%
IC95%
%
IC95%
%
IC95%
Brasil
Sexo
Masculino
-
-
40,6
39,6-41,6
44,1
43,1-45,2
26,7
25,9-27,6
Feminino
-
-
22,6
22,1-23,2
25,5
24,6-26,4
9,3
8,8-9,8
Ambos os sexos
31,2
30,5-32,0
34,5
33,8-35,3
18,0
17,5-18,6
Idade (anos)
13-15
-
-
31,5
31,0-32,1
34,5
33,7-35,3
18,1
17,5-18,8
16-17
-
-
29,0
26,6-31,5
34,7
33,1-36,3
17,8
16,9-18,7
Escore de bens e serviços *
≤ 1
-
-
22,3
19,3-25,6
27,3
25,3-29,5
15,8
14,1-17,7
2
-
-
27,2
25,7-28,7
30,2
28,7-31,7
17,5
16,0-19,0
3
-
-
31,2
29,8-32,7
33,8
32,6-35,0
16,6
15,8-17,4
4
-
-
34,7
33,8-35,5
36,6
35,6-37,5
19,3
18,5-20,1
Capitais
Sexo
Masculino
55,8
54,7-56,8
43,6
42,2-45,0
45,6
44,3-47,0
26,5
25,4-27,6
Feminino
31,6
30,5-32,8
25,0
24,0-26,0
27,1
26,0-28,3
9,8
9,1-10,6
Ambos os sexos
43,1
42,2-43,9
34,1
33,2-35,0
36,2
35,2-37,3
18,2
17,5-19,0
Idade (anos)
13-15
43,1
42,2-44,1
34,2
33,2-35,1
35,8
34,6-36,9
18,8
17,8-19,8
16-17
42,6
40,5-44,7
33,5
31,6-35,5
41,5
39,4-43,7
17,2
16,2-18,3
Escore de bens e serviços *
≤ 1
41,2
39,0-43,4
27,2
24,8-29,7
34,3
30,5-38,4
18,1
15,2-21,4
2
39,4
37,7-41,2
29,9
28,2-31,7
38,3
35,7-41,0
16,7
15,0-18,6
3
43,7
41,8-45,6
33,6
31,8-35,5
35,2
33,4-37,1
17,1
16,0-18,2
4
45,8
44,4-47,1
35,6
34,6-36,6
36,3
35,2-37,5
19,0
18,2-20,0
Interior
Sexo
Masculino
-
-
39,7
38,1-41,4
43,7
42,4-45,0
26,8
25,7-27,9
Feminino
-
-
22,0
21,1-22,9
25,0
23,8-26,1
9,1
8,5-9,7
Ambos os sexos
-
-
30,4
29,2-31,6
34,0
33,1-35,0
17,9
17,3-18,6
Idade (anos)
13-15
-
-
30,8
29,9-31,6
34,1
33,1-35,2
17,9
17,1-18,8
16-17
-
-
27,9
23,8-32,3
33,0
31,2-35,0
18,0
16,8-19,1
Escore de bens e serviços *
≤ 1
-
-
21,7
17,5-26,6
26,6
24,4-28,9
15,6
13,7-17,7
2
-
-
26,7
24,5-29,0
28,8
27,2-30,5
17,6
15,9-19,5
3
-
-
30,6
28,2-33,1
33,4
32,0-34,9
16,5
15,5-17,5
4
-
-
34,3
32,9-35,7
36,6
35,4-37,9
19,4
18,4-20,5
IC95%: intervalo de 95% de confiança. Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 6 Tabela 2 Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
7 7 Tabela 2 (continuação)
Variáveis
PeNSE
2009 (n = 61.434)
2012 (n = 106.480)
2015 (n = 100.110)
2019 (n = 124.898)
x *
IC95%
x *
IC95%
x *
IC95%
x *
IC95%
Atividade física em deslocamento ativo
Masculino
-
-
56,4
54,3-58,5
87,7
83,3-92,2
43,0
40,9-45,1
Feminino
-
-
59,5
56,4-62,6
89,1
84,9-93,4
45,2
43,1-47,4
Ambos os sexos
-
-
58,0
55,5-60,6
88,5
84,6-92,3
44,1
42,4-45,8
Atividade física de lazer **
Masculino
-
-
158,4
148,5-168,4
152,5
148,7-156,3
165,8
161,7-169,9
Feminino
-
-
85,3
82,1-88,4
76,5
73,5-79,5
95,2
91,6-98,8
Ambos os sexos
-
-
119,9
115,2-124,7
113,3
110,6-115,9
136,5
133,3-139,6
Atividade física acumulada
Masculino
-
-
275,8
267,7-283,9
300,5
293,9-307,1
207,4
202,7-212,1
Feminino
-
-
184,3
176,9-191,7
204,6
199,3-209,9
109,2
105,8-112,5
Ambos os sexos
-
-
227,7
220,1-235,2
251,0
246,1-255,8
158,2
154,7-161,7
IC95%: intervalo de 95% de confiança. * Média em atividade física expressa em minutos/semana;
** Atividade física de lazer (ou na escola, mas fora das aulas regulares). IC95%: intervalo de 95% de confiança. * Média em atividade física expressa em minutos/semana; * Média em atividade física expressa em minutos/semana; ** Atividade física de lazer (ou na escola, mas fora das aulas regulares). ** Atividade física de lazer (ou na escola, mas fora das aulas regulares). Tabela 2 Média em atividade física, expressa em minutos por semana, segundo distribuição geográfica, domínios da atividade física e sexo, conforme Pesquisa
Nacional de Saúde do Escolar (PeNSE) de 2009, 2012, 2015 e 2019. Média em atividade física, expressa em minutos por semana, segundo distribuição geográfica, domínios da atividade física e sexo, conforme Pesquisa
Nacional de Saúde do Escolar (PeNSE) de 2009, 2012, 2015 e 2019. Variáveis
PeNSE
2009 (n = 61.434)
2012 (n = 106.480)
2015 (n = 100.110)
2019 (n = 124.898)
x *
IC95%
x *
IC95%
x *
IC95%
x *
IC95%
Brasil
Educação física escolar
Masculino
-
-
62,5
59,0-66,0
61,2
59,5-63,0
60,5
58,8-62,2
Feminino
-
-
41,0
37,2-44,7
41,1
39,6-42,6
36,0
34,4-37,6
Ambos os sexos
-
-
51,3
47,2-55,3
50,9
49,5-52,3
48,4
46,9-49,8
Atividade física em deslocamento ativo
Masculino
-
-
57,4
56,0-58,7
87,4
83,9-91,0
43,5
41,8-45,2
Feminino
-
-
60,4
58,4-62,3
88,0
84,5-91,4
45,6
43,9-47,4
Ambos os sexos
-
-
58,9
57,3-60,5
87,7
84,6-90,8
44,5
43,1-45,9
Atividade física de lazer **
Masculino
-
-
160,5
154,5-166,4
154,2
151,2-157,2
167,1
163,8-170,5
Feminino
-
-
86,7
84,8-88,8
78,7
76,2-81,2
97,8
94,9-100,8
Ambos os sexos
-
-
122,0
119,1-124,8
115,4
113,2-117,5
138,5
135,9-141,0
Atividade física acumulada
Masculino
-
-
280,6
275,6-285,5
302,1
296,8-307,4
207,9
204,2-211,7
Feminino
-
-
188,3
183,6-193,0
207,2
203,0-211,5
110,5
107,7-113,2
Ambos os sexos
-
-
232,4
227,7-237,0
253,3
249,4-257,2
159,2
156,4-162,0
Capitais
Educação física escolar
Masculino
67,8
65,2-70,5
68,5
65,9-71,2
62,3
59,4-65,1
63,2
61,0-65,4
Feminino
48,4
45,9-50,9
46,8
44,6-49,0
46,2
43,9-48,5
38,2
36,0-40,3
Ambos os sexos
57,6
55,2-60,0
57,4
55,2-59,7
54,1
51,8-56,4
50,7
48,9-52,5
Atividade física em deslocamento ativo
Masculino
82,3
78,8-85,7
60,5
58,1-62,9
86,5
82,2-90,7
45,2
43,0-47,3
Feminino
87,9
83,4-92,4
63,3
60,1-66,6
84,1
79,3-88,9
47,1
44,3-49,8
Ambos os sexos
85,2
81,6-88,8
62,0
59,6-64,3
85,3
81,2-89,4
46,1
44,0-48,1
Atividade física de lazer **
Masculino
253,0
247,2-258,8
167,2
162,4-171,9
159,7
155,3-164,0
171,5
167,1-175,9
Feminino
114,1
109,0-119,1
92,1
88,8-95,3
86,2
82,1-90,2
107,2
103,6-110,7
Ambos os sexos
179,9
175,5-184,3
128,8
125,7-132,0
122,3
118,9-125,6
145,3
142,0-148,7
Atividade física acumulada
Masculino
397,5
390,7-404,3
296,3
289,9-302,7
307,4
300,2-314,5
209,7
205,1-214,3
Feminino
247,0
241,1-252,9
202,4
197,7-207,1
215,9
210,8-221,1
114,9
110,8-119,0
Ambos os sexos
318,4
313,4-323,4
248,4
243,9-252,8
260,9
255,7-266,1
162,7
159,1-166,2
Interior
Educação física escolar
Masculino
-
-
60,7
54,8-66,5
60,9
58,8-63,0
59,8
57,7-61,9
Feminino
-
-
39,3
33,3-45,4
39,6
37,8-41,3
35,4
33,4-37,3
Ambos os sexos
-
-
49,4
42,8-56,1
49,9
48,2-51,6
47,7
45,9-49,4
(continua) Cad. Resultados 8 Tabela 3 Média e percentis da atividade física, expressos em minutos por semana, segundo distribuição geográfica, domínios de
atividade física e atividade física acumulada, conforme Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009, 2012, 2015
e 2019. Média e percentis da atividade física, expressos em minutos por semana, segundo distribuição geográfica, domínios de
atividade física e atividade física acumulada, conforme Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009, 2012, 2015
e 2019. Resultados Foram utilizados dados de 392.922 adolescentes, na faixa etária de 13 a 17 anos, a maioria sendo do
sexo feminino e pardos. A descrição das características sociodemográficas e comportamentais constam
no Material Suplementar (https://cadernos.ensp.fiocruz.br/static//arquivo/suppl-e00063423_6796. pdf). Há uma flutuação descendente no percentual de ativos fisicamente entre a primeira e a última edi
ção da PeNSE, com diferenças estatisticamente significantes. Os resultados mostram que, no Brasil, o
percentual de ativos fisicamente foi de 31,2% (IC95%: 30,5-32,0) na PeNSE/2012 e houve uma queda
abrupta para 18% (IC95%: 17,5-18,6) na PeNSE/2019. A flutuação foi semelhante nos municípios do
interior: de 30,4% (IC95%: 29,2-31,6) na PeNSE/2012 para 17,9% (IC95%: 17,3-18,6) na PeNSE/2019. A tendência de reduzir o percentual de ativos fisicamente com flutuação descendente também foi
observada nas capitais: de 43,1% (IC95%: 42,2-43,9) na PeNSE/2009 para 18,2% (IC95%: 17,5-19,0)
na PeNSE/2019 (Tabela 1; Figura 1). A flutuação observada no percentual de ativos fisicamente também ocorreu na média em atividade
física: (i) no Brasil, de 232,4 minutos/semana (IC95%: 227,7-237,0) na PeNSE/2012 para 159,2 minu
tos/semana (IC95%: 156,4-162,0) na PeNSE/2019; (ii) nos municípios do interior, de 227,7 minutos/
semana (IC95%: 220,1-235,2) para 158,2 minutos/semana (IC95%: 154,7-161,7); e (iii) nas capitais, de
318,4 minutos/semana (IC95%: 313,4-323,4) para 162,7 minutos/semana (IC95%: 159,1-166,2). Nas
capitais, ao longo de dez anos, a redução foi de 50% na média em atividade física (Tabela 2), represen
tando o caminho inverso das diversas recomendações sobre atividade física para adolescentes. Por sexo, as meninas tiveram um percentual de ativos fisicamente menor em relação aos meninos
em todas as edições, estatisticamente significante para Brasil, capitais e interior e entre inquéritos
(Tabela 1). Elas também têm percentual de ativos fisicamente menor no lazer. Entretanto, na educação
física escolar, apesar de haver uma redução, a variação é baixa (9 minutos/semana) entre os inquéri
tos, mesmo que o tempo de atividade física das meninas seja inferior a 50 minutos/semana e o dos
meninos seja superior a 60 minutos/semana (Tabela 2). Isso contribui para que o percentual de ativos
fisicamente das meninas seja 47% menor que o dos meninos, conforme regressão de Poisson (Tabela
4). Ou seja, quando há autorrelato de não ter tido aulas de educação física, as meninas têm maior pre
valência; e quando os escolares referem ter tido três ou mais aulas, os meninos têm percentual maior
de participação (Figura 2). Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. Resultados Média
P10
P25
P50
P75
P90
Brasil
PeNSE/2009
-
-
-
-
-
-
PeNSE/2012
Educação física escolar
51,3
0
0
35
85
110
Atividade física em deslocamento ativo
58,9
0
0
35
105
175
Atividade física de lazer
122,0
0
0
50
195
455
Atividade física acumulada
232,4
15
70
175
355
540
PeNSE/2015
Educação física escolar
50,9
0
0
45
70
130
Atividade física em deslocamento ativo
87,7
0
0
50
140
230
Atividade física de lazer
115,4
0
0
50
195
390
Atividade física acumulada
253,3
30
90
200
375
550
PeNSE/2019
Educação física escolar
48,4
0
0
35
70
110
Atividade física em deslocamento ativo
44,5
6
6
37
44
108
Atividade física de lazer
138,5
0
25
85
210
350
Atividade física acumulada
159,2
6
37
103
231
411
Capitais
PeNSE/2009
Educação física escolar
57,6
0
0
45
90
135
Atividade física em deslocamento ativo
85,2
0
0
50
150
250
Atividade física de lazer
179,9
0
0
90
295
480
Atividade física acumulada
318,4
45
110
250
475
695
PeNSE/2012
Educação física escolar
57,4
0
0
45
90
135
Atividade física em deslocamento ativo
62,0
0
0
35
105
175
Atividade física de lazer
128,8
0
0
65
195
455
Atividade física acumulada
284,4
25
85
195
380
550
PeNSE/2015
Educação física escolar
54,1
0
0
45
90
130
Atividade física em deslocamento ativo
85,3
0
0
50
140
230
Atividade física de lazer
122,3
0
0
65
195
390
Atividade física acumulada
260,9
35
100
215
385
545
PeNSE/2019
Educação física escolar
50,7
0
0
45
85
110
Atividade física em deslocamento ativo
46,1
6
6
37
44
111
Atividade física de lazer
145,3
5
30
100
220
385
Atividade física acumulada
162,7
6
37
107
235
416
Interior
PeNSE/2009
-
-
-
-
-
-
PeNSE/2012
Educação física escolar
49,4
0
0
30
85
110
Atividade física em deslocamento ativo
58,1
0
0
35
105
175
Atividade física de lazer
119,9
0
0
50
195
455
Atividade física acumulada
227,7
15
65
175
350
535
(continua) Cad. Resultados Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
9 Tabela 3 (continuação) Média
P10
P25
P50
P75
P90
PeNSE/2015
Educação física escolar
49,9
0
0
35
70
125
Atividade física em deslocamento ativo
88,5
0
0
50
140
230
Atividade física de lazer
113,3
0
0
45
195
390
Atividade física acumulada
251,0
30
85
195
375
550
PeNSE/2019
Educação física escolar
47,7
0
0
35
70
110
Atividade física em deslocamento ativo
44,1
6
6
37
44
93
Atividade física de lazer
136,5
0
25
75
210
350
Atividade física acumulada
158,2
6
36
100
230
410 O escore socioeconômico revela que são fisicamente ativos, com diferença estatisticamente sig
nificante, aqueles que têm todos os bens do escore, sendo que em 2012 e 2015 essa diferença ocorreu
de forma progressiva nos quatro níveis de posse (Tabela 1). Não houve diferença entre as duas faixas
etárias (13-15 anos e 16-17 anos). Quando comparamos o percentual de ativos fisicamente por meio do IC95% entre municípios
das capitais e do interior, encontramos diferença estatisticamente significativa nas PeNSE/2012 e
PeNSE/2015. Nas capitais e no interior, há diferença estatística entre os inquéritos (Tabela 1). Nossas análises revelam que a variação no deslocamento, aferida pela mediana, foi de 15 minutos
entre 2009 e 2019. Porém, 90% (P90) dos adolescentes em 2009 não atingiam as recomendações de ati
vidade física de intensidade moderada à vigorosa nesse domínio, mas eram mais ativos (250 minutos/
semana) do que foram os de 2019 (108 minutos/semana), conforme Tabela 3. Em média, a diferença
da PeNSE/2019 quanto ao deslocamento, em relação aos inquéritos anteriores, é estatisticamente
significativa (Tabela 2). A atividade física no lazer também é estatisticamente significativa entre todos
os inquéritos e mantém comportamento decrescente, com redução, em média, de 40 minutos, mesmo
que em 2019 tenha ocorrido um aumento em relação a 2012 e 2015 (Tabela 2). A mudança no comportamento sedentário foi estatisticamente significativa nos acompanhados
por meio do tempo assistindo televisão e tempo sentado. Assistir TV por mais de duas horas por dia
foi informado por 79,8% (IC95%: 79,2-80,4) em 2009 e por 46% (IC95%: 45,1-46,9) em 2019, revelan
do uma redução de 50%. Ter o hábito de ficar sentado (tempo sentado) por mais de três horas saltou
de 50,1% (IC95%: 48,9-51,3) em 2009 para 54% (IC95%: 53,1-54,9) em 2019. Discussão Com relação ao Brasil, houve redução do percentual de ativos fisicamente (13,9% entre meninos e
13,3% entre meninas) no período de 2012 a 2019 (Figura 1), aproximando-se da prevalência encontra
da por Ferrari et al. 22 relativa a atingir a recomendação da OMS 13,19 de praticar atividade física por
tempo igual ou superior a 60 minutos/dia. Entretanto, nas capitais, a queda foi vertiginosa na década
estudada, pois o percentual de meninos ativos fisicamente recrudesceu 29,3%, e de meninas, 21,8%,
de maneira que, ao fim do período, os ativos fisicamente representaram menos de 30% (meninos) e
10% (meninas) (Figura 1). Tais reduções são reforçadas com a questão de não ter tido aula de educação física na última sema
na, pois essa afirmação esteve mais presente no autorrelato de meninos (+1,1%) e meninas (+1,6%). Concomitantemente, ter tido três ou mais aulas foi menos relatado por meninos (-4,5%) e meninas
(-4,1%) (Figura 2). Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 10 Tabela 4 Regressão de Poisson * analisando a associação entre o percentual de fisicamente ativos, inquéritos e variáveis
demográficas, socioeconômicas e comportamentais da Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009, 2012,
2015 e 2019. RP
IC95%
Valor de p
Inquéritos
PeNSE/2009
1,00
PeNSE/2012
0,76
0,75-0,77
< 0,001
PeNSE/2015
0,78
0,77-0,79
< 0,001
PeNSE/2019
0,42
0,42-0,43
< 0,001
Variáveis demográficas
Sexo
Masculino
1,00
Feminino
0,53
0,53-0,54
< 0,001
Idade (anos)
13-15
1,00
16-17
0,93
0,92-0,95
< 0,001
Cor da pele
Branca
1,00
Parda
1,05
1,03-1,06
< 0,001
Amarela
1,01
1,00-1,03
0,08
Preta
1,01
0,99-1,02
0,31
Indígena
1,07
1,05-1,10
< 0,001
Variável socioeconômica
Escore de bens e serviços
≤ 1
1,00
2
1,09
1,07-1,12
< 0,001
3
1,15
1,13-1,18
< 0,001
4
1,27
1,25-1,30
< 0,001
Variáveis comportamentais
Comportamento sedentário
Tempo assistindo televisão (horas/dia)
< 2
1,00
≥ 2
1,01
1,00-1,02
0,07
Tempo sentado (horas/dia)
< 3
1,00
≥ 3
0,95
0,94-0,96
< 0,001
IC95%: intervalo de 95% de confiança; RP: razão de prevalência. * Modelo ajustado para sexo, idade, cor da pele, escore de bens e serviços, comportamento sedentário relacionado ao
tempo assistindo televisão e comportamento sedentário relacionado ao tempo sentado. Discussão ressão de Poisson * analisando a associação entre o percentual de fisicamente ativos, inquéritos e variáveis
ográficas socioeconômicas e comportamentais da Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009 2012 Regressão de Poisson * analisando a associação entre o percentual de fisicamente ativos, inquéritos e variáveis
demográficas, socioeconômicas e comportamentais da Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009, 2012,
2015 e 2019 IC95%: intervalo de 95% de confiança; RP: razão de prevalência. * Modelo ajustado para sexo, idade, cor da pele, escore de bens e serviços, comportamento sedentário relacionado ao
tempo assistindo televisão e comportamento sedentário relacionado ao tempo sentado. i
ç ;
p
* Modelo ajustado para sexo, idade, cor da pele, escore de bens e serviços, comportamento sedentário relacionado ao
tempo assistindo televisão e comportamento sedentário relacionado ao tempo sentado. Um dos caminhos a seguir é, indubitavelmente, promover a atividade física no cotidiano das pes
soas, sobretudo de crianças e adolescentes, sendo uma forma eficiente para enfrentar a pandemia da
inatividade física 23, com seus altos percentuais de prevalência na população – adultos 20,23,24, crianças
e adolescentes 9,10,12,25,26. Assim, é possível consolidar hábitos saudáveis antes dos padrões comporta
mentais se tornarem resistentes às mudanças. Nesse contexto, a escola pode contribuir para elevar o percentual de ativos fisicamente e reduzir a
inatividade física. Tal assertiva encontra respaldo nos benefícios gerados pela atividade física na vida
dos indivíduos, que incluem melhoria dos resultados acadêmicos 27,28,29, redução dos riscos cardio
vasculares 29,30,31, melhoria da saúde musculoesquelética 30,32 e dos perfis lipídico e metabólico 33. Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
11 Figura 1 Figura 1 Tendência temporal do percentual de ativos fisicamente no Brasil, nas capitais e no interior nas edições da Pesquisa Nacional de Saúde do Escolar
(PeNSE) de 2009 * 2012 2015 e 2019 Tendência temporal do percentual de ativos fisicamente no Brasil, nas capitais e no interior nas edições da Pesquisa Nacional de Saúde do Escolar
(PeNSE) de 2009 *, 2012, 2015 e 2019. Tendência temporal do percentual de ativos fisicamente no Brasil, nas capitais e no interior nas edições da Pesquisa Nacional de Saúde do Escolar
(PeNSE) de 2009 *, 2012, 2015 e 2019. inua) Infelizmente, nos últimos seis anos, o Brasil foi na contramão do mundo, reduzindo a prática de
educação física escolar a uma aula por semana em muitas redes de ensino. Isso afeta a quantidade de
estímulos aos quais os adolescentes estão expostos e os afasta da prática de atividade física fora da
escola. Ou seja, a educação física escolar não tem o objetivo de tornar os adolescentes exaustivamente
ativos durante a aula, mas sim expô-los à cultura corporal do movimento e, nesse cenário, incentivar
a realização de atividades físicas em outros contextos. Porém, é necessário reverter o número total de aulas semanais ofertadas – apenas 11,3%
(PeNSE/2015) 26 e 8,9% (PeNSE/2019) 10 dos adolescentes brasileiros tiveram três ou mais aulas de
educação física nos últimos sete dias 9,25. Em estudo anterior, foi demonstrado que há forte correlação
(respectivamente, rh0 = -0,84 e rh0 = -0,81) entre não ter tido aula de educação física nos últimos sete
dias e o percentual de escolares ativos 34, tanto na PeNSE/2009 quanto na PeNSE/2012. Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 12 Figura 1 (continuação) Figura 2
Percentual de adolescentes que não tiveram aula ou tiveram três ou mais aulas de educação física nos últimos sete dias, distribuído por sexo, conforme
Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009 *, 2012, 2015 e 2019. * A PeNSE/2009 coletou dados apenas nas 27 capitais brasileiras. * A PeNSE/2009 coletou dados apenas nas 27 capitais brasileiras. Figura 2 Se considerarmos que todas as aulas foram ministradas, que as 28 semanas letivas na educação
brasileira transcorreram normalmente, a diferença encontrada representa que, de 13 a 17 anos, as
meninas perderam, em média, 49 horas de aulas de educação física – exatamente no período em que
poderiam ter uma aula sobre e com o movimento corporal igual a de seus pares, desenvolver habili
dades motoras e adquirir conhecimento e hábito da atividade física para toda a vida. Marramarco 35 analisou o desenvolvimento de habilidades motoras de uma população que apre
sentava fatores ambientais favoráveis ao pleno desenvolvimento (assistência à saúde, alimentação
adequada e condições de higiene), baixo índice de desnutrição e de preocupação em relação ao sobre
peso e à obesidade. Todavia, mesmo em condições propícias, as meninas apresentaram resultados
inferiores aos dos meninos, tanto nas habilidades de locomoção como nas capacidades de controle de
objeto e no coeficiente de motricidade ampla. Não é de nosso interesse dissertar sobre a puberdade e as alterações típicas dessa etapa da vida dos
indivíduos. Entretanto, alguns aspectos precisam ser elencados, já que, nas aulas de educação física,
a exclusão se dá pelo grau de habilidade e força dos participantes. A força começa a se diferenciar na
puberdade, que pode ocorrer entre 8 e 13 anos de idade nas meninas. A habilidade motora fundamental inclui as capacidades motoras básicas (força, velocidade e
resistência), necessárias para tarefas específicas do movimento, e precisa ser lapidada para promover
o desenvolvimento motor. Experimentar-se motoramente fornece uma abundância de informações
e percepções sobre si mesma e sobre o mundo que a cerca, contribuindo para que a criança se desen
volva cognitiva e fisicamente, progrida sequencialmente de um estágio a outro, influenciada pelo
amadurecimento e pelo conhecimento. Não se trata exclusivamente da maturação, mas sim de opor
tunidades de prática, encorajamento e instruções, que são cruciais para o desenvolvimento de padrões
maduros de movimentos fundamentais 36. Encontramos respaldo em estudo desenvolvido por Kremer et al. 37, que utilizaram acelerometria
para mensurar a intensidade e duração dos esforços físicos nas aulas de educação física, concluindo
que os meninos (44,1%) estiveram em atividade física de intensidade moderada à vigorosa por mais
tempo que as meninas (21%; p < 0,01). Fortes et al. Figura 2 Percentual de adolescentes que não tiveram aula ou tiveram três ou mais aulas de educação física nos últimos sete dias, distribuído por sexo, conforme
Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009 *, 2012, 2015 e 2019. Percentual de adolescentes que não tiveram aula ou tiveram três ou mais aulas de educação física nos últimos sete dias, distribuído por sexo, conforme
Pesquisa Nacional de Saúde do Escolar (PeNSE) de 2009 *, 2012, 2015 e 2019. * A PeNSE/2009 coletou dados apenas nas 27 capitais brasileiras. * A PeNSE/2009 coletou dados apenas nas 27 capitais brasileiras. * A PeNSE/2009 coletou dados apenas nas 27 capitais brasileiras. Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
13 13 Nossa análise mostra que há, na educação física escolar, diferença estatisticamente significante
entre a PeNSE/2009 (57,6 minutos/semana; IC95%: 55,2-60,0) e a PeNSE/2019 (48,4 minutos/sema
na; IC95%: 46,9-49,8). A análise também reporta diferença na participação das meninas nas aulas
desse componente curricular, que estão, aproximadamente, 20 minutos aquém da prática realizada
pelos meninos em todos os inquéritos no Brasil, nas capitais e no interior – em percentual de ativos
fisicamente (Tabela 1), em média de atividade física (Tabela 2) e reforçada pela regressão de Poisson
(Tabela 4). A atividade física mostra uma grande variação no domínio do lazer. Entre 2012 e 2019, em média,
houve aumento de 15,1 minutos/semana nas capitais e 9,9 minutos/semana no interior entre as
meninas, enquanto os meninos tiveram incremento de 4,3 minutos/semana nas capitais e 7,4 minu
tos/semana no interior (Tabela 2). Entretanto, quando analisamos apenas as capitais, de 2009 a 2019,
houve um decréscimo de 6,9 minutos/semana entre as meninas e de alarmantes 81,5 minutos/semana
entre meninos. Sabe-se que o lazer envolve a existência de praças e parques próximos da residência, instalações
adequadas e condições seguras para deslocamento (violência da região, iluminação), mas o que justi
fica a atividade física das meninas, na educação física escolar, ser cerca de 21 minutos/semana mais
breve do que a dos meninos? A educação física escolar não é um lugar democrático, inclusivo, acessí
vel a todos, partilhado e, principalmente, pedagogicamente orientado? Figura 2 38 concluíram que a maioria das aulas são definidas
como “aula livre”, o esporte é o conteúdo prioritário das aulas e a ênfase estava na ação dos professores
sob a forma de observação e/ou outras tarefas. Soma-se a isso o declínio acentuado no percentual de
ativos fisicamente que ocorre na adolescência quando comparado à infância 39. Estamos diante de
uma situação complexa, que precisa de políticas públicas, formações e orientações que conduzam à
reversão da inatividade física. Contudo, estudo realizado por Ferrari et al. 22 mostrou que o ambiente escolar está associado
ao aumento da atividade física de diversas maneiras: (i) associação positiva entre a atividade física
total – obtida somando todos os domínios da atividade física – e a existência de infraestrutura nas
escolas: quadras poliesportivas (três ou mais), piscina em condições de uso, pistas de corrida/atletismo Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 14 e bicicletários; (ii) infraestrutura na escola e no entorno (quadras poliesportivas, piscina disponível
em condições de uso, semáforos com limite de velocidade ao redor da escola e faixas de pedestres)
está positivamente associada às aulas de educação física; e (iii) a existência de bicicletários, de faixas
de pedestres e a limitação de velocidade ao redor da escola foram positivamente associadas ao deslo
camento ativo. Assim, é importante fortalecer o sistema de monitoramento da atividade física dos adolescentes,
que representa uma forma eficaz de analisar os efeitos de diferentes estímulos, resultantes dos exer
cícios físicos (movimento corporal planejado, organizado e repetitivo) 40 e de outras formas de ativi
dade física (por exemplo, deslocamentos para a escola). Esse sistema também permite compreender
como as formas institucionalizadas estão sendo ofertadas aos adolescentes (por exemplo, educação
física escolar ou políticas públicas esportivas) e, inclusive, acompanhar o desenvolvimento da pande
mia de inatividade física, que conduz a diversos desfechos em saúde, entre eles a obesidade, DCNT
que gera consequências na idade adulta, a curto e longo prazo, incluindo mortalidade precoce e
morbidade física. Adolescentes fisicamente ativos aumentam a probabilidade de serem adultos ativos, contribuindo
para o balanço energético (consumo e gasto), reduzem a probabilidade de desenvolver obesidade e
doenças relacionadas à obesidade na fase adulta e, o mais importante, equilibram o balanço energé
tico durante a adolescência, pois estão protagonizando a prevenção e a profilaxia da obesidade e de
doenças relacionadas nessa fase do ciclo vital. Figura 2 Com relação ao comportamento sedentário, sabe-se que há pouco mais de 20 anos ele foi reconhe
cido como problema de saúde pública e, ao mesmo tempo, é um modulador das taxas de prevalência
das DCNT. Isso ocorre porque o comportamento sedentário influencia a redução do percentual de
ativos fisicamente, somado à facilidade que os indivíduos têm de usufruir das benesses das novas
tecnologias (assistir TV, jogar videogames, navegar pela Internet, por exemplo), ao mesmo tempo que
as formas de trabalho, baseadas na força física, foram abrandadas com as revoluções industrial e tec
nológica, sendo substituídas pelo maior tempo despendido em trabalho intelectual – normalmente,
tempo sentado. A maneira como se mensura o comportamento sedentário ainda precisa evoluir para que se pos
sa afirmar com exatidão se ações caracterizadas como sedentárias contribuem ou não para que os
indivíduos usufruam das benesses que a sociedade conquistou ao longo dos últimos séculos sem se
expor às DCNT. É especialmente importante entender que há videogames que exigem a execução de
movimentos pelo praticante, portanto, reduzindo o impacto de uma atividade que é considerada pre
judicial à saúde dos indivíduos e, quiçá, revertendo os efeitos deletérios do tempo adicional sentado. Considera-se como limitação neste estudo a análise da PeNSE/2009, pois refere-se exclusiva
mente às 27 capitais brasileiras, sem aferição da atividade física e demais condições de saúde dos
adolescentes no interior. Assim, a tendência temporal das capitais inclui as quatro edições da PeNSE
(2009, 2012, 2015 e 2019), mas o interior do Brasil está restrito às edições de 2012, 2015 e 2019. Con
sequentemente, a análise de Brasil também exclui a PeNSE/2009. Colaboradores 1. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde – 2013. Percepção
do estado de saúde, estilos de vida e doenças
crônicas – Brasil, Grandes Regiões e Uni
dades da Federação. Rio de Janeiro: Instituto
Brasileiro de Geografia e Estatística; 2014. 1. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde – 2013. Percepção
do estado de saúde, estilos de vida e doenças
crônicas – Brasil, Grandes Regiões e Uni
dades da Federação. Rio de Janeiro: Instituto
Brasileiro de Geografia e Estatística; 2014. C. A. M. Soares contribuiu com a concepção do
estudo, redação, análises estatísticas e interpreta
ção dos resultados; e aprovou a versão final. O. A. A. Leão contribuiu com a redação, revisão, análises
estatísticas e interpretação dos resultados; e apro
vou a versão final. M. P. Freitas contribuiu com a
redação, revisão, análises estatísticas e interpretação
dos resultados; e aprovou a versão final. P. C. Hal
lal contribuiu com a redação, revisão e análises dos
dados; e aprovou a versão final. M. B. Wagner con
tribuiu com a concepção do estudo, redação, revisão
e análises dos dados; e aprovou a versão final. 2. Malta DC, Andrade SSCA, Oliveira TP, Moura
L, Prado RR, Souza MFM. Probability of pre
mature death for chronic non-communicable
diseases, Brazil and Regions, projections to
2025. Rev Bras Epidemiol 2019; 22:e190030. 3. World Health Organization. World health sta
tistic 2020: monitoring health for the SDGs
– Sustainable Development Goals. Genebra:
World Health Organization; 2020. Informações adicionais 4. Ministério da Saúde. Plano de Ações Estra
tégicas para o Enfrentamento das Doenças
Crônicas e Agravos não Transmissíveis no
Brasil, 2021-2030. Brasília: Ministério da
Saúde; 2021. ORCID: Carlos Alex Martins Soares (0000-0002-
8113-3010); Otávio Amaral de Andrade Leão
(0000-0002-5253-7665); Matheus Pintanel Freitas
(0000-0001-7993-3978); Pedro Curi Hallal (0000-
0003-1470-6461); Mário Bernardes Wagner (0000-
0002-3661-4851). 5. World Health Organization. Global action
plan for the prevention and control of non
communicable diseases 2013-2020. Genebra:
WHO Press; 2013. 6. World Health Organization. Global action
plan on physical activity 2018-2030: more
active people for a healthier world. Genebra:
WHO Press; 2018. TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
15 15 É imprescindível que o número de aulas seja ampliado para, no mínimo, três vezes na semana, e
que os escolares sejam estimulados à prática de atividade física fora do ambiente escolar. Além disso,
mudanças metodológicas, didaticamente conscientes e pedagogicamente igualitárias, devem ser con
duzidas, pois parece inaceitável querermos promover a interação entre meninas e meninos se elas são
preteridas nas aulas de educação física, muito provavelmente em razão das habilidades motoras dos
meninos, que são, em nossa sociedade, estimulados ao esporte, à aventura, ao lúdico em ambientes
abertos, enquanto as meninas fantasiam a vida de donas do lar. Ampliamos a lacuna no desenvolvi
mento motor ao considerarmos que todas as questões culturais desaparecem quando meninas e meni
nos se juntam na quadra/pátio/ginásio da escola e não percebemos que três horas por semana não
impedirão o desenvolvimento social e afetivo dos adolescentes. Portanto, é provável que a solução seja
separar as turmas por sexo e permitir que as meninas se apropriem da cultura corporal do movimento
e desenvolvam habilidades motoras que lhes têm sido negadas ao longo dos anos. Conclusão Em nossos achados, dois aspectos merecem atenção redobrada dos gestores do Ministério da Saúde e
das Secretarias Estaduais e Municipais de Saúde: a queda abrupta da prevalência de atividade física e
o resultado da inequidade entre os sexos no percentual de ativos fisicamente. Primeiro, a média em atividade física regrediu de 318,4 minutos/semana (2009) para 159,2 minu
tos/semana, queda de aproximadamente 50% em dez anos, e o Brasil está no nível de países conside
rados desenvolvidos, como demonstrado no resultado do HBSC. Porém, tal processo foi progressivo
e os gestores públicos tiveram oportunidades de rever as ações de promoção da atividade física em
2012 (232,4 minutos/semana) ou em 2015 (253,3 minutos/semana), pois uma parcela significativa
já não estava cumprindo as recomendações de 300 minutos/semana em atividade física de inten
sidade moderada à vigorosa. O segundo aspecto é o resultado entre sexos nas aulas de educação
física, pois as meninas estão, em média, 21 minutos/semana aquém do tempo de prática dos meninos
nesse domínio. Cad. Saúde Pública 2023; 39(10):e00063423 Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
15 Agradecimentos Strong WB, Malina RM, Blimkie CJR, Dan
iels SR, Dishman RK, Gutin B, et al. Evidence
based physical activity for school-age youth. J
Pediatr 2005; 146:732-7. 14. Department of Health and Human Services. Physical activity guidelines for Americans. 2a
Ed. Washington DC: Department of Health
and Human Services; 2018. 33. Lazzoli JK, Nóbrega ACL, Carvalho T, Oliveira
MAB, Teixeira JAC, Leitão MB, et al. Atividade
física e saúde na infância e adolescência. Rev
Bras Med Esporte 1998; 4:107-9. 15. Ministério da Saúde. Guia de atividade física
para a população brasileira. Brasília: Ministé
rio da Saúde; 2021. 34. Soares CAM, Hallal PC. Interdependência
entre a participação em aulas de Educação
Física e níveis de atividade física de jovens
brasileiros: estudo ecológico. Rev Bras Ativ Fis
Saúde 2015; 20:588-97. 16. Azevedo Junior M, Araújo C, Pereira F. Ativi
dades físicas e esportivas na adolescência:
mudanças de preferências ao longo das últi
mas décadas. Rev Bras Educ Fís Esporte 2006;
20:51-8. 35. Marramarco CA. Relação entre o estado nutri
cional e o desempenho motor de crianças do
município de Farroupilha-RS [Dissertação de
Mestrado]. Florianópolis: Centro de Educação
Física, Fisioterapia e Desportos, Universidade
do Estado de Santa Catarina; 2007. 17. Baptista F, Silva AM, Santos DA, Mota J, San
tos R, Vale S, et al. Livro verde da actividade
física. Lisboa: Instituto do Desporto de Portu
gal; 2011. 18. Florindo A, Hallal P. Epidemiologia da ativi
dade física. São Paulo: Atheneu; 2011. 36. Gallahue DL, Ozmun JC, Goodway JD. Com
preendendo o desenvolvimento motor: bebês,
crianças, adolescentes e adultos. 7a Ed. Porto
Alegre: AMGH; 2017. 19. World Health Organization. Global recom
mendations on physical activity for health. Genebra: World Health Organization; 2010. 37. Kremer M, Reichert F, Hallal P. Intensidade
e duração dos esforços físicos em aulas de
Educação Física. Rev Saúde Pública 2012; 46:
320-6. 20. Sallis JF, Bull F, Guthold R, Heath GW, Inoue
S, Kelly P, et al. Progress in physical activity
over the Olympic quadrennium. Lancet 2016;
388:1325-36. 38. Fortes M, Azevedo M, Kremer M, Hallal P. A
Educação Física escolar na cidade de Pelotas,
RS: contexto das aulas e conteúdos. Rev Educ
Fís 2012; 23:69-78. 21. Souza-Júnior PRB, Freitas MPS, Antonaci GA,
Szwarwald CL. Desenho da amostra da Pes
quisa Nacional de Saúde 2013. Epidemiol Serv
Saúde 2015; 24:207-16. 39. Cossio-Bolaños MA, Viveros-Flores A, Cas
tillo-Retamal M, Vargas-Vitoria R, Gatica P,
Gómez-Campos R. Agradecimentos Ao Conselho Nacional de Desenvolvimento Cientí
fico e Tecnológico (CNPq) pela concessão de bolsa
de doutorado a C. A. M. Soares. 7. World Health Organization. Noncommuni
cable diseases: country profiles 2018. Genebra:
World Health Organization; 2018. 7. World Health Organization. Noncommuni
cable diseases: country profiles 2018. Genebra:
World Health Organization; 2018. Cad. Saúde Pública 2023; 39(10):e00063423 Soares CAM et al. 16 25. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar – 2009. Rio de Janeiro: Instituto Brasileiro de Geogra
fia e Estatística; 2009. 8. Marques A, De Matos MG. Adolescents’ physi
cal activity trends over the years: a three-co
hort study based on the Health Behaviour in
School-aged Children (HBSC) Portuguese sur
vey. BMJ Open 2014; 4:e006012. 26. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar – 2015. Rio de Janeiro: Instituto Brasileiro de Geogra
fia e Estatística; 2016. 9. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar – 2012. Rio de Janeiro: Instituto Brasileiro de Geogra
fia e Estatística; 2013. 27. Hallal PC, Bertoldi AD, Gonçalves H, Victora
CG. Prevalência de sedentarismo e fatores as
sociados em adolescentes de 10-12 anos de
idade. Cad Saúde Pública 2006; 22:1277-87. 10. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional de Saúde do Escolar: 2019. Rio de Janeiro: Instituto Brasileiro de Geogra
fia e Estatística; 2021. 28. Hillman CH, Erickson KI, Kramer AF. Be
smart, exercise your heart: exercise effects on
brain and cognition. Nat Rev Neurosci 2008;
9:58-65. 11. Reilly JJ, Barnes J, Gonzalez S, Huang WY,
Manyanga T, Tanaka C, et al. Recent secular
trends in child and adolescent physical activity
and sedentary behavior internationally: analy
ses of Active Healthy Kids Global Alliance
Global Matrices 1.0 to 4.0. J Phys Act Health
2022; 19:729-36. 29. Sardinha LB, Marques A, Martins S, Palmeira
A, Minderico C. Fitness, fatness, and academ
ic performance in seventh-grade elementary
school students. BMC Pediatr 2014; 14:176. 12. WHO Europe. Health Behaviour in School-
aged Children (HBSC) study: international re
port from the 2013/2014 survey. Copenhage:
WHO Regional Office for Europe; 2016. 30. Howley ET, Franks BD. Manual do condicio
namento físico. Porto Alegre: Artmed; 2008. 31. Morris JN, Heady JA, Raffle PAB, Roberts CG,
Parks JW. Coronary heart-disease and physical
activity of work. Lancet 1953; 262:1111-20. 13. World Health Organization. WHO guidelines
on physical activity and sedentary behaviour. Genebra: WHO Press; 2020. 32. Resumen This study aimed to analyze the physical activ
ity trend of Brazilian schoolchildren and the as
sociations with demographic, socioeconomic, and
behavioral variables by using the Brazilian Na
tional Survey of School Health (PeNSE) in its
four editions – 2009, 2012, 2015, and 2019. Data
from students (13-17 years old) participating
in the four editions of the PeNSE (n = 392,922)
were used. We describe the percentage of active,
mean, and percentile values of moderate to vigor
ous intensity physical activity in minutes/week. Poisson’s regression was adjusted for gender, age,
skin color, goods score, and sedentary behavior
(≥ 2 hours/day watching TV and ≥ 3 hours/day
sitting time). As a limitation, the PeNSE/2009
sample refers only to the Brazilian capital cit
ies. The percentage of active students decreased
from 43.1% in 2009 to 18.2% in 2019. The mean
moderate to vigorous intensity physical activ
ity of PeNSE/2009 (mean = 318.4 minutes/week;
95%CI: 313.4-323.4) decreased 50% in 2019. In
physical education, the weekly average in moder
ate to vigorous intensity physical activity of girls
is less than 50 minutes and boys is greater than 60
minutes in the four editions of PeNSE, also 22.7%
of girls reported (PeNSE/2019) not having taken
physical education classes whereas the same thing
is reported by 19.7% of boys. Sedentary behavior
reduced regarding time watching TV, but sitting
time increased by 50.1% (95%CI: 48.9-51.3) and
54% (95%CI: 53.1-54.9) between PeNSE/2009
and PeNSE/2019. As a consequence of the drop
in physical activity levels, public policies that pro
mote physical activity are necessary, including in
creasing physical education classes at school to at
least three times a week. El objetivo de este estudio fue analizar la tenden
cia de la actividad física del alumnado brasileño
y sus asociaciones con las variables demográficas,
socioeconómicas y de comportamiento mediante
la Encuesta Nacional de Salud del Escolar
(PeNSE) en sus cuatro ediciones (de 2009, 2012,
2015 y 2019). Se utilizaron los datos del alumnado
(13-17 años) que participó en las cuatro ediciones
de la PeNSE (n = 392.922). Se describieron el por
centaje de valores activos, la media y los valores
percentiles de actividad física de intensidad mo-
derada a vigorosa en minutos/semana. La re
gresión de Poisson se ajustó según sexo, edad, color
de la piel, puntuación de activos y comportamiento
sedentario (≥ 2 horas/día para ver televisión y ≥
3 horas/día sentado). Agradecimentos Patrones de actividad físi
ca en adolescentes en función del sexo, edad
cronológica y biológica. Nutr Clín Diet Hosp
2015; 35:41-7. 22. Ferrari G, Rezende LFM, Florindo AA, Mielke
GI, Peres MFT. School environment and phys
ical activity in adolescents from São Paulo city. Sci Rep 2021; 11:18118. 23. Kohl HW, Craig CL, Lambert EV, Inoue S, Al
kandari JR, Leetongin G, et al. The pandemic
of physical inactivity: global action for public
health. Lancet 2012; 380:294-305. 40. Riebe D, Ehrman JK, Liguori G, Magal M. Di
retrizes do ACSM para os testes de esforço e
sua prescrição. 10a Ed. Rio de Janeiro: Guana
bara Koogan; 2018. 24. Hallal PC, Andersen LB, Bull FC, Guthold R,
Haskell W, Ekelund U, et al. Global physical ac
tivity levels: surveillance progress, pitfalls, and
prospects. Lancet 2012; 380:247-57. Cad. Saúde Pública 2023; 39(10):e00063423 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
17 TENDÊNCIA TEMPORAL DE ATIVIDADE FÍSICA EM ADOLESCENTES BRASILEIROS
17 Adolescent; Physical Activity; Risk Factors; Time
Series Studies Adolescente; Actividad Física; Factores de Riesgo;
Estudios de Series Temporales Recebido em 02/Abr/2023
Versão final reapresentada em 26/Jun/2023
Aprovado em 04/Jul/2023 Cad. Saúde Pública 2023; 39(10):e00063423 Resumen Como limitación, la mues
tra de la PeNSE/2009 se refiere únicamente a las
capitales brasileñas. El porcentaje de activos dis
minuyó del 43,1% en 2009 al 18,2% en 2019. La
media de actividad física de intensidad moderada
a vigorosa de la PeNSE/2009 (media = 318,4
minutos/semana; IC95%: 313,4-323,4) tuvo una
reducción de un 50% en 2019. En educación física,
el promedio semanal en actividad física de intensi
dad moderada a vigorosa de las niñas fue menos de
50 minutos, y el de los niños llegó a 60 minutos en
las cuatro ediciones de la PeNSE, además, el 22,7%
de las niñas y el 19,7% de los niños (PeNSE/2019)
informaron no haber tomado clases de educación
física. Hubo una disminución en el comporta
miento sedentario de ver televisión, pero el tiempo
sentado aumentó del 50,1% (IC95%: 48,9-51,3)
al 54% (IC95%: 53,1-54,9) entre la PeNSE/2009
y la PeNSE/2019. El descenso de los niveles de
actividad física lleva a la necesidad de desarro-
llar políticas públicas que promuevan la activi
dad física, incluido el incremento de las clases de
educación física en las escuelas al menos tres veces
por semana. Adolescent; Physical Activity; Risk Factors; Time
Series Studies Adolescente; Actividad Física; Factores de Riesgo;
Estudios de Series Temporales Adolescente; Actividad Física; Factores de Riesgo;
Estudios de Series Temporales Cad. Saúde Pública 2023; 39(10):e00063423
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Improved tools and strategies for the prevention and control of arboviral diseases: A research-to-policy forum
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POLICY PLATFORM OPEN ACCESS Citation: Olliaro P, Fouque F, Kroeger A, Bowman
L, Velayudhan R, Santelli AC, et al. (2018)
Improved tools and strategies for the prevention
and control of arboviral diseases: A research-to-
policy forum. PLoS Negl Trop Dis 12(2):
e0005967. https://doi.org/10.1371/journal. pntd.0005967 Abstract Editor: Pattamaporn Kittayapong, Faculty of
Science, Mahidol University, THAILAND
Published: February 1, 2018
Copyright: © 2018 Olliaro et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Pattamaporn Kittayapong, Faculty of
Science, Mahidol University, THAILAND
Published: February 1, 2018 Method A research-to-policy forum was convened by TDR, the Special Programme for Research
and Training in Tropical Diseases, with researchers and representatives from ministries of
health, in order to review research findings and discuss their implications for policy and
research. Funding: The underlying research and the
"Research to Policy Forum" were supported by TDR
core-funding and by funds made available under
the umbrella of an EU financed consortium IDAMS
(International Research Consortium for Dengue
Risk Assessment, Management and Surveillance,
grant number m281803). The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. * s.runge-ranzinger@t-online.de Background Research has been conducted on interventions to control dengue transmission and respond
to outbreaks. A summary of the available evidence will help inform disease control policy
decisions and research directions, both for dengue and, more broadly, for all Aedes-borne
arboviral diseases. Copyright: © 2018 Olliaro et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Improved tools and strategies for the
prevention and control of arboviral diseases:
A research-to-policy forum Piero Olliaro1, Florence Fouque1, Axel Kroeger1,2, Leigh Bowman3, Raman Velayudhan4,
Ana Carolina Santelli5, Diego Garcia6, Ronald Skewes Ramm7, Lokman H. Sulaiman8,
Gustavo Sanchez Tejeda9, Fabiàn Correa Morales9, Ernesto Gozzer10, Ce´sar
Basso Garrido11, Luong Chan Quang12, Gamaliel Gutierrez13, Zaida E. Yadon14,
Silvia Runge-Ranzinger15* 1 UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases
(TDR), World Health Organization, Geneva, Switzerland, 2 Global Health Department, Centre for Medicine
and Society/Anthropology, Freiburg University, Freiburg im Breisgau, Germany, 3 Department of Vector
Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 4 Department of Control of
Neglected Tropical Diseases (WHO/NTD), World Health Organization, Geneva, Switzerland, 5 Dengue
Control Program, Ministry of Health, Brasilia, Brazil, 6 Department of Communicable Diseases, Ministry of
Health, Bogota, Colombia, 7 Program for the Prevention and Control of Dengue, Ministry of Health, Santo
Domingo, Dominican Republic, 8 Department of Public Health, Ministry of Health, Kuala Lumpur, Malaysia,
9 Centro Nacional de Programas Preventivos y Control de Enfermedades (CENAPRECE), Ministry of Health,
Mexico City, Mexico, 10 Universidad Peruana Cayetano Heredia, Lima, Peru, 11 Facultad de Agronomia,
Universidad de la Repu´blica, Montevideo, Uruguay, 12 Department for Disease Control and Prevention,
Pasteur Institute, Ho Chi Minh City, Vietnam, 13 PAHO/AMRO, World Health Organization, Washington, DC,
United States of America, 14 PAHO/AMRO, World Health Organization, Rio de Janeiro, Brazil, 15 Institute of
Public Health, University of Heidelberg, Heidelberg, Germany a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 virus) to dengue outbreaks. A range of variables that, according to the literature, either indi-
cate risk of forthcoming dengue transmission or predict dengue outbreaks were tested and
some of them could be successfully applied in an Early Warning and Response System
(EWARS). Entomological surveillance and vector management. A summary of the published lit-
erature shows that controlling Aedes vectors requires complex interventions and points to
the need for more rigorous, standardised study designs, with disease reduction as the pri-
mary outcome to be measured. House screening and targeted vector interventions are
promising vector management approaches. Sampling vector populations, both for surveil-
lance purposes and evaluation of control activities, is usually conducted in an unsystematic
way, limiting the potentials of entomological surveillance for outbreak prediction. Combining outbreak alert and improved approaches of vector management will help to
overcome the present uncertainties about major risk groups or areas where outbreak
response should be initiated and where resources for vector management should be allo-
cated during the interepidemic period. Conclusions The Forum concluded that the evidence collected can inform policy decisions, but also that
important research gaps have yet to be filled. Results The participants reviewed findings of research supported by TDR and others. The participants reviewed findings of research supported by TDR and others. Surveillance and early outbreak warning. Systematic reviews and country studies
identify the critical characteristics that an alert system should have to document trends reli-
ably and trigger timely responses (i.e., early enough to prevent the epidemic spread of the Surveillance and early outbreak warning. Systematic reviews and country studies
identify the critical characteristics that an alert system should have to document trends reli-
ably and trigger timely responses (i.e., early enough to prevent the epidemic spread of the Competing interests: The authors have declared
that no competing interests exist. 1 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Background As dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) continue to spread
worldwide, there is an ever-increasing need to develop and apply cost-effective, evidence-based
approaches to identify and respond to arboviral disease outbreaks. Outbreak response should
be timely (i.e., early enough to prevent the epidemic spread of the virus), coordinated among
multiple stakeholders, and should make use of existing vector control interventions against
Aedes populations—the principal vector responsible for the transmission of these viruses. Research has been conducted on interventions to control dengue transmission and out-
break preparedness, including work supported and coordinated by TDR, the Special Pro-
gramme for Research and Training in Tropical Diseases. A substantial body of information
has been generated over 13 years, and a summary of the available evidence would presently be
helpful both to the research and disease control communities. It also remains crucial to estab-
lish the various capacity-strengthening needs that countries require to test, deploy, and moni-
tor interventions. Indeed, such information could be used to inform policy decisions as well as
identify knowledge gaps (see Fig 1). Accordingly, this article provides the necessary summary complemented by valuable input
from a research-to-policy forum held in August 2016 in Geneva, Switzerland. The forum
reviewed the output of research on tools for early dengue outbreak detection and response, as
well as improved approaches to dengue vector control that have been developed and tested by
TDR with its partner research institutions and country control programmes, against the back-
ground of the broader research context. Specifically, this paper reviews the output of research on (1) outbreak preparedness of den-
gue epidemics and (2) innovative vector control interventions to control Aedes–borne disease
transmission. 2 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Fig 1. Evidence generation to inform policy decisions and further research requirements. IR, Implementation
Research; OR, Operational Research. Fig 1. Evidence generation to inform policy decisions and further research requirements. IR, Implementation
Research; OR, Operational Research. https://doi.org/10.1371/journal.pntd.0005967.g001 https://doi.org/10.1371/journal.pntd.0005967.g001 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Laboratory diagnosis of dengue is currently done either by detecting the virus or its compo-
nents (viral RNA by PCR, or antigens like NS1) or by detecting immune response by serologi-
cal tests. A “confirmed case” requires either virus isolation, RNA detection, antigen detection,
seroconversion for IgM, or a 4-fold rise in IgG titres; IgM positivity is considered highly sug-
gestive. The timing of testing is critical, considering that both viraemia and NS1 are confined
to the first week of illness, and IgM production is transient (lasting 5–6 months), while IgG
lasts longer [5]. Confirming dengue diagnosis is a clear challenge for countries, both at point-
of-care and at referral facilities. However, the combination of PCR or NS1 ELISA plus IgM
ELISA appears to accurately identify >90% of primary and secondary dengue cases from a sin-
gle serum specimen collected during the first 10 days of illness [6,7]. While operationally and
financially demanding, laboratory confirmation is important, both for clinical management
and outbreak identification: it increases the specificity of the information captured by the sur-
veillance system, contributes to syndromic surveillance (e.g., increased numbers of laboratory
requests), and may generate serotype- or genotype-specific data as a potential additional out-
break alarm signal. Syndromic surveillance [8] (developed as an additional, often context-specific tool for early
outbreak alert) is not limited only to clinical syndromes but may include increased numbers of
school absenteeism, increased laboratory requests—as described above—or an increased pro-
portion of positive laboratory results in the inter-epidemic period. These alarm signals can be
then integrated into a risk assessment tool (see below). Indeed, enhanced surveillance should
aim to combine tools that complement routine reporting, not to replace it [9]. One question is whether and how what we know about dengue outbreak detection can be
applied to Zika and chikungunya. For dengue, we have years of historical incident case data
and can construct an “endemic channel.” We do not have this information for the other two,
which have so far occurred in naïve populations; therefore, syndromic and sentinel-based
approaches seem the best option at this point in time until comprehensive, historic datasets
become available. Outbreak preparedness and response. A range of variables that either indicate risk of
forthcoming dengue transmission or predict dengue outbreaks have been suggested through-
out the literature [10,11,12]. The combination of these variables, or alarm indicators, for use in
early warning systems is the next natural step. Summary of findings of TDR-supported research on dengue
outbreak detection and innovative vector interventions
Outbreak preparedness of dengue epidemics Effective dengue, chikungunya and Zika surveillance. Routine reporting of notifiable
diseases in national disease surveillance systems is the backbone of epidemiological informa-
tion; it is used to monitor the spatial and temporal distribution of different clinical expressions
of diseases, to determine the risk and priority areas for interventions, and to trigger outbreak
alerts. Dengue-endemic country surveillance and response systems were systematically reviewed
to identify what corrective actions should be undertaken and how countries should be sup-
ported. Dengue surveillance was found to suffer from significant delays and marked underre-
porting, especially for nonhospitalized dengue cases [1]. The comparative analysis of national
dengue contingency plans [2] revealed weak overall outbreak governance due to poor clarity
of stakeholder roles, weak surveillance systems, inadequate use of routinely generated data and
additional alerts in tandem, absence of outbreak definition, and absence of structured early-
response mechanisms. Systematic reviews and country studies highlighted the critical characteristics of an efficient
alert system to trigger responses: it should be sensitive to predict or detect outbreaks in a
timely manner; specific to avoid unnecessary false alerts; and timely to trigger early response
[3]. In order for systems to meet these requirements, they should make use of a simplified and
standardized case classification [4], be supported by laboratories using standardized and qual-
ity-controlled assays, include active/enhanced/syndromic surveillance, and either incorporate
additional alarm signals or increase data quality and/or timeliness [3]. 3 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 An Early Warning and Response System
(EWARS) has since been developed following a period of retrospective country dataset analysis
[13], modelling, and prospective field evaluation (Fig 2). This recent research has also included
the development of a “staged” response system, which gradually introduces greater interven-
tion resources in response to increasing certainty of forthcoming outbreaks. The outbreak
warning system (EWS)—a combination of statistical (STATA) and database management
(Microsoft Excel) software—will be freely available via the publication Early Warning and
Response System (EWARS) for Dengue Outbreak: Operational Guide (WHO-TDR)[14]. These
tools and materials will help build capacity in dengue-endemic settings. In addition, a technical handbook (model contingency plan) has been developed. It is
intended to serve as a framework, incorporating all under the aspects of evidence described in
“Outbreak preparedness of dengue epidemics” above in order to support and guide the
national contingency-planning process [15]. Details of this framework are summarized and
published in a separate publication [9]. Following up the above-mentioned evidence, two additional areas of future development
have emerged: (1) the ability to combine qualitative and quantitative variables into a broad-
ranging early warning system, and (2) spatial risk-mapping tools that help to identify smaller
spatial areas for fine-scale interventions. 4 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Fig 2. The process: From research to operational implementation. (1) Retrospective analysis of alarm indicators for dengue outbreaks. (2) Use
of algorithms to generate prospective early warning system (EWS). (3) Prospective randomised controlled trial of early warning and response
system (EWARS). (4) Associated EWARS publications (5) Scale up of EWARS. a Bowman et al.2016; b Cluster-randomized controlled trial for
dengue early warning systems (in-prep); c Runge-Ranzinger et al. 2016; d WHO-TDR. Early Warning and Response System (EWARS) for
Dengue Outbreaks: Operational Guide (in-press); e WHO-TDR. Technical handbook. 2016. https://doi.org/10.1371/journal.pntd.0005967.g002 Fig 2. The process: From research to operational implementation. (1) Retrospective analysis of alarm indicators for dengue outbreaks. (2) Use
of algorithms to generate prospective early warning system (EWS). (3) Prospective randomised controlled trial of early warning and response
system (EWARS). (4) Associated EWARS publications (5) Scale up of EWARS. a Bowman et al.2016; b Cluster-randomized controlled trial for
dengue early warning systems (in-prep); c Runge-Ranzinger et al. 2016; d WHO-TDR. Early Warning and Response System (EWARS) for
Dengue Outbreaks: Operational Guide (in-press); e WHO-TDR. Technical handbook. 2016. https://doi.org/10.1371/journal.pntd.0005967.g002 https://doi.org/10.1371/journal.pntd.0005967.g002 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 compared to unscreened homes. These findings are corroborated by new investigations of
screening in Mexico, which have shown window and door screens to be a popular and widely-
adopted intervention that can significantly reduce domestic infestations of A. aegypti [17,18] compared to unscreened homes. These findings are corroborated by new investigations of
screening in Mexico, which have shown window and door screens to be a popular and widely-
adopted intervention that can significantly reduce domestic infestations of A. aegypti [17,18] When data were not appropriate for meta-analysis, single interventions have been
described in systematic reviews elsewhere that address peridomestic space spraying [19], teme-
phos [20], Bacillus thuringiensis israelensis [21], copepods [22], and larvivorous fish [23]. Fur-
ther work has been completed on the effectiveness of pyriproxyfen [24] and work on indoor
residual house spraying (IRS), including targeted indoor spraying, is under way. In summary,
these results indicate that as single, standalone interventions, these approaches are efficient;
however, little evidence exists with respect to reduction of disease incidence. A summary of the published literature to-date can be described as follows: (1) Vector con-
trol can be effective against Aedes populations; (2) Effectiveness against transmission has been
reported in a minority of robust study designs—implementation remains an issue; (3) Single
interventions may be largely ineffective with respect to disease reduction in the community;
(4) Complex interventions have proven very effective at reducing vector abundance as part of
community-based campaigns; (5) Early vector control implementation prior to outbreaks
would likely have greater impact at mitigating dengue cases; (6) Methodologically rigorous
studies are required to contribute further evidence towards effective vector control strategies. Systematic review on entomological surveillance. A systematic literature review [25]
examined the published evidence investigating associations between vector indices and dengue
cases. After assessment of the epidemiological study designs, all but three of the 18 studies
were classified as methodologically weak. Heterogeneity among spatial/temporal sampling and
analyses was high, perhaps demonstrating an absence of standardization for conducting such
research. Of the 13 studies that investigated associations between vector indices and dengue
cases, 4 reported positive correlations, 4 found no correlation, and 5 reported ambiguous or
unreliable associations. Of the 7 studies that measured the Breteau Index, 6 reported dengue
transmission at levels below the widely used threshold of 5. Sampling vector populations, both for surveillance purposes and evaluation of control
activities, is conducted annually worldwide. Improved vector interventions to control Aedes-borne disease transmission Systematic reviews on dengue vector management. A systematic review and meta-analy-
sis [16] evaluated the evidence of the effectiveness of vector control interventions in (a) reduc-
ing vector indices and (b) preventing dengue transmission. The searches covered all major
indexing databases throughout the period 1980–January 2013. The primary outcome was den-
gue incidence with secondary outcomes comprising a number of Aedes indices (e.g., Breteau
Index, House Index, Container Index, mosquito adults per person, pupae per person); analyses
were stratified by study design, intervention, and measures of effect and outcome. The main
findings of the meta-analysis were: (1) moderate evidence that house screening can reduce vec-
tor abundance and emerging evidence that it reduces dengue transmission; (2) strong evidence
that community-based campaigns can impact vector abundance, with emerging evidence for
impact on transmission; and (3) no robust studies on the impact of fogging on transmission,
with only one study showing an impact on Aedes albopticus. The only evidence to-date of effec-
tiveness in preventing dengue transmission is in the form of house screening; the meta-analy-
sis demonstrated a significant reduction in the odds of dengue incidence among households
with screens (three studies, pooled odds ratio: 0.22 [95% confidence interval: 0.05, 0.93]) 5 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Further research needs for contingency planning and the Early Warning
and Response System (EWARS) The indicator-based early warning system for dengue outbreaks and the translation of the
technical handbook into national policy requires further evaluation and development in the
following areas: • Implementation at the appropriate level of the health system to assess needs, user-friendli-
ness, feasibility of application, and training requirements; • Refinement and adjustment for general use; • Refinement and adjustment for general use; • Evaluation of the effectiveness at impacting transmission via early response and cost effec-
tiveness in support of country adoption; • Extension of the EWARS to include additional alarm indicators, particularly qualitative vari-
ables, in an alert algorithm, using decision trees and variable weighting; • Integration of Zika and chikungunya into the alert tool as historic data accumulate; • Scaling up implementation in current and new countries; • Scaling up implementation in current and new countries; • Strengthening in-country capacity and ensuring sustainability. Additional research will be required for adapting the alert algorithm after implementation
of new interventions, such as a dengue vaccine—preferably in combination with vector control
activities—and/or Wolbachia-infected mosquitoes. Additional research will be required for adapting the alert algorithm after implementation
of new interventions, such as a dengue vaccine—preferably in combination with vector control
activities—and/or Wolbachia-infected mosquitoes. Bringing outbreak alert and integrated vector management together:
Research needs and expected results Based on the above-mentioned research findings on (1) outbreak preparedness of dengue epi-
demics and (2) improved vector control interventions to control Aedes–borne disease trans-
mission, we identified the complementary relationship of the two components. With this in
mind we aim to develop a comprehensive intervention package including a) tools for use dur-
ing the inter-epidemic period—including window screening and community-based cam-
paigns and b) additional interventions for stepwise outbreak response following timely alerts
of defined levels. This will require further research (Fig 3). PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Further evidence of the relationship between vec-
tor indices and dengue transmission is necessary to better understand the impact of control
activities on dengue incidence. Also, the role of asymptomatic individuals in virus transmis-
sion requires further analysis. Research on house screening and targeted vector interventions. The research pro-
gramme labelled “eco-bio-social research” in five Asian and five Latin American countries has,
in its first phase, identified the complexity of determinants for vector breeding [26,27,28] and,
in its second phase, identified innovative vector control interventions delivered through part-
nership models including vector control services, communities, and others. The interventions
included protection from adult vectors through insecticide-treated window curtains or screens
and/or targeting productive oviposition sites (i.e., in those container types which produce
more than 70% of all Aedes pupae) [29,30,31]. While the intervention packages including
treated window curtains were all able to reduce vector densities significantly, reduction of den-
gue incidence could not be used as an end-point measure due to relatively small intervention
clusters. In settlements with rather compact houses (i.e., houses with few or no openings addi-
tional to windows and doors) [32,33] the effect of fixed window screens with insecticide-
treated netting materials seemed to have an even stronger and longer–lasting effect, and are
well accepted and affordable if certain cost-saving strategies are introduced [18,34,35]. Cost
savings are expected to occur with the massive industrial production of window screens, the
increased demand by the middle and upper classes, and by the use of cheaper materials
(wooden rather than aluminum frames). 6 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Further research needs identified for scaling up vector interventions The optimal strategy to deliver vector control tools needs to take into account the principles
and key elements of Integrated Vector Management (IVM), which include evidence-based
decision making, judicious use of insecticides, and social mobilization and collaboration
within the health sector and beyond [36]. Such a strategy requires further research, with the
following specific needs: • Robust study designs to produce evidence of effectiveness against transmission. • Health system research focusing on implementation issues. • Complex intervention strategies targeting larvae and adult mosquitos as part of community. based-campaigns, including delivery of several interventions from an operational
perspective. 7 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 Fig 3. Priority research areas in the EWARS and improved vector control programme. https://doi.org/10.1371/journal.pntd.0005967.g003 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
February 1, 2018 From research to policy support Following the research-to-policy forum, evidence supporting policy uptake of interventions
were extracted with a view of informing guidelines and policy recommendations, including
the process being undertaken by WHO to update the 2009 Global Dengue Guide. Specific con-
siderations regarding the translation of research findings into policy were as follows: Fig 3. Priority research areas in the EWARS and improved vector control programme. More specifically, we also need to: More specifically, we also need to: • Identify the best practices and strategies for a successful partnership model, including social
(community) participation. • Identify the best practices and strategies for a successful partnership model, including social
(community) participation. • Analyse the enabling and the limiting factors for successful inter-sectoral work at the munic-
ipality level. 8 / 13 • Develop and evaluate effective and cost-effective methods to promote social participation in
urban environments, using modern communication technologies. • Develop and evaluate effective and cost-effective methods to promote social participation in
urban environments, using modern communication technologies. • Identify decision makers’ perceptions and biases on the risks, costs, and benefits of improved
interventions. • Identify decision makers’ perceptions and biases on the risks, costs, and benefits of improved
interventions. • Develop a framework that facilitates decision-making regarding how successful small-scale
pilot research projects should be taken to scale. • Develop a framework that facilitates decision-making regarding how successful small-scale
pilot research projects should be taken to scale. Research needs for defining the priority areas for the application of
improved vector control interventions during the inter-epidemic period
and outbreaks DENV, ZIKV and CHIKV transmission mainly occur in urban and periurban areas—usually
consisting of large and heterogeneous districts. National programme managers, district health,
and control programme staff urgently require a tool that identifies priority areas for action,
particularly where the interventions should start, and ideally includes a rapid diagnostic test to
distinguish between the different Aedes-borne diseases. In the presence of predictive outbreak
alarms, a method to focus interventions spatially in a targeted fashion would likely increase the
efficiency of available resources. Indeed, the absence of such a tool is a major concern often
expressed by national programme managers; after receiving an alarm signal at the district
health office, staff need clear guidance on where to intervene with early response actions, par-
ticularly for highly focal vector control measures such as fogging. A Geographical Information System (GIS) risk mapping tool that includes appropriate evi-
dence-based variables such as vector densities, historical clustering of cases, and/or population
movements (yet to be developed and tested) could potentially overcome these difficulties. Research needs include: • Developing and pilot testing using GIS software; • Field evaluation of the application in high-risk areas; • Feasibility, cost, and acceptance studies; • Integration and combined use of the GIS application with EWARS. Research on improved vector management approaches: Implications for
policy Complex intervention strategies targeting larvae and adults as part of community-based cam-
paigns are effective, although it is difficult to disentangle the contribution of each individual
component. It is now important to assess how these interventions can be best delivered and
evaluate their impact from an operational perspective. Also necessary are studies that can tease
apart the individual impacts of interventions that comprise complex community-based
campaigns. According to the available data on successes of house screening in reducing indoor and out-
door vector densities—as well as some evidence to suggest an impact on transmission—house
screening seems to be a promising measure to limit the transmission of Aedes-borne arboviral
infections, provided suitable house structure, coverage, acceptability, and sustainability. Addi-
tionally, evidence exists in support of complex community-based campaigns to reduce the cir-
culating Aedes population suggesting that these can directly translate into an impact on disease
transmission, although further studies are required. Research on risk mapping tool: Implications for policy A shift away from global risk maps (which show the burden of dengue and other Aedes-borne
diseases in countries and regions) towards fine-scale risk maps (which visualize, for opera-
tional purposes, the infection risk at sub-district level called “localities” or “health areas” or
“PHC areas,” etc.) is required to better focus vector control interventions. The addition of
fine-scale variables, such as adult mosquito abundance and entomo-virological parameters,
will facilitate this process. Human movement and contact-tracing studies are also crucial to
understand the role of human behaviour in the transmission of Aedes-borne diseases (see also
Vazquez-Prokopec (2017) [37]). Finally, studying the impact upon prevailing herd immunity
of newly introduced vaccines becomes now a necessary addition. Together, such advance-
ments will enable national control services to focus and prioritise areas for intervention, both
in-between and during epidemic periods. Vector surveillance data (generally larval/pupal surveys) are frequently captured using non-
standardised methods. These data are often used for monitoring the success of an intervention
rather than generating time-series data to monitor fluctuations in vector abundance. Further-
more, adult mosquito data are not routinely captured and less so investigated for the presence/
absence of viruses. Together, these factors have limited the predictive ability of vector dynam-
ics for epidemic transmission and contribute to the lack of evidence of a correlation between
vector density and disease incidence. A transition to capturing adult mosquito data may be a
better option. Moreover, establishing the number of adult mosquitos positive for virus will
enable the production of fine spatial scale risk maps for targeting of interventions. Research on outbreak detection and response (EWARS) and contingency
planning: Implications for policy The technical handbook for contingency planning, along with the operational guide describing
the steps of establishing the EWARS at national, state, or even district level are being consid-
ered by several ministries of health. The first year of implementation, monitoring, and evalua-
tion will be crucial to understanding the benefits and limitations of this system. The feedback
will influence the direction of the EWARS, whether it can be recommended for general use or
whether further adaptations are necessary. 9 / 13 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0005967
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https://openalex.org/W1998404111
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English
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PRIVATE CAPITAL FLOWS TO LOW‐INCOME COUNTRIES: THE ROLE OF DOMESTIC FINANCIAL SECTOR
|
Journal of business economics and management
| 2,010
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cc-by
| 7,846
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Received 9 December 2009; accepted 17 September 2010 Abstract. The relationship between private capital flows and growth has been examined
extensively in the literature, yet numerous controversies still remain. The study examines
the relationships among private capital flows (foreign direct investment, portfolio invest-
ment and foreign debt), financial development and economic performance in a sample
of 16 low-income developing countries over the period 1988–2006, by employing gen-
eralized method of moments (GMM) panel data analysis. We find that private capital
flows have a positive impact on growth in low-income countries with well-developed
financial sector but have a negative effect in the presence of poor financial sector develop-
ment. Well-developed financial sectors are ones that are themselves crucial for economic
growth. Our results indicate that private capital flows would be more effective if they were
more systematically conditional on well-developed financial systems. Keywords: private capital flows, stock market, growth, panel data analysis. Reference to this paper should be made as follows: Choong, C.-K.; Lam, S.-Y.; Yusop, Z. 2010. Private Capital Flows to Low-Income Countries: The Role of Domestic Financial
Sector, Journal of Business Economics and Management 11(4): 598–612. Reference to this paper should be made as follows: Choong, C.-K.; Lam, S.-Y.; Yusop, Z. 2010. Private Capital Flows to Low-Income Countries: The Role of Domestic Financial
Sector, Journal of Business Economics and Management 11(4): 598–612. Journal of Business Economics and Management
www.jbem.vgtu.lt
2010, 11(4): 598–612 Journal of Business Economics and Management
www.jbem.vgtu.lt
2010, 11(4): 598–612 Chee-Keong Choong1, Siew-Yong Lam2, Zulkornain Yusop3 1Centre for Economic Studies, Faculty of Business and Finance,
Universiti Tunku Abdul Rahman (Perak Campus), Jalan Universiti, Bandar Barat,
31900 Kampar, Perak Darul Ridzuan, Malaysia
2Centre for Business and Management, Faculty of Business and Finance,
Universiti Tunku Abdul Rahman (Perak Campus), Jalan Universiti, Bandar Barat,
31900 Kampar, Perak Darul Ridzuan, Malaysia
3Department of Economics, Faculty of Economics and Management,
Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
E-mails: 1choongck@utar.edu.my (corresponding author); 2lamsy@utar.edu.my;
3zulyusop@econ.upm.edu.my 1. Introduction In recent years, there has been a revival of interest on the nature and role of private
capital flows and their impact on investment and economic growth of host countries
(Andersen and Tarp 2003; Albuquerque 2003; Soto 2003; Mody and Murshid 2005;
Giovanni 2005; Khamfula 2007; Pazienza and Vecchione 2009; Tvaronavičienė et al. 2008; Tvaronavičienė and Kalašinskaitė 2010; Weng et al. 2010). In developing coun-
tries, this interest has been fueled by the reappearance of huge private capital inflows ISSN 1611-1699 print / ISSN 2029-4433 online doi: 10.3846 / jbem. 2010.29 Journal of Business Economics and Management, 2010, 11(4): 598–612 since the early 1990s, through a process of rapid financial sector liberalization (Blejer
2006; Bordo and Meissner 2006; Eller et al. 2006). The findings of the research between private capital flows and economic growth, how-
ever, have been mixed. On the one hand, some studies conclude that private capital
inflows raise the efficiency of recipient country such as stimulating capital accumula-
tion (de Mello 1996, 1997; Adams 2009), improving resource allocation (Reisen and
Soto 2001), interacting with human capital (Borensztein et al. 1998; Wang and Wong
2009), promoting international trade (Balasubramanyam et al. 1996; Basu and Guariglia
2007; Liu et al. 2009) and deepening domestic financial sector (Hermes and Lensink
2003; Alfaro et al. 2004; Durham 2004; Azman-Saini et al. 2010). On the other hand,
counterevidence also exists and argues that: “There is a growing agreement that exces-
sive build-up of short-term debt was a proximate cause of the recent crises…” (Rodrik
and Velasco 1999); “… short-term capital inflows can be counterproductive as they
may hinder economic growth through externalities emanated both during the surges
and sudden reversals” (Baharumshah and Thanoon 2006: 81); and “… private capital
flows do not help but do not hurt either economic growth in developing countries” (Soto
2003: 218). In short, the effects of private capital flows on economic growth still remain
ambiguous. Bearing this in mind, therefore, the study aims to investigate the role of domestic fi-
nancial sector in examining the linkages between private capital flows (foreign direct
investment (FDI), portfolio investment and foreign debt) and economic growth in the
selected low-income countries from 1988 to 2006, using generalized method of mo-
ments (GMM) panel data model. 2 Greater financial sector efficiency should result in an overall reduction of transaction costs. As a
result, cost of borrowing (capital cost) might decline, as interest margins shrink. If these gains are
being forwarded to the investors, the cost of borrowing in the markets will decline and promote
investments and economic growth (Levine 1997). 1 A well-developed financial system provides fertile ground for the allocation of resources, better
monitoring, better information symmetries, and economic growth (King and Levine 1993).ii 1. Introduction A number of studies point out the importance of
domestic financial system in attracting the private capital flows (Reisen and Soto 2001;
Hermes and Lensink 2003; Alfaro et al. 2004; Dumludag 2009). For example, Reisen
and Soto (2001: 12–13) concluded that “Foreign saving … has been shown to contrib-
ute to growth only if the banking system is well-capitalised; otherwise “good” risks
will be underfinanced and “bad” risks overfinanced”1. Moreover, the extent of direct
participation in local exchanges and gains due to the presence of private capital flows
(financial liberalization) depends on market investability manifested by financial market
breadth, depth, liquidity, efficiency, regulation, information, removal of perceived barri-
ers (risks), transparency of investment and repatriation rules (Errunza 2001; Ucal et al. 2010)2. This would mean that a minimum level of financial development must be met
before a country is in conformity to attract private capital flows in pursuit of enhanc-
ing its economic growth (Hermes and Lensink 2003; Alfaro et al. 2004; Durham 2004;
Azman-Saini et al. 2010). 599 C.-K. Choong et al. Private capital flows to low-income countries: the role of domestic financial sector Growth of developing countries, especially low-income groups depends on a large
extent on their own financial sector development3. Albuquerque (2003: 380) reveals: Growth of developing countries, especially low-income groups depends on a large
extent on their own financial sector development3. Albuquerque (2003: 380) reveals:
“… the relatively large proportion of FDI in private capital flows to less developed
countries or low-income countries reflects their poor financial status rather than any
comparative advantage”. Therefore, we investigate a new about private capital flows:
these capital flows do affect economic growth in the low-income countries; however,
their impact is conditional on the development of domestic financial system. “… the relatively large proportion of FDI in private capital flows to less developed
countries or low-income countries reflects their poor financial status rather than any
comparative advantage”. Therefore, we investigate a new about private capital flows:
these capital flows do affect economic growth in the low-income countries; however,
their impact is conditional on the development of domestic financial system. While most studies on link between private capital flows, financial development and
economic growth focus on the middle-income countries and high-income countries,
there is a dearth of evidence on low-income countries as financial market and sys-
tem in these countries are less developed. 1. Introduction Questions remain regarding the relevance
for researchers of previous literature that domestic financial system enhances the effect
of private capital flows on growth in low-income countries. By focusing on this low-
income group, we could identify the role of financial development in influencing the
link between private capital flows and growth. In other words, this study tends to find
that financial systems may have a different impact on growth in earlier stages of devel-
opment. It is believed that low-income countries with well-developed financial sector
benefit directly more from private capital flows, and this environment accelerates the
growth rate of economic. The remainder of the paper is organized as follows: In Section 2, we present the panel
data model used in this study. In Section 3, we discuss the impact of private capital
flows on growth with and without interaction with financial sector development. The
fourth section contains concluding remarks ad policy recommendations. 4 This method is fully described in Arellano and Bond (1991), Arellano and Bover (1995), and Blundell
and Bond (1998). 3 In this regard, Levine (1997), Andersen and Tarp (2003), and Wachtel (2003) have provided com-
prehensive surveys on the relationship between financial development and economic growth. 3 In this regard, Levine (1997), Andersen and Tarp (2003), and Wachtel (2003) have provided com-
prehensive surveys on the relationship between financial development and economic growth.
4 This method is fully described in Arellano and Bond (1991), Arellano and Bover (1995), and Blundell
and Bond (1998). 2. Panel data regression models: generalized
method of moments (GMM) The study uses recently developed dynamic panel generalized method of moments
(GMM) techniques to examine the interactions among different sorts of private capital
flows, financial development and economic growth in the 16 low-income countries in
the period of 1988–20064. Following standard growth equation, we construct the fol-
lowing dynamic panel data model, as suggested by Arellano and Bond (1991): ,
1
,
2
,
3
,
4
,
5
,
6
,
,
+
+
+
+
+
*
,
∆
= α + β ∆
β ∆
β ∆
β ∆
β ∆
β ∆
∆
+ τ + η + ε
i t
i t
Di t
Fi t
i t
i t
Fi t
t
i
i t
y
l
k
k
fd
X
fd
k
(1) (1) where yi,t be the logarithm of real GDP per capita growth rate in a country i at time
t, l is the natural log of labour force; kD and kF represent the natural log of domestic
capital stock and foreign capital flows respectively; fd represents the natural log of 600 Journal of Business Economics and Management, 2010, 11(4): 598–612 chosen financial development indicator; X is a set of macroeconomic variables that are
generally accepted to be important to explain economic growth; and ε is a normally
distributed error term. Then Equation 1 can be simplified as follows: Then Equation 1 can be simplified as follows: ,
,
1
,
1
,
,
i t
i t
i t
i t
t
i
i t
y
y
y
X
−
−
−
= −α
+ β
+ τ + η + ε
,
(2) (2) where
,
,
1
,
(
)
i t
i t
i t
y
y
y
−
−
= ∆
is the growth rate in real per capital GDP; α is a param-
eter reflecting the convergence speed; Xi,t is a set of explanatory variables, including
a measure of financial development, labour, domestic capital stock, national saving,
inflation, foreign capital flows and the interaction term, with associated parameter β; ηi
captures unobserved country-specific effects; τt is a period-specific effect common to
all countries; and εit is disturbance term. According to Arellano and Bond (1991), there is a strong autoregressive structure in
the residual term. 2. Panel data regression models: generalized
method of moments (GMM) This is not a surprise because the model is using annual data and
business-cycle effects may propagate for more than one year. In order to deal with
this problem, these business-cycle effects can be taken into account by assuming that
1
it
it
it
−
µ
= ρµ
+ ε , where|
| 1
ρ < , and εit is white noise disturbance term. After rearrang-
ing terms, Equation 2 becomes: ,
,
,
2
,
,
1
1
,
(1
)
(1
)
(1
)
−
−
−
=
−α + ρ
−ρ
−α
+
β −ρ
β + τ −ρτ
+
−ρ η + ε
i t
i t
i t
i t
i t
t
t
i
i
y
y
y
X
Xi First-differences are required in order to eliminate the country-specific effects. First-differences are required in order to eliminate the country-specific effects. From Equation 3, the lagged difference in per capita GDP is correlated with disturbance
term, which may produce an endogeneity of the explanatory variables, X. Besides, Blun-
dell and Bond (1998) argue that persistence in the explanatory variables may adversely
affect the small-sample and asymptotic properties of the difference estimator, therefore,
the difference estimator is further combined with an estimator in levels to produce a
system estimator. In dealing with this econometric problem, it is required the use of in-
struments. Arellano and Bond (1991) have proposed few steps to overcome the problem. The first step is to eliminate the time effect, τt by subtracting from each variable by its
cross average in period t. After that, the variables are transformed into first differences
to eliminate the individual effect as follows: ,
,
1
,
2
,
,
1
,
(1
)
(1
)
. −
−
−
∆
=
−α + ρ ∆
+ ρ
−α ∆
+ ∆
β + ρ∆
β + ∆ε
i t
i t
i t
i t
i t
i t
y
y
y
X
X
(4) (4) Arellano and Bond (1991) essentially propose estimating Equation 4 with GMM using
lagged levels of the endogenous variables as instruments. Nevertheless, the selection
of instruments is important. 2. Panel data regression models: generalized
method of moments (GMM) The GMM difference estimator uses the lagged levels of
the explanatory variables as instruments under the condition that the disturbance term
is not serially correlated and that the levels of the explanatory variables are weakly ex-
ogenous – that is, they are uncorrelated with future error terms. If the condition that the
explanatory variables are weakly exogenous is not hold, which is likely to be happen in
the present context as the higher economic growth may promotes more capital inflows, 601 C.-K. Choong et al. Private capital flows to low-income countries: the role of domestic financial sector both Xit and Xit–1 are correlated with disturbance term in Equation 4. Therefore, only
levels of variables lagged 2 years or more may be used as instruments. Then, the following moment conditions are used to calculate the difference estimator: both Xit and Xit–1 are correlated with disturbance term in Equation 4. Therefore, only
levels of variables lagged 2 years or more may be used as instruments. , the following moment conditions are used to calculate the difference estimator ,
,
,
1
[
(
)]
0
i t s
i t
i t
E y
−
−
ε
−ε
=
for
2,
3,...,
s
t
T
≥
=
,
(5)
,
,
,
1
[
(
)]
0
i t s
i t
i t
E X
−
−
ε
−ε
=
for
2,
3,...,
s
t
T
≥
=
. (6) (5) (6) This is a necessary way in the estimation as the equation in levels uses the lagged dif-
ferences of the explanatory variables as instruments under two conditions. First, the
error term is not serially correlated. Second, although there may be correlation between
the levels of the explanatory variables and the country-specific error term, there is no
correlation between the difference in the explanatory variables and the error term. This yields the following stationarity properties: This is a necessary way in the estimation as the equation in levels uses the lagged dif-
ferences of the explanatory variables as instruments under two conditions. First, the
error term is not serially correlated. Second, although there may be correlation between
the levels of the explanatory variables and the country-specific error term, there is no
correlation between the difference in the explanatory variables and the error term. 5 Arellano and Bond (1991) have called this test statistic as m2 test. For the test statistic, if the re-
siduals eit were first-order correlated, then
,
2
i t
y
− would be correlated with Deit and it could not be
used as an instrument. The same is true with any variable from
it
X
that is temporarily correlated
with eit. 2. Panel data regression models: generalized
method of moments (GMM) This yields the following stationarity properties: ,
,
[
]
[
]
i t
p
i
i t q
i
E y
E y
+
+
η
=
η and
,
,
[
]
[
]
i t
p
i
i t q
i
E X
E X
+
+
η
=
η
for all p and q. (7 (7) The additional moment conditions for the regression in levels are: The additional moment conditions for the regression in levels are: ,
,
1
,
[
)(
)
0 for
1
i t s
i t s
i
i t
E y
y
s
−
−−
−
η + ε
=
= ,
(8)
,
,
1
,
[
)(
)
0 for
1
i t s
i t s
i
i t
E X
X
s
−
−−
−
η + ε
=
= . (9) (8) (9) In summary, the GMM system estimator is obtained using the moment conditions in
Equations 5, 6, 8, and 9. Following Blundell and Bond (1998), the validity of the in-
struments used in these regressions is examined with two different statistics. The first
is Sargan (or overidentifying restrictions) test aims to examine the null hypothesis that
the instruments used are not correlated with the residuals. The second test is proposed
by Arellano and Bond (1991), which examines the hypothesis that the residuals from the
estimated regressions is first-order correlated but not second-order correlated5. In summary, the GMM system estimator is obtained using the moment conditions in
Equations 5, 6, 8, and 9. Following Blundell and Bond (1998), the validity of the in-
struments used in these regressions is examined with two different statistics. The first
is Sargan (or overidentifying restrictions) test aims to examine the null hypothesis that
the instruments used are not correlated with the residuals. The second test is proposed
by Arellano and Bond (1991), which examines the hypothesis that the residuals from the
estimated regressions is first-order correlated but not second-order correlated5. 3. Data sources Low-income Countries based on the 2005 World Bank Income Classification
Low Income Countries
Total
Bangladesh, Benin, Cameroon, Cote d’Ivoire, India, Kenya, Mauritania, Nicaragua,
Niger, Nigeria, Pakistan, Papua New Guinea, Rwanda, Senegal, Togo, Zimbabwe
16 Table 1. Data Sources Table 2. Low-income Countries based on the 2005 World Bank Income Classification
Low Income Countries
Total
Bangladesh, Benin, Cameroon, Cote d’Ivoire, India, Kenya, Mauritania, Nicaragua,
Niger, Nigeria, Pakistan, Papua New Guinea, Rwanda, Senegal, Togo, Zimbabwe
16 3. Data sources Databases on the various categories of foreign capital flows to low-income countries
from 1988 to 2006 are employed for the study. The sources of the variables used in
this study are summarized in Table 1. The low-income countries chosen are based on
the World Bank’s income classification 2008. The selection of country and period were
determined exclusively by data availability. This results in 16 low-income countries as
shown in Table 2 to examine the relationship between private capital flows, financial
development and economic growth. 602 Journal of Business Economics and Management, 2010, 11(4): 598–612 Table 1. Data Sources
Variable
Data Source
Real GDP per capita growth rate (GDPGR)
World Development Indicators, World Bank
Capital stock (CAP)
World Development Indicators, World Bank
Labour force (LF)
World Development Indicators, World Bank
Saving as % of GDP (NSAV)
World Development Indicators, World Bank
Inflation (INF)
World Development Indicators, World Bank
Foreign direct investment (FDI)
World Development Indicators, World Bank
Portfolio investment (PI)
World Development Indicators, World Bank
Foreign debt (DEBT)
World Development Indicators, World Bank
Central Bank Assets to GDP ratio (CBAGDP)
Beck et al. (2000a) World Bank database
Deposit money bank assets to GDP ratio
(DBAGDP)
Beck et al. (2000a) World Bank database
Private credit by deposit money bank to GDP
ratio (PCGDP)
Beck et al. (2000a) World Bank database
Table 2. Low-income Countries based on the 2005 World Bank Income Classification
Low Income Countries
Total
Bangladesh, Benin, Cameroon, Cote d’Ivoire, India, Kenya, Mauritania, Nicaragua,
Niger, Nigeria, Pakistan, Papua New Guinea, Rwanda, Senegal, Togo, Zimbabwe
16 Table 1. Data Sources
Variable
Data Source
Real GDP per capita growth rate (GDPGR)
World Development Indicators, World Bank
Capital stock (CAP)
World Development Indicators, World Bank
Labour force (LF)
World Development Indicators, World Bank
Saving as % of GDP (NSAV)
World Development Indicators, World Bank
Inflation (INF)
World Development Indicators, World Bank
Foreign direct investment (FDI)
World Development Indicators, World Bank
Portfolio investment (PI)
World Development Indicators, World Bank
Foreign debt (DEBT)
World Development Indicators, World Bank
Central Bank Assets to GDP ratio (CBAGDP)
Beck et al. (2000a) World Bank database
Deposit money bank assets to GDP ratio
(DBAGDP)
Beck et al. (2000a) World Bank database
Private credit by deposit money bank to GDP
ratio (PCGDP)
Beck et al. (2000a) World Bank database
Table 2. 4.1. The relationship between private capital flows,
financial development and economic growth Equations 1, 2 and 3 in Table 3 report the estimates of private capital flow and economic
growth regressions for the low-income countries when the interaction term between
private capital flows (FDI, portfolio and foreign debt) and financial development is not
included, while Equations 4, 5 and 6 included the interaction term. Overall, the signs of
the capital stock (CAP), labour force (LF) and national saving (NSAV) are positive and
significant in most of the regressions. The findings are consistent with a priori, which
shows that low-income countries have benefited from their national saving, capital stock
and human capital development in promoting the economic growth rate. On the other hand, the sign of the inflation rate is negative and statistically significant
associated with GDP per capita growth rate in most regressions. Referring to the fi-
nancial development indicator (CBAGDP), this variable is positively associated with
economic growth in all regressions and it is statistically significant at 10 percent sig-
nificance level or better. This implies that financial development is crucial to promote
economic growth when the countries have well-developed banking and financial sector. 603 C.-K. Choong et al. Private capital flows to low-income countries: the role of domestic financial sector Table 3. Private Capital Flows, Financial Development (CBAGDP) and Economic Growth
in Low-Income Countries, 1988–2006
Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 4.1. The relationship between private capital flows,
financial development and economic growth 6
Constant
–0.025*
(–1.696)
0.020
(1.454)
–0.127
(–1.457)
–0.035**
(–2.139)
–0.040
(–0.547)
–0.036*
(–1.930)
GDPGRt-1
–0.042*
(–1.954)
–0.123***
(–2.759)
0.047*
(1.857)
–0.689***
(–2.746)
–0.158*
(–1.768)
–0.211***
(–2.864)
CAP
1.075***
(19.141)
0.597***
(3.743)
0.078***
(3.044)
0.044*
(1.883)
0.101*
(1.714)
0.019
(0.976)
INF
–0.047**
(–2.185)
–0.162**
(–1.958)
0.016
(0.518)
–0.053**
(–2.047)
–0.310***
(–2.772)
0.019
(0.976)
LF
0.912***
(16.204)
0.460***
(4.224)
0.035*
(1.743)
0.205**
(2.334)
0.025**
(2.348)
0.044**
(2.198)
NSAV
0.959***
(16.212)
0.070***
(3.551)
1.257***
(22.104)
0.217***
(3.944)
0.036*
(1.678)
0.116*
(1.701)
CBAGDP
0.597***
(3.743)
0.191*
(1.751)
0.179***
(6.211)
1.101***
(18.695)
0.035*
(1.743)
0.044**
(2.198)
FDI
–0.045*
(–1.768)
0.051**
(2.331)
PI
–0.090***
(–3.200)
0.044*
(1.883)
DEBT
–0.282***
(–3.157)
0.046*
(1.917)
FDI*CBAGDP
0.036*
(1.678)
PI*CBAGDP
0.025**
(2.348)
DEBT*CBAGDP
0.116*
(1.701)
Sargan test
10.71
[0.446]
10.33
[0.612]
14.41
[0.413]
13.06
[0.411]
6.80
[0.791]
8.24
[0.702]
A-B test 1st Order
–1.88*
[0.079]
–8.01***
[0.001]
–2.95**
[0.001]
–2.78**
[0.004]
–5.16***
[0.001]
–1.88*
[0.086]
A-B test 2nd Order
–0.99
[0.341]
–0.85
[0.412]
–0.67
[0.552]
–0.96
[0.361]
–0.98
[0.392]
–0.89
[0.390]
Observations
304
304
304
304
304
304
Notes: Dependent variable is real GDP per capita growth rate. All the variables are taken in differences and lagged one period. The Sargan Chi-square statistic tests the null hypothesis of no correlation between the
instruments and residuals. The Arellano and Bond (A-B) Z-statistic tests the null hypothesis that the residuals are
fi
d
l
d (A B
1st O d )
d h
id
l
d
d
l
d 604 Journal of Business Economics and Management, 2010, 11(4): 598–612 The significant and positive association between the financial development and the de-
velopment of the real economy is consistent with the empirical studies such as Roubini
and Sala-i-Martin (1992), King and Levine (1993), Benhabib and Spiegel (2000), and
Beck et al. (2000b). The significant and positive association between the financial development and the de-
velopment of the real economy is consistent with the empirical studies such as Roubini
and Sala-i-Martin (1992), King and Levine (1993), Benhabib and Spiegel (2000), and
Beck et al. (2000b). Looking at the impact of the private capital flows, the signs of private capital flow
variables are negative and significant in all equations when the interaction term is not
included. The results demonstrate that foreign debt may hurt the low-income countries
than help to promote their economic performance, as the estimated coefficient is the
highest as compared to other capital flows. 4.1. The relationship between private capital flows,
financial development and economic growth The negative effect of these capital flows
is in line with the results previously estimated by Reisen and Soto (2001) and Levine
(2001). Interestingly, while the estimates for private capital flows are negative (Equations 1–3),
the coefficient of national saving are positive, which suggesting that these sorts of capi-
tal flows are less productive than national saving, and thus there are less spillover effects
from these foreign capitals. This is consistent with the findings reported in firm-level
studies by Aitken and Harrison (1999), Haddad and Harrison (1993), Vissak (2009),
and Zeng et al. (2009), which indicating that low-income countries have not enough
“absorptive capacity” in transferring the advantages embodied in private capital inflows
into the positive economic growth. One possible explanation for these results may be the failure to capture contingency ef-
fects in the relationship between private capital flows and economic growth and the rela-
tionship between private capital flows and growth may be contingent on other countries’
absorptive capabilities such as domestic financial systems and laws and institutional
reforms (Brock and Urbonavicius 2008). To determine the validity of the hypothesis that
well-developed domestic financial system would help to benefit more from private capi-
tal flows, the interaction term is included. From Equations 4 to 6 in Table 3, it is found
that the coefficients of the private capital flows and the interaction term are positive and
statistically significant in all regressions at 10 percent significance level or better. The
positive sign of the interaction term does support the notion that domestic financial sys-
tem is effectively transforming the negative effect of all private capital flows on growth
into positive impact in low-income countries. It is concluded that the effect of private
capital flows on growth is greatly influenced through the domestic financial system. In checking the validity of the instruments used, both Sargan and Arellano-Bond test
statistics show that the instruments used are no-overidentifying restrictions and the re-
siduals are independent or white noise, and hence, suitable for the estimations6. 6 See Newey and McFadden (1994: 2231) for details on this test. 4.2. Further analysis of the relationship between private capital flows,
financial development and economic growth The relationship between private capital flows, financial development and economic
growth may be further investigated by using alternative indicators of financial develop-
ment. Two alternative measures of financial development are used to gauge different 6 See Newey and McFadden (1994: 2231) for details on this test. 605 C.-K. Choong et al. Private capital flows to low-income countries: the role of domestic financial sector functions of financial intermediary in the system, namely: deposit money bank assets to
GDP ratio (DBAGDP) and private credit by deposit money bank to GDP ratio (PCGDP). The first indicator measures the degree of monetization and the relative significance of
particular financial institutions. The second indicator takes into account the credits to
private sector only and isolates the credits channeled to public sector and credits from
central bank. The results among private capital flows, two financial development measures and growth
are reported in Tables 4 and 5. The results reveal that FDI is statistically significant at
10 percent significance level or better and it has a positive impact on growth, either in-
cluded or excluded the interaction term. Moreover, the coefficient of the interaction term
is positive and significant. Hence, it is obvious that FDI flows have an unambiguously
positive effect on growth in the low-income countries, which is in line with Amdam
et al. (2007), and Basti and Bayyurt (2008). Looking at both portfolio investment and foreign debt (without interaction term) as
reported in Tables 4 and 5, it is noted that these capital flows are negatively associated
with economic growth. However, the variable flows are positive and significant after
the inclusion of interaction term. This implies an interesting situation that although
portfolio investment and foreign debt are negatively associated with economic growth,
the well-developed financial system would change this negative impact to positive im-
pact on growth. This finding is consistent with the results reported in Table 3. Again,
this provides additional evidence to support the notion that the negative impact of pri-
vate capital flows can be transferred into positive if the domestic financial system has
reached a certain minimum level of development. Table 4. Private Capital Flows, Financial Development (PCGDP) and Economic Growth
in Low-Income Countries, 1988–2006
Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 4.2. Further analysis of the relationship between private capital flows,
financial development and economic growth 6
Constant
–0.014
(–0.626)
–0.083
(–0.140)
0.023
(0.032)
0.130**
(2.054)
–0.127***
(-2.864)
–0.105
(–0.264)
GDPGRt-1
0.048**
(2.415)
–0.003
(–0.140)
0.041
(0.579)
–0.407***
(–4.715)
–0.087***
(–2.842)
0.118**
(2.107)
CAP
0.045*
(1.884)
0.103*
(1.786)
0.115*
(1.748)
0.120
(1.402)
0.100**
(2.443)
0.117**
(2.050)
INF
–0.163*
(–1.801)
–0.005
(–0.264)
–0.051**
(–2.331)
–0.127***
(–2.864)
0.014
(0.860)
–0.046*
(–1.917)
LF
0.035*
(1.758)
0.037*
(1.726)
0.032***
(3.081)
0.094**
(2.308)
0.117*
(1.801)
0.011
(0.158)
NSAV
0.055***
(2.772)
0.051**
(2.302)
0.028**
(2.556)
0.051**
(2.569)
0.072*
(1.689)
0.026*
(1.832)
PCGDP
0.047**
(2.329)
0.045*
(1.924)
0.008**
(2.201)
0.046**
(2.308)
0.014*
(1.951)
0.015**
(1.980)
FDI
0.117*
(1.713)
0.166**
(2.452) ble 4. Private Capital Flows, Financial Development (PCGDP) and Economic Growth
in Low-Income Countries, 1988–2006 606 607
Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 6
PI
–0.052***
(–2.639)
0.062***
(3.308)
DEBT
–0.052***
(–2.639)
0.088***
(4.362)
FDI* PCGDP
0.100**
(2.461)
PI* PCGDP
0.052***
(2.621)
DEBT*
PCGDP
0.050**
(2.501)
Sargan test
12.26
[0.962]
17.12
[0.837]
17.93
[0.813]
19.73
[0.725]
16.41
[0.852]
14.10
[0.931]
A-B test 1st
Order
–1.99*
[0.045]
–1.83*
[0.075]
–2.98**
[0.008]
–1.74*
[0.082]
–2.37**
[0.036]
–2.46*
[0.031]
A-B test 2nd
Order
–0.89
[0.365]
–0.99
[0.342]
–0.62
[0.470]
–0.76
[0.422]
–0.93
[0.301]
–0.66
[0.468]
Observations
304
304
304
304
304
304
Note: Refer to Table 3. Table 5. Private Capital Flows, Financial Development (DBAGDP) and Economic Growth
in Low-Income Countries, 1988–2006
Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 6
Constant
3.632**
(2.662)
0.029
(0.411)
–0.044***
(–3.030)
–0.080
(–0.067)
–0.069***
(–3.237)
3.926**
(2.700)
GDPGRt-1
0.015**
(2.061)
0.036
(0.825)
–0.027
(–1.495)
–0.107*
(–1.719)
0.065
(0.068)
–0.263***
(–4.262)
CAP
0.172***
(4.333)
0.037***
(2.644)
1.117***
(21.080)
2.216*
(1.964)
0.021***
(2.865)
0.030*
(1.672)
INF
–0.118**
(–2.167)
–0.051***
(–2.644)
–0.061
(–0.043)
–0.072*
(–1.759)
–3.568***
(–2.877)
–1.508
(–1.278)
LF
0.101*
(1.850)
0.029
(1.320)
0.108***
(2.698)
1.647**
(2.293)
0.109
(1.531)
0.032***
(3.012)
NSAV
0.033**
(2.307)
0.194***
(3.474)
0.053***
(2.618)
0.048**
(2.302)
0.023**
(2.281)
0.057***
(2.743)
DBAGDP
0.132**
(3.155)
0.086**
(2.176)
0.454*
(1.886)
2.771**
(2.126)
0.792*
(1.918)
2.321*
(1.780)
FDI
0.026*
(1.816)
0.085**
(2.379)
PI
–0.052***
(-2.639)
0.090**
(2.255)
End of Table 4
Journal of Business Economics and Management, 2010, 11(4): 598–612 Journal of Business Economics and Management, 2010, 11(4): 598–612 Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 4.2. Further analysis of the relationship between private capital flows,
financial development and economic growth 6
PI
–0.052***
(–2.639)
0.062***
(3.308)
DEBT
–0.052***
(–2.639)
0.088***
(4.362)
FDI* PCGDP
0.100**
(2.461)
PI* PCGDP
0.052***
(2.621)
DEBT*
PCGDP
0.050**
(2.501)
Sargan test
12.26
[0.962]
17.12
[0.837]
17.93
[0.813]
19.73
[0.725]
16.41
[0.852]
14.10
[0.931]
A-B test 1st
Order
–1.99*
[0.045]
–1.83*
[0.075]
–2.98**
[0.008]
–1.74*
[0.082]
–2.37**
[0.036]
–2.46*
[0.031]
A-B test 2nd
Order
–0.89
[0.365]
–0.99
[0.342]
–0.62
[0.470]
–0.76
[0.422]
–0.93
[0.301]
–0.66
[0.468]
Observations
304
304
304
304
304
304
Note: Refer to Table 3. End of Table 4 Table 5. Private Capital Flows, Financial Development (DBAGDP) and Economic Growth
in Low-Income Countries, 1988–2006
Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 6
Constant
3.632**
(2.662)
0.029
(0.411)
–0.044***
(–3.030)
–0.080
(–0.067)
–0.069***
(–3.237)
3.926**
(2.700)
GDPGRt-1
0.015**
(2.061)
0.036
(0.825)
–0.027
(–1.495)
–0.107*
(–1.719)
0.065
(0.068)
–0.263***
(–4.262)
CAP
0.172***
(4.333)
0.037***
(2.644)
1.117***
(21.080)
2.216*
(1.964)
0.021***
(2.865)
0.030*
(1.672)
INF
–0.118**
(–2.167)
–0.051***
(–2.644)
–0.061
(–0.043)
–0.072*
(–1.759)
–3.568***
(–2.877)
–1.508
(–1.278)
LF
0.101*
(1.850)
0.029
(1.320)
0.108***
(2.698)
1.647**
(2.293)
0.109
(1.531)
0.032***
(3.012)
NSAV
0.033**
(2.307)
0.194***
(3.474)
0.053***
(2.618)
0.048**
(2.302)
0.023**
(2.281)
0.057***
(2.743)
DBAGDP
0.132**
(3.155)
0.086**
(2.176)
0.454*
(1.886)
2.771**
(2.126)
0.792*
(1.918)
2.321*
(1.780)
FDI
0.026*
(1.816)
0.085**
(2.379)
PI
–0.052***
(-2.639)
0.090**
(2.255) e 5. Private Capital Flows, Financial Development (DBAGDP) and Economic Growth
in Low-Income Countries, 1988–2006 607 C.-K. Choong et al. Private capital flows to low-income countries: the role of domestic financial sector Variable
Equ. 1
Equ. 2
Equ. 3
Equ. 4
Equ. 5
Equ. 6
DEBT
–0.133**
(–2.408)
0.052***
(2.649)
FDI* DBAGDP
0.065**
(2.415)
PI* DBAGDP
0.047***
(3.108)
DEBT* DBAGDP
0.131***
(2.879)
Sargan test
12.23
[0.851]
17.61
[0.862]
16.88
[0.889]
13.36
[0.841]
19.69
[0.622]
15.46
[0.776]
A-B test 1st Order
–1.93*
[0.052]
–2.96***
[0.004]
–2.26**
[0.021]
–2.39**
[0.023]
–1.76*
[0.077]
–2.19**
[0.025]
A-B test 2nd Order –0.88
[0.362]
–0.91
[0.323]
–0.89
[0.376]
–0.99
[0.331]
–0.93
[0.321]
–0.83
[0.411]
Observations
304
304
304
304
304
304
Note: Refer to Table 3. End of Table 5 5. Conclusions and policy implications In this paper, we have investigated the effect of different sorts of private capital flows
(FDI, portfolio investment and foreign debt) on economic growth in the selected low-
income countries for the period of 1988–2006. We found that FDI has a positive effect
on economic growth in the low-income countries while portfolio investment and total
foreign debt have negative and significant impacts on economic growth. Our inter-
pretation for the negative sign for these private capital flows is that low degree of the
financial sector development in the low-income countries leads to misallocation of these
private capital flows, which reduces and even reverses their impacts on economic per-
formance. Hence, well-developed financial system is of importance and the transition of
the local financial market is a must in dealing with the presence of private capital flows. To support this idea, we allowed interaction of all private capital flows (FDI, foreign
debt and portfolio investment) with different measures of financial sector development. When these private capital flows were interacted with financial development indica-
tors, even though the sign of both portfolio investment and foreign debt remain nega-
tive and significant in the regressions, the interaction terms are generally positive and
significant, which implies the importance of financial sector development in benefiting
from private capital flows. Our findings are different from the previous findings. Arteta
et al. (2001), for example, do not find any significant linkages among financial opening,
level of financial depth and income level in a panel of countries. On the other hand,
Klein and Olivei (1999) also reveal that the presence of private capital flows only sig-
nificant in OECD countries only, but not for developing countries. Similarly, Edwards
(2001) shows that financial liberalisation had a positive effect on growth only in more
developed countries, supporting the hypothesis of the role of well-functioning financial 608 Journal of Business Economics and Management, 2010, 11(4): 598–612 institutions. Our findings show that making private capital flows more systematically
conditional on the development of domestic financial sector would tend to increase its
impact on growth. This explains why the impact of private capital flows on growth is
not all the time positive. A crucial starting point in designing policies to optimize the benefits from private capital
flows is to have a basic understanding of a country’s comparative advantage and de-
velopment objectives. 5. Conclusions and policy implications This helps in absorbing the benefits embodied in private capital
flows effectively (United Nations Conference on Trade and Development 2002). Even
though it is important for low-income developing countries to attract more foreign pri-
vate capital flows, they should be careful in dealing with the presence of these capital
inflows since the nature of these private capital flows are quite different. It is recom-
mended that low-income countries, or emerging economies give priority to foreign
direct investment (FDI) as this is the most preferred capital flow contributing to the
economic growth. Acknowledgements This paper is a product of an on-going research (entitled: Foreign Direct Invest-
ment, Economic Growth and Institutional Innovations: The Cross-National Evidence
(FRGS/1/10/SSK/UTAR/03/2)) sponsored by Fundamental Research Grant Scheme
(FRGS), Ministry of Higher Education (MOHE), Malaysia. Views expressed in this
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ment 11(3): 396–414. doi:10.3846/jbem.2010.19 Zeng, S. X., Wan, T. W.; Tam, V. W. Y. 2009. Towards FDI and Technology Spillover: A Case
Study in China, Transformations in Business and Economics 8(1): 50–62. PRIVATAUS KAPITALO SRAUTŲ ĮTAKA MAŽAS PAJAMAS GAUNANČIOMS
ŠALIMS: FINANSINIO VIDAUS SEKTORIAUS VAIDMUO C.-K. Choong, S.-Y. Lam, Z. Yusop Santrauka Ryšys tarp kapitalo srautų ir jų augimo yra intensyviai tiriamas. Pastebėta, kad daug prieštaravimų tarp
jų vis dar išlieka. Šiame tyrime nagrinėjamas santykis tarp privataus kapitalo srautų (įeinančių užsienio
investicijų, portfelinių investicijų ir užsienio skolų) finansinės plėtros ir ekonominės veiklos pasirink-
tose šešiolikoje mažas pajamas gaunančių besivystančių šalių 1988–2006 m., duomenų analizei taikant
apibendrinamąjį momentų metodą (GMM). Nustatyta kad privataus kapitalo srautai turi teigiamą įtaką
augimui mažas pajamas turinčiose šalyse su gerai išplėtotu finansiniu sektoriumi, tačiau daro neigiamą
poveikį toms šalims, kurių finansinis sektorius yra skurdus. Gerai išvystyti ekonominiai sektoriai yra
tie, kurie vaidina lemiamą vaidmenį ekonominiam augimui. Rezultatai parodė, kad privataus kapitalo
srautai būtų efektyvesni, jei jie sistemiškiau priklausytų nuo gerai išsivysčiusių finansinių sistemų. Reikšminiai žodžiai: privataus kapitalo srautai, akcijų rinka, augimas, duomenų analizė. Chee-Keong CHOONG. An Assistant Professor and deputy dean at Faculty of Business and Finance,
Universiti Tunku Abdul Rahman, Malaysia. He completed his doctorate degree at the Faculty of Eco-
nomics and Management, Universiti Putra Malaysia (UPM), Malaysia in 2007. He teaches Econom-
ics, Business Finance, Statistics for Economics and Management, Basic Econometrics, Econometrics,
Research Methods and Business Research Methodology. He has published widely in various refereed
journals, which include Energy Policy, Japan and the World Economy, Singapore Economic Review,
Pacific Economic Bulletin, African Journal of Business Management, and Journal of the Asia Pacific
Economy. His current research interests are in the fields of applied macroeconomics, international
economics, monetary economics and financial economics. Siew-Yong LAM. A lecturer from Faculty of Business and Finance, Universiti Tunku Abdul Rahman
(UTAR), Malaysia. She has published a number of journals such as Global Economic Review, ICFAI
Journal of Managerial Economics, Icfai Journal of Service Marketing and Banker’s Journal Malaysia. Her research interests are in the areas of Relationship Marketing, Consumer Behaviour and manage-
rial economics. Zulkornain YUSOP. An Associate Professor and deputy dean at Faculty of Economics and Manage-
ment, Universiti Putra Malaysia. He teaches International Economics, International Trade and Current
Issues in International Economics. Topics for research include FDI, private capital flow, capital flight and
Economic Integration. He has published a number of journals such as International Journal of Business
and Society, Journal of International Food and Agribusiness Marketing, World Review of Entrepreneur-
ship, Management and Sustainable Development, The Singapore Economic Review, The ICFAI Jour-
nals, ASEAN Economic Bulletin, Journal of the Asia Pacific Economy and Applied Economics Letters. 612
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Article: Andersen, M.F., Nielsen, K. orcid.org/0000-0001-9685-9570 and Ajslev, J.Z.N. (2021) The
relational fit in organizational interventions—what can organizational research learn from
research in psychotherapy? International Journal of Environmental Research and Public
Health, 18 (15). 8104. ISSN 1660-4601 https://doi.org/10.3390/ijerph18158104 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence
allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the
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https://creativecommons.org/licenses/ This is a repository copy of The relational fit in organizational interventions—what can
organizational research learn from research in psychotherapy?. This is a repository copy of The relational fit in organizational interventions—what can
organizational research learn from research in psychotherapy?. White Rose Research Online URL for this paper:
https://eprints.whiterose.ac.uk/176716/ Version: Published Version Version: Published Version
Citation: Andersen, M.F.; Nielsen, K.;
Ajslev, J.Z.N. The Relational Fit in
Organizational Interventions—What
Can Organizational Research Learn
from Research in Psychotherapy? Int. J. Environ. Res. Public Health 2021,
18, 8104. https://doi.org/10.3390/
ijerph18158104 Keywords:
organizational interventions; evaluation; effect; psychotherapy; implementation;
mental health; relational fit Academic Editors: Irene Jensen and
Christina Björklund Academic Editors: Irene Jensen and
Christina Björklund Malene Friis Andersen 1,*, Karina Nielsen 2
and Jeppe Zielinski Nguyen Ajslev 1 1
National Research Centre for the Working Environment, Department of Psychosocial Work Environment,
2100 Copenhagen, Denmark; jza@nrcwe.dk
2
Institute of Work Psychology, Management School, University of Sheffield, Sheffield S10 1FL, UK;
k.m.nielsen@sheffield.ac.uk
*
Correspondence: mfa@nrcwe dk 1
National Research Centre for the Working Environment, Department of Psychosocial Work Environmen
2100 Copenhagen, Denmark; jza@nrcwe.dk
2
Institute of Work Psychology, Management School, University of Sheffield, Sheffield S10 1FL, UK;
k.m.nielsen@sheffield.ac.uk *
Correspondence: mfa@nrcwe.dk Abstract: There is a growing interest in organizational interventions (OI) aiming to increase employ-
ees’ well-being. An OI involves changes in the way work is designed, organized, and managed. Studies have shown that an OI’s positive results are increased if there is a good fit between context
and intervention and between participant and intervention. In this article, we propose that a third
fit—the Relational Fit (R-Fit)—also plays an important role in determining an intervention’s outcome. The R-Fit consists of factors related to (1) the employees participating in the OI, (2) the intervention
facilitator, and (3) the quality of the relation between participants and the intervention facilitator. The concept of the R-Fit is inspired by research in psychotherapy documenting that participant
factors, therapist factors, and the quality of the relations explain 40% of the effect of an intervention. We call attention to the importance of systematically evaluating and improving the R-Fit in OIs. This
is important to enhance the positive outcomes in OIs and thereby increase both the well-being and
productivity of employees. We introduce concrete measures that can be used to study and evaluate
the R-Fit. This article is the first to combine knowledge from research in psychotherapy with research
on OIs. The Relational Fit in Organizational
Interventions—What Can Organizational Research
Learn from Research in Psychotherapy? Malene Friis Andersen 1,*, Karina Nielsen 2
and Jeppe Zielinski Nguyen Ajslev 1 Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by
emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk
https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk
https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk
https://eprints.whiterose.ac.uk/ International Journal of
Environmental Research
and Public Health International Journal of
Environmental Research
and Public Health 1. Introduction Received: 1 June 2021
Accepted: 27 July 2021
Published: 30 July 2021 In recent years, there has been a growing interest in organizational interventions
(OIs) aiming to increase the well-being of employees [1–3]. This interest derives, at least
partly, from knowledge that poor well-being is costly for organizations and societies [4,5]
and is related to poor life quality for the individual [6,7]. OIs can be defined as planned,
behavioral, theory-based actions that aim to improve employee health and well-being
by changing the way work is designed, organized, and managed [8]. The growth in
OIs has been accompanied by an increasing interest in how to evaluate OIs [8]. Until
recently, most evaluations have focused on the intervention’s effects [9]; however, there is
an increasing interest in processual factors and conditions affecting the implementation
and effect of OIs [2,3]. Process evaluations of OIs have revealed factors and conditions that
should be taken into consideration to understand why OIs succeed or fail and why the
same type of interventions can have different outcomes across organizational settings [10]. One explanation for this is that no two organizations are alike when it comes to factors
and conditions such as organizational structures, tasks, change readiness, possibilities,
and challenges [11]. But some under-researched factors that are of importance to the
outcome of an OI exist. In this paper, we argue that the person or persons that drive
and facilitate the OI—labeled Intervention Facilitator (IF) in this article—also affect the Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). Int. J. Environ. Res. Public Health 2021, 18, 8104. https://doi.org/10.3390/ijerph18158104 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 8104 2 of 12 2 of 12 outcome of an OI, that this also goes for the participants in the intervention, and not
least for the relation between the IF and the participants in the intervention. In this
paper, we present a theoretical framework for how researchers may understand the role
of IFs and the relation between the IFs and the participants in an OI. The Importance of Fit in Organizational Interventions Frameworks for the process evaluation of OIs identify three central themes in an appropri-
ate process evaluation: (1) the intervention design and implementation, (2) the intervention
context, and (3) participants’ mental models of the intervention and their work situa-
tion [12]. Recently, it has been suggested that OIs are more likely to bring about in-
tended outcomes if there is a good “fit” between context and intervention as well as a
good fit between the intervention and the persons participating in the intervention [13]. The concept of person–intervention fit stems from the organizational psychology theory
of Person–Environment fit (P–E fit), which has been defined as the “compatibility be-
tween an individual and a work environment that occurs when their characteristics are
well-matched” [14]. In relation to organizational interventions, fit includes the need to match and adjust
the intervention to existing practices and procedures within organizations and the need to
consider, for example, participants’ perceived readiness for change and educational levels. One type of fit is the Person–Job fit (P–J fit), which concerns the fit between individuals’
skills and competencies and the demands of the job [15]. In a review of OI processes,
Nielsen and Noblet called for studies on the role of the IF as well as on the relation between
the IF and the participants [16]. This call is echoed in recent debates focused on the role of
facilitation and facilitators within implementation science [17,18]. p
Although the importance of the IF has been considered, the role of IFs and the degree
to which they influence the implementation and effect of an intervention is not well
described in existing literature on OIs [16]. To the best of our knowledge, only one study
has focused on the fit between facilitators in organizational interventions and their skills
and knowledge. In a study where employees were appointed facilitators within their
own departments, the researchers found that those facilitators who were dissatisfied with
their jobs but felt they possessed the necessary competencies to fulfil the facilitator role
reported higher levels of job satisfaction post-intervention [19]. In the present paper, we
answer these calls by proposing a framework for examining how IFs, the participants in the
intervention, and the relation between the IF and the participants may affect the outcome
of an OI. 1. Introduction This is a conceptual
paper that integrates research and theory from the fields of both OI and psychotherapy,
as the research field in psychotherapy can teach us much about which components in
an intervention especially affect its outcome. We hope that our conceptual paper will
inspire future empirical evaluations of OIs. In order to enhance the chances for this, we
introduce modified concrete measures that can be used to study, validate, or reject the
theses presented here. The Importance of Fit in Organizational Interventions We suggest that future evaluation research on OIs should look systematically into
what we conceptualize as the Relational Fit, i.e., the fit between the IFs and the participants. This knowledge is important to be able to develop a more thorough understanding of
why OIs fail or succeed in bringing about the intended outcomes: improving the work
conditions and health of employees. Our conceptualization of the relational fit is based on research studies on OIs and the well-
established research field of psychotherapeutic interventions. For decades, the psychotherapeutic
field has systematically explored the importance and effect of relations in therapeutic
interventions, and we believe it may confer knowledge that could help researchers in OIs
to evaluate relational fit in future studies. By integrating knowledge from research on
psychotherapy with research on OIs, we present important factors in relational fit in OIs. Inspired by measures used in psychotherapy, we provide concrete suggestions for how
future studies may explore and evaluate the relational fit and its impact on the outcome of
OIs. Our inspiration to use the concept of “relational fit” is derived from research on the Int. J. Environ. Res. Public Health 2021, 18, 8104 3 of 12 3 of 12 two above-mentioned fits. But the term “fit” is also used—although rarely—in research in
psychotherapy regarding the fit between patient and treatment. In this article, we therefore
incorporate the knowledge from psychotherapy into the concept of “fit” derived from the
research on OIs. The relational fit should be considered as an extension of the two fits presented
by Nielsen and Randall [13]: context-intervention and person-intervention, as shown in
Figure 1. Figure 1. Three important fits in an Organizational Intervention (OI). Figure 1. Three important fits in an Organizational Intervention (OI). As can be seen in Figure 1, we use the term “participant” instead of “person”, which
is the term used by Nielsen and Randall [13]. The reason for this is that we find the term
“person” too broad for the purpose of our article, and we want to emphasize that we focus
on and write about two different players: those who participate in the intervention and
those who facilitate it. This paper is a first step in bringing together the research fields of OI and psychother-
apy. We first introduce major findings from research in psychotherapy, which highlight the
importance of common factors for positive outcomes in therapeutic interventions. The Importance of Fit in Organizational Interventions Second,
we unfold the relational fit between IFs and participants in OIs by presenting key findings
from research in psychotherapy and OIs. Instead of presenting the major findings from
each field in separate sections, we have chosen to integrate the major findings from both
fields in the following three sections: (1) Intervention facilitators, (2) Participant factors,
and (3) Quality of relation. In this way, we hope to give the best introduction to what might
be the most effective components in both psychotherapy and OIs. p
p y
py
Our ambition is that future empirical studies will test our framework and explore
the effect and importance of the relational fit in OIs and thereby increase the chances for
positive outcomes in OIs 2. What Affects the Outcome of Psychotherapy? Back in 1961, Jerome Frank published his famous book Persuasion and Healing in
which he argued that change in patients during psychotherapy occurs when factors that
are common to all forms of psychotherapy operate in concert [20]. Frank’s work has
been a great inspiration for research in understanding what affects the outcome of psy-
chotherapy. A number of psychotherapeutic traditions exist, each of which has their own
therapeutic methods and techniques (e.g., psychodynamic therapy, cognitive behavioral
therapy, etc.) [21]. Despite variations, these methods and techniques share some common
characteristics: there is a therapist, a client (or a group), and a relation between the two. These common characteristics are labelled common factors and refer to aspects of psy-
chotherapy that are important in all therapeutic schools: a good therapist, an engaged
client, and a high-quality relation between therapist and client [22]. Through decades of studies, researchers in psychotherapy have refined their eval-
uation methods to achieve more detailed knowledge of the extent to which different
intervention components can explain the effects of a therapeutic invention [23,24]. Re-
search has revealed that specific therapeutic methods and techniques can explain only
approximately 15% of the effect of the intervention (e.g., reduction in symptoms, increase in
quality of life), while extra-therapeutic changes (spontaneous remission, events that are not Int. J. Environ. Res. Public Health 2021, 18, 8104 4 of 12 related to the therapy) account for 40% of the effect. The clients’ motivation and expecta-
tions of the therapy can explain 15% of the success or failure of the therapeutic intervention. The remaining 30% can be ascribed to common factors (therapeutic alliance and therapist
characteristics) [23–25]. In other words, common factors account for a greater portion of
the effect than the specific therapeutic method and is one of the strongest predictors of
success or failure in psychotherapy [22,26]. Although there are differences between OIs and psychotherapeutic interventions,
they also share a number of features: they both aim to change the thoughts and actions of
individuals and/or groups, they both enhance well-being, and they both address patterns
and dynamics at the individual and group levels in order to create awareness of behavior
that facilitates or hinders well-being [27–30]. 3. The Relational Fit in OI Translating the insights from the research on common factors in psychotherapy,
it is important to discuss three different but interrelated components of the relational
fit. First, as in the case with the therapist, the IFs’ competences and abilities may influence
how the OI is implemented and in what way participants are involved in the intervention. Second, similar to the client, participants’ expectations, self-efficacy, experiences, etc., may
affect their motivation and ability to participate in and use the intervention, as well as
their motivation and ability to integrate the intervention in their work. The third is the
quality of the relation between IFs and participants, as the quality of this relation can
influence the motivation of participants for engaging actively in OIs. In the following
presentation of the literature, we have chosen to mainly include reviews or meta-analyses
that give an overview of the literature on common factors. Thus, we have not included
primary studies in the following unless there are no existing reviews or meta-analyses on
the relevant subjects. 2. What Affects the Outcome of Psychotherapy? Even though the relation between IFs and participants in OIs might not be as deep
or intimate as the relation between therapists and clients, we argue that the relation also
plays a central—but so far widely neglected—role in the implementation and outcome of
an intervention. 3.1. Intervention Facilitators Even though there is almost always an IF who facilitates and implements the interven-
tion in cooperation with the participants, very little is known about how the IFs influence
the success or failure of such interventions. However, a few studies have pointed out
relevant competencies and roles for the IF. Nielsen et al. suggested that IFs should “possess
expertise in process consultation and knowledge about occupational health issues” [31],
and Peiró et. al. (2007) in Nielsen et al. pointed to the following competencies that external
consultants should embrace: change management skills, expert knowledge of psychosocial
risk factors, awareness of regulations and laws, and practical skills in conducting risk as-
sessment and evaluation [31]. A few studies in OIs have suggested that IFs’ communication
skills are important as these could influence participants’ perception of the motives and
objectives of the intervention—and thereby their commitment to engage in intervention
activities [12,32–34]. To the best of our knowledge, no studies have looked systematically
and empirically into the above-mentioned aspects of evaluation for OIs, even though
especially qualitative process evaluations of OIs have shown that participants perceive IFs
as important for the implementation of the OI [3]. Another unanswered question in the literature is whether internal or external IFs
may be more suited for supporting OIs. Former research has shown that the IF role
has usually been managed by external or internal consultants, or the manager [12,33,35],
but we know little about the pros and cons of the IF being a manager, an external consultant,
or an internal consultant [35,36]. It has been suggested that external consultants may be
more objective and avoid taking sides [37], but others have argued that internal IFs might
more successfully sustain the possible positive effect of the OI [33,35]. Berta et al. [17], Int. J. Environ. Res. Public Health 2021, 18, 8104 5 of 12 Semmer [10], and Nielsen and Randall [13] called for systematic research into the role of
IFs in OIs, and Nielsen has called for more research into how managers as IFs can “make
or break an intervention” [33]. In pursuing a more systematic evaluation of the role of the IF, inspiration may be
drawn from research in psychotherapy as this research field has already looked into this. 3.1. Intervention Facilitators In their book “The Great Psychotherapy Debate”, Wampold and Imel write that we need
to look at therapists in the same way as we do other professionals: some lawyers win
more cases than others and some teachers inspire their students to achieve better grades
than others. Therefore, researchers should continually explore what characterizes the more
successful therapist so others can learn from them [38]. We argue that the same goes for
IFs: if we want to improve the effect of OIs, we need to know what characterizes the most
successful IFs. It is well-documented that the therapist’s empathy enhances the chances of clients
profiting from individual psychotherapy [30,39]; Kivligham et al. have also found that
therapists’ level of empathy increases the group members’ level of engagement in group
therapy [40]. Furthermore, in a review, Ackerman and Hilsenroth identified 11 therapist
characteristics and attributes as well as 11 therapist generic techniques (how the therapist
more specifically helps the client to reflect and change thoughts and behavior) that affect the
therapeutic alliance and outcome of an intervention [41]. The 11 personal characteristics and
attributes are: flexible, experienced, honest, respectful, trustworthy, confident, interested,
alert, friendly, warm, and open; the 11 therapist techniques related to the characteristics
and attributes are: exploration, depth, reflection, supportive, notes past therapy success,
accurate interpretation, facilitates expression of affect, active, affirming, understanding,
and attends to clients’ experience. Whereas the existing OI research has suggested that IFs’
knowledge- and communication-related competencies are important, psychotherapeutic
evidence has also documented that relational and sociable characteristics are important
in increasing the effect of an intervention. To provide a starting point for research on
the importance of these characteristics to the outcome of OIs, we adapted Ackerman and
Hilsenroth’s therapist characteristics and techniques [41] in the formulation of 11 questions
for the mapping of the IF’s characteristics and techniques (Table 1). We have prepared
11 questions aimed at identifying whether participants in OIs feel and experience that IFs
possess and apply these techniques in their implementation and facilitation practice. The qualities outlined above may not necessarily be equally important for Ifs. How-
ever, we suggest that asking these questions may provide a starting point for future
quantitative as well as qualitative explorative research on the role of IFs in the OI. 3.2. Participant Factors It has been suggested that the following three participant factors may play a role in
the implementation and effect of OI: (1) mental models, (2) self-efficacy, and (3) readiness
for change. Research on OIs points to the importance of participants’ mental models of
(1) the intervention program and (2) the intervention activities and how they influence
the motivation to participate in the intervention [2,42]. Participants’ mental models are
important as they may influence how participants understand and react to the intervention
and how they perceive their own and the management’s responsibility in the working
environment [12,43]. It has also been shown in qualitative process evaluations how partici-
pants’ views on the feasibility and acceptability of the intervention method influence their
motivation to implement the intervention [2,3]. Participants’ self-efficacy (how much one
believes in one’s competencies) is hypothesized to affect the outcome of an intervention,
as the level of self-efficacy can influence the employees’ belief in their own capability to
engage actively in the intervention and implement the suggested improvement of working
conditions [44]. Furthermore, it is suggested that participants’ readiness for change may
influence how motivated they are for participating in the intervention [12,31]. As can be
seen, what in therapy is labeled “participant factors” has, to a certain degree, been the topic Int. J. Environ. Res. Public Health 2021, 18, 8104 6 of 12 of investigation in OI research. But there is no tradition for systematically addressing this
in the evaluation and understanding of the outcome of OIs. of investigation in OI research. But there is no tradition for systematically addressing this
in the evaluation and understanding of the outcome of OIs. Table 1. (Re)conceptualization of therapist characteristics and techniques into the relational fit: IF characteristics. Ackerman and Hilsenroth’s Review of Therapist
Characteristics and Techniques
Relational Fit Items—Intervention Facilitator
Characteristics and Techniques
Personal attributes
Technique
The participants are asked the following questions (we suggest using a
5-point Likert scale): To which degree do you experience that . . . 3.2. Participant Factors Flexible
Exploration
the IF contributes to investigating several sides of work-related issues
Experienced
Depth
the IF asks relevant questions to get to the center of issues
Honest
Reflection
the IF shows an interest in improving our working environment
Respectful
Supportive
the IF encourages reflections and suggestions in a respectful way
Trustworthy
Notes past therapy success
the IF is attentive to the progress made in improving the work environment
Confident
Accurate interpretation
the IF understands work-related problems and suggestions
for possible solutions
Interested
Facilitates expression
the IF actively includes participants’ different perspectives
Alert
Active
the IF is actively present in the process
Friendly
Affirming
the IF is friendly and appreciative
Warm
Understanding
the IF understand us
Open
Attends to client’s experience
the IF is curious about our experiences in work-related problems
IF = Intervention Facilitator. While researchers in OIs have only suggested the importance of participant factors [13],
researchers in psychotherapy have both theoretically and empirically studied which indi-
vidual factors and variables affect outcomes [22,26]. We suggest that several aspects drawn
from psychotherapeutic research may hold explanatory value and could be incorporated
in future evaluations of OIs. First, research in psychotherapy has documented a relation
between the client’s expectations of therapy and the outcome [30,45,46]. Second, client
factors such as openness [47], motivation, and the ability to establish stable relationships,
to verbalize, and to cooperate lead to more successful outcomes [48]. Third, the client’s attach-
ment style and social competencies influence the client’s ability to develop a strong alliance
and therefore indirectly influence how much the client profits from the intervention [49]. More than 161 patient characteristics have been studied to investigate the extent
they can affect psychotherapy outcomes [39]. As one of the most well-documented pa-
tient characteristics is the patients’ expectations and their belief that the therapy will
work [30,39], we utilized the Credibility/Expectance Questionnaire (CEQ) developed by
Devilly and Borkovec [50] as a main source of inspiration for measuring the effect partic-
ipants have on the outcome. The CEQ is widely used in contemporary psychotherapy
research and has been shown to account for approximately one-third of the variance in
treatment outcomes [45,51]. In order to make the CEQ operational for OIs, we present our
reconceptualization in Table 2. We have modified the original questions from the CEQ
and adjusted them for OIs. 3.3. Quality of Relation Studies on OIs have highlighted that interventions may fail to achieve their objectives
if participants and IFs disagree on which work-related problems the intervention should
address, what the goal of the intervention is, and/or which methods should be used in the
intervention [52,53]. Furthermore, an OI may fail to achieve its intended outcome if there
is considerable distance between participants’ and IFs’ perceptions of how work-related
problems should be addressed [54]. However, none of these factors are included in current
evaluation frameworks [12,43]. Research in psychotherapy has systematically documented that the relation influences
the outcome of the therapy, and that the quality of the relation shapes the client’s positive
or negative mental models of the psychotherapeutic intervention [22,24,55]. A strong
alliance in psychotherapy is characterized by a positive and respectful emotional bond,
by appreciation, mutual agreement on treatment goals, and a feeling that problems are
addressed and managed in relevant ways [30,46,56,57]. From research in psychotherapy,
we know that a strong alliance and particularly agreement on the tasks and goal of the
therapy increase the chances of the client engaging in healthier actions and improving
their well-being [30]. A strong alliance increases the likelihood of the client accepting the
intervention and being confident that treatment has a positive effect [30,58]. Research has
shown that the therapist’s contribution to the alliance has a greater impact on the outcome
than the client’s contribution [21], and a meta-analytic review concluded that the quality
of the alliance is associated with the client’s perceptions of the therapist’s empathy and
genuineness [57]. g
We propose that an evaluation of the emotional bond, appreciation, and mutual agree-
ment on methods and goals may likewise be important for researchers in OIs. In this
regard, the Working Alliance Inventory (WAI) presents a well-established paradigm and
may also be fruitful in the evaluation of OIs. WAI consists of 36 survey items responded to
by both participants and therapists and has shown consistent predictive capacity in relation
to counseling outcomes [26,59]. In recent years, the WAI has been successfully modified
to fit other areas of therapy or counselling such as physiotherapy, stuttering treatment,
and physical rehabilitation [60–62]. The WAI may also—in a modified version—be transfer-
able to investigating the quality of the relation between the IF and participants in OIs. 3.2. Participant Factors As can be seen in Table 2, we have chosen to replace “feel”
with “think”, as this term may have a higher face validity and may be more recognizable
for participants in OIs. We hope that future studies of OIs will use, validate, and qualify
our suggestions. gg
Applying this modified instrument along with a qualitative exploration of participants’
expectations of and motivation for OIs may provide useful insights into which different
types of OIs and IFs fit different participant groups best. Knowledge derived from this
type of analysis will enable the matching of both the intervention and the IFs with the
participants’ needs and conditions. 7 of 12 Int. J. Environ. Res. Public Health 2021, 18, 8104 Table 2. (Re)conceptualization of the CEQ into relational fit: Participant factors. Devilly and Borkovec:
Credibility/Expectancy Questionnaire (CEQ)
Relational Fit Items—Participant Factor
How logical does the therapy offered
to you seem
Are the OI’s aims and objectives clear to you? How successfully do you think this treatment
will be in reducing your symptoms
How successful do you think the OI will be in
improving your working environment? How confident would you be in
recommending this treatment to a friend
How confident would you be in
recommending this OI to another
team or organization? How much improvement in your symptoms
do you think will occur
How much improvement in the working
environment do you think will occur? How much do you really feel that therapy will
help you to reduce your symptoms
How much do you really think that the OI will
improve your working environment? How much improvement in your symptoms
do you really feel will occur
How much improvement in your working
environment do you really think will occur? OI = Organizational Intervention. Table 2. (Re)conceptualization of the CEQ into relational fit: Participant factors. 3.3. Quality of Relation Since
the WAI in the original form consists of 36 items, it may be too long for participants in OIs
to complete. Hence, in Table 3 we suggest a modification, taking its departure from Hatcher
and Gillaspy’s [63] revised short version of the WAI. This version has validated the use of
12 items of the WAI, with the highest capability to predict client outcomes. We have modi- Int. J. Environ. Res. Public Health 2021, 18, 8104 8 of 12 fied the original questions from the Working Alliance Inventory—Short Revised (WAI-SR)
Subscales and adjusted them to a work context and to an organizational intervention. fied the original questions from the Working Alliance Inventory—Short Revised (WAI-SR)
Subscales and adjusted them to a work context and to an organizational intervention. fied the original questions from the Working Alliance Inventory—Short Revised (WAI-SR)
Subscales and adjusted them to a work context and to an organizational intervention. Table 3. (Re)conceptualization of the WAI-SR into relational fit: Quality of relation. 3.3. Quality of Relation (Re)conceptualization of the WAI-SR into relational fit: Quality of relation. We suggest that these items may provide a starting point for studying the importance
of the quality of the relation between the IF and the participants in OIs. 3.3. Quality of Relation Working Alliance Inventory—Short Revised (WAI-SR) Subscales
Relational Fit Items—Quality of Relation
Goal Scale
Goal scale
The therapist and I are working towards mutually agreed upon goals
IF supports us in working towards agreed upon goals to improve our
working environment
We agree on what is important for me to work on
The IF and the team agree on what is important for us to work on
The therapist and I collaborate on setting goals for my therapy
The IF and the team collaborate on setting goals for the OI
We have established a good understanding of the kind of changes that
would be good for me
The IF and the team have established a good understanding of the kind
of changes in our working environment that would be good for us
Task Scale
Task scale
What I am doing in therapy gives me new ways of looking at my
problem
What the IF and the team are doing in the process is giving us new ways
of looking at our work-related problems and challenges in
our working environment
I feel that the things I do in therapy will help me to accomplish the
changes that I want
The things the IF and the team do in relation to the OI will help us to
accomplish the work-related changes that we want
As a result of these sessions I am clearer as to how I might
be able to change
As a result of the activities we engage in with the IF, I am clearer as to
how I might contribute to the desired change in
the working environment
I believe the way we are working with my problem is correct
I believe the way we are working with the IF on our work-related
problems is correct
Bond Scale
Bond scale
I believe the therapist likes me
I believe that the IF likes the participants in the intervention
The therapist and I respect each other
The IF and the participants respect each other
I feel that the therapist appreciates me
I feel that the IF appreciates us
I feel the therapist cares about me even when I do things that he/she
does not approve of
I feel the IF cares about us even when we do things that he/she does not
approve of
IF = Intervention Facilitator, OI = Organizational Intervention. Table 3. 4. Discussion In the present paper, we introduced a new important fit—the relational fit—in OIs. The relational fit focuses on the characteristics, attributes, and techniques of IFs and
participants as well as on the quality of their relation. The relational fit complements
existing “fits” literature (organization–intervention fit and person–intervention fit) in OI
research [13]. We showed how research on OIs can benefit from psychotherapeutic research
in understanding and exploring the importance of IFs and the relational fit between IFs
and participants; we also proposed that this can help us understand why interventions
succeed or fail. Furthermore, we suggested measures that may be used to systematically
evaluate relational fit in future studies. Such evaluations may provide valuable insights into
designing the OIs and also help us to prepare the IFs for the running and implementing of
OIs. We proposed that evaluations of the relational fit between IFs and participants in OIs
may take inspiration from three adjusted surveys and measurement methods presented in
this paper (1) Ackerman and Hilsenroth’s review of therapist characteristics and techniques,
(2) Devilly and Borkovec credibility/expectancy questionnaire (CEQ), and (3) The Working
Alliance Inventory—Short Revised (WAI-SR) Subscales), which were developed for use in
psychotherapy. We also argued that adjusted versions of these could be a good starting
point. In this article, we focused on adjusted quantitative questionnaires. It might, however,
also be relevant to conduct qualitative research on the presented subjects. The advantage Int. J. Environ. Res. Public Health 2021, 18, 8104 9 of 12 of the quantitative methods here is that they allow the relational fit to be analyzed with
outcome variables [64]. of the quantitative methods here is that they allow the relational fit to be analyzed with
outcome variables [64]. A study has shown that IFs who had undergone training in how to manage and
implement an intervention increased the positive effect of an OI more successfully than
IFs who did not receive training [65]. Knowledge on the relational fit and important IF
competences and techniques could be useful in the optimal training of Ifs, with special
focus on their ability to develop good relations with the participants, thereby potentially
increasing positive outcomes of OIs. Knowledge on which participant factors and to what
degree these affect the relation to the IF will also help to better match IFs and participants,
which could also help the IF to meet the needs and conditions of the participants. 4. Discussion p
p
p
Our arguments for the importance of a high-quality relation between the IF and
participants in OIs go hand in hand with one of the main findings and general recom-
mendation in the literature on intervention method in OIs, namely that OIs should be
participatory [66,67]. This recommendation has frequently been voiced, as participatory
interventions seem to be most successful in improving the working environment and the
health of employees [31]. The participatory element in these approaches is highlighted as
securing the relevance of interventions through the activation and consideration of the
practical knowledge and know-how of managers, employees, and other potentially relevant
actors [68]. Qualitative studies have also pointed to the importance of the IF in participatory
interventions. Conversation analyses of the dialogue between IF and participants showed
that the IF’s way of facilitating the intervention had an effect on whether the participants
participated actively or were passive in the intervention workshops, depending on the IFs’
facilitation styles [69,70]. Studies have shown how IFs varied in how much they welcomed
the participants’ work improvement suggestions, how open they were to suggestions,
and whether they themselves played an active role in supporting or rejecting the concrete
suggestions [69,70]. We therefore propose that a high-quality relation between IFs and par-
ticipants produces an increase in motivation and the willingness to actively participate in
the intervention by, for example, expressing criticism of the organization and the working
environment, or by presenting improvement ideas, taking risks, and suggesting changes. A high-quality relation could therefore be important and even necessary to unleash the
potential of participation as a working mechanism in OIs. In this paper, we argued that the research field of psychotherapy may bring us closer to
answering the call from Nielsen and Randall for the development of evaluation models and
methods that can “identify how the potential effects of interventions on health and well-
being are moderated and mediated by intervention processes” (p. 602) [12]. The degree
to which the results and measurement methods from psychotherapy are transferable to
organization interventions, and whether common factors have the same explanatory power
in OIs have yet to be explored. Therefore, we hope that researchers will be inspired to
look more systematically into the relational fit in future evaluations of OIs. 4. Discussion Knowledge
on relevant and important characteristics, attributes, and techniques of IFs would be of
great relevance to practice, as it may permit the training of IFs and facilitate their ability
to develop relations of high quality, thereby increasing the chances of the OIs’ success. We argue that knowledge on the importance of a good relational fit will help decision
makers when planning and deciding on an OI to improve employees’ well-being. They
need to be aware that it is insufficient to select an evidence-based intervention that fits
the organization. It may be just as important—or even more important—to select highly
qualified and trained IFs as well as to continuously ensure that there is a good fit between
the IF and the employees participating in the intervention. 5. Conclusions Many organizations initiate OIs to improve employees’ mental well-being. From research,
we know that the effect of these interventions is limited and not sufficient for researchers
and organizations to be able to choose and implement an evidence-based intervention. In this article, we have argued that it might be equally important to secure a good fit Int. J. Environ. Res. Public Health 2021, 18, 8104 10 of 12 10 of 12 between the person or persons facilitating the intervention and the employees participating
in the intervention. We labelled this fit the Relational Fit. Based on research from the fields
of OI and psychotherapy, we have shown the importance of future studies addressing and
evaluating the R-fit, and we hope that researchers will be inspired by the three concrete
measures presented here to further study and evaluate the R-Fit. Knowing that there is an
increase in mental health problems, it is especially important to optimize the OIs that aim
at improving the mental well-being of employees. We hope that with this article, we have
managed to play a small but important role in this. Author Contributions: Conceptualization, M.F.A., J.Z.N.A. and K.N.; methodology, M.F.A., J.Z.N.A. and K.N.; investigation, M.F.A., J.Z.N.A. and K.N.; original draft preparation, M.F.A.; writing—
review and editing, M.F.A., J.Z.N.A. and K.N. All authors have read and agreed to the published
version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Montano, D.; Hoven, H.; Siegrist, J. Effects of organisational-level interventions at work on employees’ health: A systematic
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Export Potential as the Competitiveness Indicator of the Agri-Food Complex
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International journal of engineering & technology
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Abstract The paper deals with some theoretical approaches to determining the essence of the export potential, the features of its formation and
development in the agri-food complex, the impact on the growth of competitiveness. This made it possible to clarify the definition of the
export potential of the agri-food complex as an aggregate of competitive advantages. The specifics of the development of the export po-
tential in the context of globalization and the development of international integration are revealed. The dynamics and structure of ex-
ports of food products and agricultural raw materials are analyzed; the physical capacity of the domestic markets of basic food products,
the dynamics, and trends of its change are estimated. Excessive growth in the volume of production of agricultural products can lead to
oversaturation of the domestic market, a decrease in the profitability of domestic producers, worsening the financial situation in the in-
dustry. Russia has a significant potential for integration into the world agri-food system. However, the main share of the export of the
agri-food complex is made up of agricultural raw materials, primarily grains and oilseeds, which has an adverse effect on the structure of
the complex and the rate of its development. The diversification of exports, the growth of exports of products with high added value will
contribute to the sustainable development of agriculture and food industries. The development of the export potential of the Russian agri-
food complex should be based not only on the active use of national competitive advantages but also on the formation of competitive
advantages based on the international division of labor as well. The export-oriented strategy for the development of Russia's agri-food
complex needs to be justified in the context of food security policy, which implies resolving the contradiction between national interests
related to the saturation of domestic markets, and attitudes toward growing competitiveness in the world food markets, as well as the
development and realization of the export potential. Keywords: Agri-Food Complex, Export Potential, Globalization, Competitiveness, Competitive Advantages; economic space in which the state borders become more transpar-
ent, and national-state interests are blurred by the interests of
transnational firms. The competitiveness of the country is con-
nected with the competitiveness of national companies both in the
domestic and foreign markets. However, the country cannot lead
in all spheres of production at the same time. Natalia Anatolyevna Yakovenko1*, Irina Anatolyevna Rodionova2, Irina Serafimovna Ivanenko1, Natalia Arkadev-
na Kireeva3, Antonina Mikhailovna Sukhorukova3 1Institute of Agrarian Problems of the Russian Academy of Sciences, Moskovskaya Street, 94, Saratov, 410012, Russia
2Saratov State Agrarian University, Teatralnaya Square, 1, Saratov, 410012, Russia
3Saratov Socio-Economic Institute of Plekhanov Russian University of Economics, Radishcheva Street, 89, Saratov, 410003, Russia
*Corresponding author E-mail: yakovenko.n.a@bk.ru Abstract It is competitive in
those industries or segments where the combination of competitive
advantages is most favorable. It should also be noted that "... countries do not succeed in individual industries but in groups of
industries connected by vertical and horizontal links. The econo-
my of the country is a set of such groups. Their composition and
sources of competitive advantage (or reasons for its absence) re-
flect the level of development of the national economy" [1, p. 94]. In conditions of strengthening the influence of external factors, the
competitiveness of multisectoral complexes, including the agri-
food one, can be estimated using the following indicators and their
combinations: labor productivity; specific pay for labor; capital
intensity; science intensity and the technical level of products; a
set of knowledge and scientific reserves necessary for the inde-
pendent development of products and their reproduction; the vol-
ume of technological reserves for the implementation of scientific
and design development; the degree of export orientation or im-
port dependence of the industry; the degree of conformity of the
level of development of the industry to the overall level of devel- International Journal of Engineering & Technology, 7 (4.38) (2018) 654-658 International Journal of Engineering & Technology, 7 (4.38) (2018) 654-658 Copyright © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits
use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Engineering & Technology Website: www.sciencepubco.com/index.php/IJET 3. Results The formation and development of the export potential of Russia's
agri-food complex are aimed at increasing the competitiveness of
the complex in the context of expanding and deepening engage-
ment with the global economy. The export potential of the agri-
food complex is a combination of competitive advantages, includ-
ing natural and climatic conditions, resource security, high techno-
logical and technical level, structural balance, etc., which form the
opportunity to produce goods and services that are competitive in
world markets and export them under certain conditions (ensuring
the country's food security, lack of commodity and monetary defi-
cit, etc.). The export potential is based on production, personnel,
innovation, financial and other potentials and contributes to their
effective use due to the resulting synergetic effect [9]. g y
g
[ ]
When estimating and forecasting the export potential of the agri-
food complex and its implementation, it is necessary to take into
account the features of the research object. The agri-food complex
is a complicated socio-ecological and economic system; the coun-
try's food security, social stability of the society, sustainable de-
velopment of rural areas depends on its functioning. This forms a
number of restrictions for the realization of the export potential in
the world food market. First, the diversity of products, their quali-
ty characteristics, production technologies; all this implies a high
differentiation of markets, their characteristics, and market condi-
tions. Secondly, the development of the agri-food complex is
closely interrelated with the security of the country, first of all, the
food industry. Therefore, the export of food and agricultural prod-
ucts should be linked with the saturation of the domestic market,
an increase in the standard of living of the population. The processes of globalization have intensified the competitive
struggle in the world food markets. The result of the increased
competition was the volatility of the world food markets, their
instability and unpredictability. In these conditions, there is a need
in the system of state regulation to shift priorities to support the
export of food and agricultural products by developing methods
for promoting exports, financing activities to enter foreign markets. It is necessary to develop measures for structural policies aimed at
adapting Russian producers to the changes taking place in the
world agri-food system. The expansion and deepening of global
value chains are very active. 1. Introduction The studies are based on theoretical and methodological positions
contained in the works by foreign scholars, such as Smith [4],
Ricardo [5], Porter [6], Tracy [7], Scherer and Ross [8], North and
others. Theoretical and methodological approaches to managing
the formation and realization of the export potential, the analysis
of the impact of the processes of the international division of labor
and integration on the development of the Russian economy are
set forth in the works of such Russian scientists as Bagrinovsky,
Glaziev,
Ivanter,
Seifullaeva,
Butayev,
Vardomsky,
Skatershchikova, Petrakova, Fatkhutdinov, Uzyakova and others. Problems of competitiveness growth and formation of competitive
advantages, export-oriented development of Russia's agri-food
sector, interaction of intensification of inter-industry relations,
exports of food and agricultural products and prospects for the
development of the agri-food complex are studied in the works by
Altukhov, Anfinogentova, Krylatykh, Ksenofontova, Usachev,
Uzun, and others. 3. Results The creation of global value chains
allows many countries to improve the economic situation within
the country. The most effective are those countries that form the
modern business environment, the investment climate, and the
corresponding infrastructure [10, p. 31]. Qualitatively, a new stage
in the development of Russia's agri-food complex should be char-
acterized not just by the growth of export operations, but also by
reaching a higher level of production cooperation and integration
with the formation of national intersectoral segments of global
reproductive systems. The processes of globalization and integra-
tion form the advantages resulting from the deepening of speciali-
zation, the rapid spread of scientific, technical, economic
knowledge, management experience, the expansion of innovation
investment, the internationalization of financial and information
markets, the reduction of transaction costs, structural changes in
the economy. However, the dynamic changes occurring both in the world and in
the national food market, especially the realization of the export
potential of the agri-food complex, require further in-depth study. The paper is devoted to some peculiarities of the formation and
realization of the export potential of the Russian agri-food com-
plex; these peculiarities are considered to be fundamental ones. 1. Introduction The current stage in the development of the world economic sys-
tem is characterized by the aggravation of international competi-
tion, which led to an increase in the dynamism and changes in the
correlation of conditions and factors of the functioning of national
markets, and macroeconomic instability. In a market economy,
one of the key categories that most effectively characterizes the
effectiveness of the functioning of the market is competitiveness. Competitiveness is a complex multi-level concept, the analysis
and evaluation of which should be linked to a specific competitive
field (the area of competition) and its level. All the variety of
competitive relations that arise in the sphere of the economy are
divided into three levels: microlevel – specific types of products,
manufactures, enterprises; mesolevel – branches, corporate associ-
ations of enterprises and firms of conglomerate type; macrolevel –
national economic complexes. The micro- and mesolevel of the
competitive field can have both a national and an international
scale. At each level of competitiveness, their specific tasks are
solved and their demands on the subject are made. The globalization of economic processes transforms the problem
of competitiveness both at the macrolevel and at the meso- and
microlevels. The world economy gradually turns into a geo- 655 International Journal of Engineering & Technology opment of the national economy; the dynamics of prices for prod-
ucts; expansion of the industry – the degree of use of its products
in various sectors of the economy. The development of the export
potential as the ability of the national economy to produce goods
that are competitive in world markets, their export in sufficient
quantities at world prices and its actual sale becomes one of the
main indicators of the country's competitiveness. In their works,
Guerson et al. have revealed the dependence of growth of gross
domestic product on the specialization of exports and their struc-
ture [2]. The studies by Sachs and Warner proved a direct relation-
ship between low economic growth and the country's primary
export specialization [3]. This makes it necessary to study the
essence of the export potential, its formation, influence on the
development prospects of the Russian economy and its separate
sectors, analyze the dynamics and structure of exports, and esti-
mate the possibility of inclusion in global food chains. 1. Introduction The inten-
sification of the competitive advantages of the Russian agri-food
complex in the international division of labor, the build-up of the
export potential will contribute to its stable and sustainable growth. The studies are based on theoretical and methodological positions
contained in the works by foreign scholars, such as Smith [4],
Ricardo [5], Porter [6], Tracy [7], Scherer and Ross [8], North and
others. Theoretical and methodological approaches to managing
the formation and realization of the export potential, the analysis
of the impact of the processes of the international division of labor
and integration on the development of the Russian economy are
set forth in the works of such Russian scientists as Bagrinovsky,
Glaziev,
Ivanter,
Seifullaeva,
Butayev,
Vardomsky,
Skatershchikova, Petrakova, Fatkhutdinov, Uzyakova and others. Problems of competitiveness growth and formation of competitive
advantages, export-oriented development of Russia's agri-food
sector, interaction of intensification of inter-industry relations,
exports of food and agricultural products and prospects for the
development of the agri-food complex are studied in the works by
Altukhov, Anfinogentova, Krylatykh, Ksenofontova, Usachev,
Uzun, and others. opment of the national economy; the dynamics of prices for prod-
ucts; expansion of the industry – the degree of use of its products
in various sectors of the economy. The development of the export
potential as the ability of the national economy to produce goods
that are competitive in world markets, their export in sufficient
quantities at world prices and its actual sale becomes one of the
main indicators of the country's competitiveness. In their works,
Guerson et al. have revealed the dependence of growth of gross
domestic product on the specialization of exports and their struc-
ture [2]. The studies by Sachs and Warner proved a direct relation-
ship between low economic growth and the country's primary
export specialization [3]. This makes it necessary to study the
essence of the export potential, its formation, influence on the
development prospects of the Russian economy and its separate
sectors, analyze the dynamics and structure of exports, and esti-
mate the possibility of inclusion in global food chains. The inten-
sification of the competitive advantages of the Russian agri-food
complex in the international division of labor, the build-up of the
export potential will contribute to its stable and sustainable growth. 2. Methods This requires, as many experts note, the
development of a balanced policy aimed at developing the export
potential, increasing the export of products with high added value,
deepening the level of processing of agricultural products, expand-
ing sales markets. The study of separate markets of basic food
products in Russia confirms these conclusions (Table 1). The es-
timation of the physical capacity of the Russian domestic market
was carried out taking into account the achievement of rational
consumption norms and was calculated as the difference between
the rational and actual volume of consumption of the population. Since 2010, there has been an overproduction in the markets of
potatoes, vegetable oil, and eggs. The capacity of the markets for
meat and meat products, fruits, vegetables and cucurbits crop de-
creases. The potential capacity of the meat products has decreased
in 2016 compared to 2010 by 48.6%. The tendency to decrease the
capacity of domestic markets is mainly due to the growth in sup-
ply. Fig. 1: Trend of production of the main branches of the agri-food complex,
in %
Source: calculated using Rosstat data [12] Source: calculated using Rosstat data [12] However, a significant differentiation of production indices per-
sists over the years, which is primarily due to the high dependence
of agricultural production on weather and climate factors, low
innovation and investment potential. The implementation of the
target parameters of the state agrarian policy aimed at increasing
agricultural production made it possible to solve the problem of
food security of the country in basic foodstuffs in a relatively short
period of time. The working conditions of agricultural producers
were also affected by a decrease in the level of competition from
foreign manufacturers as a result of Russia's counter-sanctions, the
growth of the capacity of agri-food markets for domestic produc-
ers, and a relative reduction in production costs due to the deval-
uation of the national currency. 2. Methods The purpose of the work is to study the interrelation between
competitiveness and export activities of the agri-food complex of
Russia, identify the main trends and priority points of export
growth, and increase the export potential of the complex based on
the formation of competitive advantages. During the research, monographic, abstract-logical and economic-
statistical methods were used. Based on the principles of classical economic theory, institutional
theory, the theory of comparative advantages of international trade,
the theory of competition, the concept of the export potential of
the agri-food complex is clarified; the features of its formation
taking into account the object of research are revealed. Russia has a significant potential for integration into the world
agri-food system. Integration processes can be connected, first,
with the inclusion of domestic producers in the global agri-food
chains, and secondly, with the growth of the level and structure of
exports. Based on economic and statistical methods, the estimation of
trends and prospects for the growth of the export potential of the
Russian agri-food complex and the capacity of the main food mar-
kets in Russia is given. Against the backdrop of a decline in the gross domestic product
and stagnation in industrial production, the agri-food sector retains
a positive development trend. The volume of production in agri-
culture in 2016 increased by 63.9% compared with 2000, in the
food industry – by 93.1% (Figure 1). In addition to some specific methods, the following scientific ap-
proaches to the analysis of the problem were used: dialectics, ab-
straction, deduction, induction, analysis, and synthesis. International Journal of Engineering & Technology 656 Fig. 1: Trend of production of the main branches of the agri-food complex,
in %
Source: calculated using Rosstat data [12] The opportunities for further development of the sectors of the
Russian agri-food complex, as noted by Russian scientists, will be
determined by reaching high levels of meeting the food require-
ments in their physical terms, bringing the domestic agri-food
markets closer to the limits of their capacity [11]. Continued
growth in production in such promising sectors as pig breeding,
poultry industry, production of oil crop and sugar can lead to the
oversaturation of the domestic market, a decrease in the profitabil-
ity of domestic producers. 2. Methods Table 1: Trend of the capacity of the markets for basic food products of the Russian Federation
Basic food products
2010
2011
2012
2013
2014
2015
2016
Milk and dairy products, million tons
11.14
11.29
10.88
11.05
11.83
12.58
13.07
Meat and meat products, thousand tons
1,428.5
1,143.68
716
574.04
584.36
878.34
734.02
Potatoes, thousand tons
-1,999.9
-2,859.2
-3,007.2
-3,013.7
-3,067.9
-3,220.6
-3,376.5
Vegetables and cucurbits crop, thousand tons
5,571.2
4,860.6
4,439.2
4,448.8
4,236.6
4,245.3
4,110.5
Fruit, thousand tons
5,999.7
5,718.4
5,584.8
5,166.4
5,259.2
5,709.2
5,578.6
Vegetable oil, thousand tons
-199.9
-214.4
-243.4
-243.9
-262.9
-234.2
-249.6
Eggs, thousand pieces
-1,285.7
-1,572.6
-2,291.2
-1,291.6
-1,314.8
-1,317.5
-1,908.5 However, the volume and structure of foreign trade in agri-food
products indicate that the export potential of the domestic agri-
food complex has not yet been fulfilled. Despite the high growth
rates, the share of food products and agricultural raw materials in
total exports in 2016 was only 6.0%, including 1.5% in the CIS
countries, and 4.5% in the far-abroad countries. Deliveries of the
products of the agri-food complex abroad were hampered by the
absence of an effective state policy of stimulating exports. High
transportation costs, shortage of port capacities, underdeveloped
production and trade infrastructure hamper the active development
of exports of agri-food products. Among the studied markets, there was an increase from 11.14
million tons in 2010 to 13.07 million tons in 2016 in the market
for dairy products. Despite the growth in milk production more
than twofold in 2016 compared to 2010, consumption amounts to
72.6% of the rational norm, which keeps the market capacity at a
high level. The main factors limiting the growth of consumer de-
mand in the dairy market are the decrease in the purchasing power
of the population's income and the level of real disposable wages. Since 2013, there has been an increase in consumer prices for
dairy products, which also leads to a drop in demand in the dairy
market. This is especially reflected in the mid-price and high-price
segments of the market. To estimate the prospects for the development of exports of food
commodities and agricultural raw materials, a polynomial trend
line was formed (taking into account the approximation), which
shows a steady upward trend. The level of reliability of the model
is quite high (the coefficient of determination R2 = 0.9438). 2. Methods However,
as some economists note, "participation of national producers in
global value chains does not guarantee a technologically "qualita-
tive" industrial development of the country" [13-14]. Currently, the export segment operates mainly under the influence
of exogenous factors of the world market and is weakly connected
with the domestic market of the country. Russian food producers
are already built into world economic ties and global chains of
creating value added but are located mainly at the lower levels
(raw materials production). The prevailing types of competitive
advantages are still their price types. When domestic producers
enter the global chains, it is necessary to take into account the
export potential of agriculture, in particular, the great opportuni-
ties for the production of environmentally friendly products. In the institutional theory with such representatives as Veblen,
Commons, and others, the concept "institute" is introduced as a set
of norms and rules of behavior that define economic relations; the
importance of institutions for the functioning of the economy,
their role and evolution are studied. Neoinstitutionalists, including
Coase, Stiglitz, Pozner, expanded the interpretation of the catego-
ry "institute" studying formal and informal rules of conduct that
establish the relationship between economic agents and organiza-
tions. "The main role that institutions play in society is to reduce
uncertainty by establishing a stable (albeit not necessarily effec-
tive) structure of interaction among people" [18, p. 21]. Export
opportunities of the country and their implementation in the world
market are investigated in institutional economics in terms of the
effectiveness of functioning of the institutions, institutional envi-
ronment and institutional structure of the economy formed in the
country. The state is the most important system-forming institu-
tion that influences the transformation in the economy and the
effectiveness of its functioning. It can both promote the creation of
effective market institutions and create an institutional structure
that does not allow the advantages of a competitive order to mani-
fest themselves due to monopoly power and other factors leading
to an increase in transaction costs. Prospects of integration of the Russian agri-food complex into the
world food economy are reflected in different ways in various
branches of¬ agricultural production. The least competitive are
intensive industries, especially livestock due to their backward
technology, high¬ costs, and low productivity¬. The cost of feed
for a production unit in Russia significantly exceeds the level of
developed countries. 2. Methods The essence of the theory of comparative advantages was revealed
by Ricardo in his work "On the Principles of Political Economy
and Taxation" [16]. He proved that interstate specialization and
foreign economic relations are beneficial if the country exports
goods for which it has comparative advantages, that is, it produces
goods with relatively lower costs compared to other countries. "Under the system of complete freedom of trade, each country
spends its capital and labor on such industries that give it the
greatest benefits" [16, pp. 75-76]. With high price volatility in the world food markets, there is a
need to diversify exports, which involves expanding the range of
goods that have competitive advantages, increasing the competi-
tiveness of the exported goods. This will require investments in
the modernization of production capacities, implementation of
modern technologies. State support for expanding export opportu-
nities is an important condition for the growing commercial pres-
ence of national business in the world market. To implement the
export potential, state support is needed for a system of measures,
including reducing barriers, resolving issues of product certifica-
tion and veterinary control, marketing support for exporters and
financial support for export operations. At present, in the economic theory, the issues of expanding world
economic ties and increasing the competitiveness of national
economies are closely interrelated with the problems of formation
and development of competitive advantages. In the theory of com-
petitive advantages developed by the American economist Porter,
the main idea is about the interconnection of four groups of factors
(or determinants) that form a competitive environment for the
functioning of firms in the country. Competitive advantages
should be constantly maintained, preserved, built up and formed. "There are striking differences among separate countries in terms
of competitiveness; no country can be competitive in all or even in
most sectors of the economy. Ultimately, countries achieve suc-
cess in particular sectors of the economy because the environment
that has developed within them is the most future-oriented and
dynamic" [17]. Qualitatively, a new stage in the development of the export poten-
tial of Russia's agri-food complex should be characterized not just
by the growth of export operations, but also by reaching a higher
level of production cooperation with the formation of national
intersectoral segments of global reproductive systems. 2. Methods If the
existing conditions are maintained in foreign and domestic mar-
kets, the continuing growth in the volume of exports of food prod-
ucts and agricultural raw materials is possible. Currently, there is a shift in priority from import substitution to
export development, the formation of an export promotion system
and financing activities to enter foreign markets. Russia is step-
ping up its participation in international trade in agri-food prod-
ucts. From 2000 to 2016, the volume of exports increased by 2.8
times (including food products and agricultural raw materials – by
10.5 times) (Figure 2). The geographical spread of exports has
increased significantly. The export of food and agricultural prod-
ucts to the CIS countries has increased 5.7-fold for the period
under study and 14.6-fold for the countries of the far abroad. g
p
The traditional goods of Russian exports in the world food mar-
kets are grain, vegetable oil, fresh fish, that is, goods with low
added value. The growth of grain exports from 2010 to 2016 in-
creased in physical terms by 2.4 times, vegetable oil – by 3 times,
fresh fish – by 1.1 times. The growth of non-primary exports was
insignificant, and there is a decrease in exports of some products. In the period under review, there is a significant decline in prices
for agricultural products and foodstuffs in the world food markets. Prices for wheat decreased from 2014 to 2016 from 242.5 dollars
per ton to 143.2 dollars per ton, that is, by 40.9%, for beef – by
20.5%, for butter – by 7.8% [12, p. 607]. In these conditions, with
the growth of the physical volume of exports of the Russian Fed- Fig. 2: Trends and structure of exports of food products and agricultural
raw materials in Russia, million dollars Fig. 2: Trends and structure of exports of food products and agricultural
raw materials in Russia, million dollars Fig. 2: Trends and structure of exports of food products and agricultural
raw materials in Russia, million dollars Fig. 2: Trends and structure of exports of food products and agricultural
raw materials in Russia, million dollars 657 International Journal of Engineering & Technology yond national borders and ensures the sale of surplus products that
cannot be sold in the domestic market. eration, there is a decrease in export earnings from the export of
agri-food products. 2. Methods The¬ inorganic branches of plant growing
stand in a better position. This is primarily grain¬ farms, especial-
ly wheat production and¬ sunflower growing. Integration into
world economic relations is impossible without the formation of a
single market space within the country, the creation of a system of
measures that hinders the establishment of interregional trade
barriers. The strategy for the development of the agri-food com-
plex of Russia means participation in the world division of labor
in agri-food production, while respecting national interests [15]. The provision of food security of the country, the search for new
sources of economic growth, the formation of competitive ad-
vantages of the agri-food complex of Russia is associated with the
strengthening of external and internal challenges. The objective
tendency of the modern development of national economies is to
change the correlation between internal and external factors, deep-
en their interdependence and interconnection, and increase the
influence of external factors. The processes of globalization lead
to a deepening of the involvement of economies and their individ-
ual sectors in the world economy system, targeting external mar-
kets for the sale of products. An increase in the share of exports
remains an important factor in the strengthening and development
of national economies. 5. Conclusion [9] Savenok EA & Pochernyai AS (2017), Eksportnyi potentsial
promyshlennogo predpriyatiya: teoreticheskie aspekty opredeleniya
[Export Potential of an Industrial Enterprise: Theoretical Aspects of
the Definition]. Izvestiya Gomelskogo gosudarstvennogo universi-
teta imeni F. Skoriny, 2(101), 173-176. Based on foregoing, it can be concluded that import substitution is
one of the main objectives of the state agricultural policy. The
study of the agri-food complex as a multifunctional system
providing multiplicative effects allowed presenting the export
potential of the complex as a set of competitive advantages. This
made it possible to identify the specifics of the formation and
realization of the export potential of the agri-food complex, its
impact on the growth of competitiveness of the main branches of
the complex, and to justify the priorities of its development. [10] Revenko LS (2015), Mirovye tovarnye rynki: tendentsii XXI veka
[World Commodity Markets: Trends of the 21st Century]. Vestnik
Sankt-Peterburgskogo universiteta, 5(3), 27-45. [11] Ksenofontov MYu, Gromova, NA & Polzikov DA (2013), Potent-
sial rosta agrarnogo sektora ekonomiki i osobennosti dol-
gosrochnoi politiki razvitiya selskogo khozyaystva [Potential for the
Growth of the Agrarian Sector of the Economy and Features of the
Long-Term Development Policy of Agriculture]. In V.V. Ivaneter,
& M.Yu. Ksenophontov (Eds.), Perspektivy razvitiya ekonomiki
Rossii: prognoz do 2030 goda. Kollektivnaya monografiya [Pro-
spects for the Development of the Russian Economy: Forecast until
2030. Multi-Authored Monograph]. Moscow: Ankil, 408. the complex, and to justify the priorities of its development. The formation of the export potential is an integral part of the
strategy to increase the competitiveness of the national agri-food
complex. Currently, the export segment of the Russian agri-food
complex operates mainly under the influence of exogenous factors
of the world market and is weakly connected with the domestic
market of the country. The export of food and agricultural prod-
ucts should be linked with the saturation of the domestic market,
an increase in the standard of living of the population. An estima-
tion of the physical capacity of the markets for basic food products
made it possible to conclude that certain internal agri-food mar-
kets are approaching their capacity limits, which requires shifting
the priority of the agri-food complex development from import
substitution to export development. [12] Rosstat. (2017). Rossiiskii statisticheskii ezhegodnik. 2017: Stat. sb. [Russian Statistical Yearbook. 2017: Statistical Compilation]. Mos-
cow, 686. 4. Discussion Classical theory, the main provisions of which were developed by
such outstanding economists as Smith, Ricardo, Say, Mill, Mar-
shall, Pigu and others, considers the content and development of
foreign economic relations and export capacity in the framework
of models of absolute and comparative advantages. In his work
called "An Inquiry into the Nature and Causes of the Wealth of
Nations", Smith says: "If any foreign country can supply us with
some commodity at a cheaper price than we are able to produce it,
it is much better to buy it for some part of the product of our own
industrial work, applied in the area in which we possess some
advantage" [4, pp. 30-31]. The essence of the theory of absolute
advantages lies in the fact that countries export goods that they
produce with the least cost, and import goods that are produced by
other countries with lower costs. The specialization of the country
(region) in the international division of labor is influenced by the
absolute advantages that it possesses in the production process in
comparison with other countries (regions). Export stimulates the
development of labor productivity by expanding the market be- Production factors, on the basis of which countries have achieved
advantages in the world market, become increasingly diverse,
dynamic and mobile. Capital is invested and moved to other coun-
tries, labor migrates to countries with the highest pay and most
favorable working conditions, scientific and technological
achievements are exported and imported. Preservation of the lead-
ing role and growth of competitiveness of states are now closely
related to their ability to produce and redistribute not only goods International Journal of Engineering & Technology 658 [3] Sachs JD & Warner AM (2001), The Curse of Natural Resources. E
E
i R
i
45 827 838 [3] Sachs JD & Warner AM (2001), The Curse of Natural Resources. European Economic Review, 45, 827-838. [3] Sachs JD & Warner AM (2001), The Curse of Natural Resources. European Economic Review, 45, 827-838. but financial, investment, innovation-technological, intellectual
and other "surplus" as well [19-21]. European Economic Review, 45, 827-838. [4] Smith A (1962), Issledovanie o prirode i prichinakh bogatstva
narodov [A Study on the Nature and Causes of the Wealth of Peo-
ples]. Moscow: Sotsekgiz, 684. 5. Conclusion [13] Tolkachev S & Teplyakov A (2017), Importozameshchenie v Ros-
sii: neobkhodimost sistemno-strategicheskogo podkhoda [Import
Substitution in Russia: The Need for a System-Strategic Approach]. Ekonomist, 8, 47-66. [14] Tolkachev SA & Teplyakov AYu (2017), Globalnyye tsepochki
stoimosti i natsionalnaya promyshlennaya kompetentnost [Global
Value Chains and National Industrial Competence]. Ekonomich-
eskoe vozrozhdenie Rossii, 1(51), 63-81. The authors have proved that Russia has a good potential for mul-
ti-vector integration into the world agri-food system. Expansion of
export opportunities is an important condition for Russia's full-
scale active participation in integration processes. Qualitatively, a
new stage in the development should be characterized not just by
the growth of export operations, but also by reaching a higher
level of production cooperation with the formation of national
intersectoral segments of global reproductive systems. Russian
food producers are already built into world economic ties and
global chains of creating value added but are located mainly at the
lower levels of raw materials production. The prevailing types of
competitive advantages are still their price types. [15] Faltzman V (2017), K kontseptsii ekonomicheskogo rosta Rossii:
Importozameshchenie, eksportozameshchenie, umerennaya globali-
zatsiya [To the Concept of Russia's Economic Growth: Import Sub-
stitution, Export Substitution, Moderate Globalization]. Ekonomist,
11, 3-8. [16] Ricardo D (1935), Sochineniya. T.2. Nachala politicheskoi
ekonomii i podatnogo oblozheniya [The Works. Vol. 2: On the
Principles of Political Economy and Taxation]. Moscow, 75-76. [17] Porter M (1993), Konkurentnye preimushchestva stran [Competi-
tive Advantages of Nations]. http://www.seinstitute.ru/Files/Veh6-
35Porter.pdf [18] North D (1997), Instituty, institutsionalnye izmeneniya i funktsion-
irovanie ekonomiki [Institutions, Institutional Change and Econom-
ic Performance]. Moscow: Fond ekonomicheskoi knigi "Nachalo". The processes of globalization and the deepening of the interna-
tional division of labor contribute to the growth of Russian exports. In these conditions, the effectiveness of implementing an export-
oriented strategy for the development of Russia's agri-food indus-
try depends on the implementation of resource-saving and pro-
gressive technologies, orientation on intensive development meth-
ods, investment support for advanced industries, equalization of
disproportions in the development of industries, strengthening
competitive advantages and building up the export potential of the
complex on this basis. [19] Wallerstein I (1974), The Modern World-System: Capitalist Agri-
culture and the Origins of the European World-Economy in the
16th Century. New York: Academic Press. [20] Wallerstein I (1980), The Modern World-System-II: Mercantilism
and the Consolidation of the European World-Economy, 1600-1750. New York: Academic Press. 4. Discussion 4] Smith A (1962), Issledovanie o prirode i prichinakh bog The deepening of the international division of labor leads to the
expansion of the reproduction process beyond national borders. The internationalization of production dictates the need for the
creation of appropriate organizational forms, norms, and rules that
allow one to overcome the limitations of national economies. This
can lead to contradictions related to the economic security of
countries, including food security. p
]
g
[5] Ricardo D (1955), Sochineniya [Writings]. Moscow: Gosudar-
stvennoe
izdatelstvo
politicheskoi
literatury. https://www.bibliofond.ru/view.aspx?id=122512 [6] Porter M (2016), Mezhdunarodnaya konkurentsiya. Konkurentnye
preimushchestva stran [International Competition. Competitive
Advantages of Countries]. Moscow: Alpina Publisher, 947. The identified problems require a new approach to the study of
export potential, its essence, structure, relations with other socio-
economic categories and terms, their systematization and compari-
sons. g
[7] Tracy M (1995), Selskoe khozyaistvo i prodovolstvie v ekonomike
razvitykh stran: Vvedenie v teoriyu, praktiku i politiku [Food and
Agriculture in a Market Economy: Introduction to Theory, Practice,
and Politics]. Saint Petersburg: Ekonomicheskaya shkola, 431. [8] Scherer FM & Ross D (1997), Struktura otraslevykh rynkov [Indus-
trial Market Structure and Economic Performance]. Moscow: Infra-
M, 698. 5. Conclusion [21] Wallerstein I (1989), The Modern World-System-III: The Second
Era of Great Expansion of the Capitalist World-Economy, 1730-
1840. San Diego: Academic Press. [1] Porter M (1993), Mezhdunarodnaya konkurentsiya [International
Competition]. Moscow: Mezhdunarodnye otnosheniya, 896.
[2] Guerson A, Parks J & Torrado M (2007), Export Structure and
Growth: A Detailed Analysis for Argentina. Policy Research Work-
ing Paper, № 4237. Washington, DC: Word Bank. [2] Guerson A, Parks J & Torrado M (2007), Export Structure and
Growth: A Detailed Analysis for Argentina. Policy Research Work-
ing Paper, № 4237. Washington, DC: Word Bank.
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A Truncated Fragment of Src Protein Kinase Generated by Calpain‐mediated Cleavage is a Mediator of Neuronal Death in Excitotoxicity.
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A Truncated Fragment of Src Protein Kinase Generated by
Calpain-mediated Cleavage Is a Mediator of Neuronal Death
in Excitotoxicity*□
S y
Received for publication,September 14, 2012, and in revised form, January 29, 2013 Published, JBC Papers in Press,February 1 M. Iqbal Hossain‡, Carli L. Roulston§¶, M. Aizuddin Kamaruddin‡, Percy W. Y. Chu‡, Dominic C. H. Ng‡1,
Gregory J. Dusting§¶, Jeffrey D. Bjorge, Nicholas A. Williamson‡, Donald J. Fujita, Steve N. Cheung**,
Tung O. Chan‡‡, Andrew F. Hill‡1, and Heung-Chin Cheng‡2 From the ‡Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia, the §O’Brien Institute, 42 Fitzroy Street,
Fitzroy, Victoria 3065, Australia, the ¶Centre for Eye Research Australia, University of Melbourne, Gisborne Street, East
Melbourne 3002, Australia, the Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of
Calgary, Health Sciences Centre, Calgary, Alberta T2N 4N1, Canada, the **Centre for Cellular and Molecular Biology, School
of Life and Environmental Sciences, Deakin University, Burwood, Victoria 3125, Australia, and the ‡‡Department of Medicine,
Center for Translational Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 nd: Abnormal regulation of calpains and Src contributes to stroke-induced brain damage. Background: Abnormal regulation of calpains and Src contributes to stroke-induced brain damage. Results: The abnormally activated calpains cleave Src to generate a truncated Src fragment capable Background: Abnormal regulation of calpains and Src contributes to stroke-induced brain damage. Results: The abnormally activated calpains cleave Src to generate a truncated Src fragment capable of directing neurons to
undergo cell death. Conclusion: A new function of Src in neuronal death is discovered. Significance: Prevention of calpain-mediated cleavage of Src is a potential therapeutic strategy to minimize stroke-induced
brain damage. suggests new therapeutic strategies with the potential to min-
imize brain damage in ischemic stroke. Excitotoxicity resulting from overstimulation of glutamate
receptors is a major cause of neuronal death in cerebral ische-
mic stroke. The overstimulated ionotropic glutamate recep-
tors exert their neurotoxic effects in part by overactivation of
calpains, which induce neuronal death by catalyzing limited
proteolysis of specific cellular proteins. Here, we report that
in cultured cortical neurons and in vivo in a rat model of focal
ischemic stroke, the tyrosine kinase Src is cleaved by calpains
at a site in the N-terminal unique domain. This generates a
truncated Src fragment of 52 kDa, which we localized pre-
dominantly to the cytosol. * This work was supported by project grants from the National Health and
Medical Research Council of Australia.
□
S This article contains supplemental Tables S1–S3 and Figs. S1–S4.
1 Supported by Future Fellowships of the Australian Research Council.
2 To whom correspondence should be addressed. Tel.: 61-3-83442254; Fax:
61-3-93481421; E-mail: heung@unimelb.edu.au. A Truncated Fragment of Src Protein Kinase Generated by
Calpain-mediated Cleavage Is a Mediator of Neuronal Death
in Excitotoxicity*□
S A cell membrane-permeable
fusion peptide derived from the unique domain of Src pre-
vents calpain from cleaving Src in neurons and protects
against excitotoxic neuronal death. To explore the role of the
truncated Src fragment in neuronal death, we expressed a
recombinant truncated Src fragment in cultured neurons and
examined how it affects neuronal survival. Expression of this
fragment, which lacks the myristoylation motif and unique
domain, was sufficient to induce neuronal death. Further-
more, inactivation of the prosurvival kinase Akt is a key step
in its neurotoxic signaling pathway. Because Src maintains
neuronal survival, our results implicate calpain cleavage as a
molecular switch converting Src from a promoter of cell sur-
vival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our dis-
covery of the neurotoxic action of the truncated Src fragment Constitutive activation and expression of Src contribute to
formation, survival, and progression of many types of cancer
(reviewed in Refs. 1 and 2). In neurons, Src exerts its neu-
rotrophic function by cooperating with Ret, the common
receptor of glial cell line-derived neurotrophic factor family
ligands to maintain their survival (3, 4). Thus, Src is a key
enzyme enhancing survival, growth, and/or proliferation of
neuronal and non-neuronal cells. In this paper, we provide evi-
dence that in addition to functioning as a prosurvival enzyme,
Src can act as a cell death mediator facilitating neuronal death
in excitotoxicity, a key process contributing to brain damage in
ischemic cerebral stroke and neurodegenerative diseases. In ischemic stroke, occlusion of a cerebral artery decreases
the supply of oxygen and glucose to brain cells located in the
region (referred to as the ischemic core) directly supplied by the
artery. Consequently, the cells die in a few minutes (5, 6). How-
ever, the area surrounding the ischemic core (referred to as the
ischemic penumbra) has a milder deficit of blood supply, due to
support from the collateral blood vessels. Neurons in ischemic
penumbra receive sufficient oxygen and nutrients to sustain
their survival (7, 8). Despite this, if restoration of the blood
supply is delayed, neurons in the ischemic penumbra eventually
die (5, 6). Thus, these neurons are the targets for neuroprotec-
tive therapy to reduce brain damage in stroke patients (5). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 14, pp. 9696–9709, April 5, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 14, pp. 9696–9709, April 5, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 14, pp. 9696–9709, April 5, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. EXPERIMENTAL PROCEDURES Primary Cortical Neuronal Culture and Treatment with
Glutamate, Glutamate Receptor Antagonists, and Calpain
Inhibitor—Primary cortical neurons were isolated from mouse
embryos collected at day 16 of gestation as detailed in the sup-
plemental material. They were maintained at 37 °C in 5% CO2
and 95% air in a humidified incubator in the neurobasal
medium containing 2.5% B-27, 0.25% GlutaMAX-1, and 1%
penicillin and streptomycin. Cells were maintained for 7 days
prior to treatment. The cultured neurons were treated with 100
M glutamate at the seventh day in vitro (DIV 7) to mimic
excitotoxicity. To examine the effect of other extracellular
agents on glutamate-treated neurons, the cultured neurons
were pretreated for 30 min with the NMDA receptor antagonist
MK801 (50 M), the GluN2B-containing NMDA receptor
antagonist ifenprodil (20 M), the antagonist for AMPA and
kainate receptor 6-cyano-7-nitroquinoxaline-2,3-dione (40
M), and calpain inhibitor calpeptin (20 M) prior to treatment
with glutamate. g
Overstimulation of NMDA receptors leads to calcium over-
load in the cytosol of the affected neurons. The sustained high
cytosolic calcium concentration constitutively activates cal-
pains, which contribute to neuronal death by catalyzing limited
proteolysis of specific cellular proteins, some of which are crit-
ical for neuronal survival (10). One notable example is the
metabotropic glutamate receptor mGluR1, which plays an
essential role in maintaining neuronal survival by activating the
prosurvival phosphatidylinositol 3-kinase (PI3K)/Akt signaling
pathway. mGluR1 employs its C-terminal tail to bind and acti-
vate PI3K (11). The overactivated calpains abolish the prosur-
vival function of mGluR1 by cleaving off its C-terminal tail. In
addition to blocking prosurvival signaling, calpains can induce
neuronal death by aberrantly regulating enzymes, such as
caspase-3 and cyclin-dependent kinase 5 (Cdk 5), to initiate
prodeath signaling in the affected neurons (12, 13). p
g
g
(
)
Members of the Src family of protein kinases (SFKs),3 such as
Src and Fyn, contribute to brain damage in stroke and
Alzheimer disease (14–18). Results of previous studies gave
conflicting conclusions on the role of Src and other SFKs in
neuronal death in stroke (15–17, 19). Using rodent models of
cerebral ischemia and hypoxia-ischemia (15–17), several
groups of researchers independently demonstrated that tar-
geted disruption of the src gene and inhibitors of SFKs signifi-
cantly reduce brain damage, suggesting that Src and/or other
SFKs contribute to neuronal death in stroke. EXPERIMENTAL PROCEDURES On the contrary,
treatment with inhibitors of SFKs induces cell death of cultured
primary cortical neurons (19), suggesting that SFK activity is
critical for neuronal survival. The conflicting suggestions aris-
ing from these studies are due to our lack of understanding of
the role of SFKs in neuronal death. Excitotoxicity, neuroinflam-
mation, and edema resulting from the breakdown of the blood
brain barrier and increased vascular permeability are the major
contributing factors of neuronal death in stroke patients (6). The main objective of our study reported in this paper is to
elucidate the role of Src in neuronal death in excitotoxicity. Our
results reveal that Src is aberrantly modified in neurons in
response to overstimulation of NMDA receptors. Furthermore,
such a modification of Src is a key event contributing to neuro-
nal death in excitotoxicity. Biochemical analyses revealed that
this modified form of Src induces neuronal death in part by
inhibiting the prosurvival kinase Akt. More importantly, in
contrast to the commonly accepted view of Src as a protoonco-
genic enzyme promoting cell growth and survival, we demon-
strate that Src is a key mediator of neuronal death in excitotox- Assays to Monitor Viability of Neurons—Neuronal cell viabil-
ity in culture was monitored by three different assays. The MTT
assay, which measures the rate of enzymatic cleavage of the
tetrazolium salt to purple formazan crystal by active mitochon-
drial reductase in viable neuronal cells, was used to monitor cell
viability. The extent of cell death of neurons in culture was
monitored also by the activity of lactate dehydrogenase released
by the damaged neurons to the culture medium (LDH release
assay). In addition to biochemical assays, live and dead neuro-
nal cells were monitored by incubation with calcein-AM and
EthD1 (ethidium homodimer-1), which stain live and dead
cells, respectively. Fluorescent microscopy reveals the number
of live calcein-containing neurons (green) and dead EthD1-
stained neurons (red). Details of the cell viability assays are
described in the supplemental material. Calpain Activity Assay—The calpain activity of neurons with
and without treatment with 100 M glutamate was measured by
monitoring the rate of cleavage of the fluorogenic substrate
Ac-Leu-Leu-Tyr-(7-amino-4-trifluoromethylcoumarin) by cal-
pain in the crude cell lysates as described in the supplemental
material. 3 The abbreviations used are: SFK, Src family protein kinase; DIV, day in vitro;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ET-1,
endothelin-1; SH2 and -3, Src homology 2 and 3, respectively; LDH, lactate
dehydrogenase; Myr, Src myristoylation signal. Neurotoxicity Caused by Calpain Cleavage of Src NMDA receptors are involved in maintenance of neuronal sur-
vival as well as other physiological functions, accounting for the
limited therapeutic potential of NMDA receptor antagonists in
reducing stroke-induced brain damage (reviewed in Ref. 9). Thus, deciphering the molecular mechanism governing neuro-
nal death resulting from overstimulation of NMDA receptors
will reveal more appropriate molecular targets for therapeutic
intervention to reduce brain damage in stroke. icity. Thus, future investigation of the molecular basis of
aberrant modification and the neurotoxic mechanism of Src
will identify new targets for therapeutic intervention to reduce
brain damage in stroke. A Truncated Fragment of Src Protein Kinase Generated by
Calpain-mediated Cleavage Is a Mediator of Neuronal Death
in Excitotoxicity*□
S One of the major causes of neuronal death in ischemic
penumbra is excitotoxicity, which is initiated mainly by over-
stimulation of N-methyl-D-aspartate (NMDA) receptors by
glutamate. However, in addition to inducing neuronal death, VOLUME 288•NUMBER 14•APRIL 5, 2013 9696
JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 14•APRIL 5, 2013 9696 Neurotoxicity Caused by Calpain Cleavage of Src The brain tissues were lysed with ice-cold lysis buffer con-
taining 50 mM Tris, pH 7.0, 1 mM EDTA, 5 mM EGTA, 1 mM
dithiothreitol, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 50
mM NaF, 40 mM sodium pyrophosphate, 0.5 mM Na3VO4, 50
mM -glycerophosphate, 0.2 mg/ml benzamidine, 0.1 mg/ml
phenymethylsufonyl fluoride, and a mixture of EDTA-free pro-
tease inhibitors and phosphatase inhibitors (Roche Applied
Science). The detailed procedures of surgical preparation and
stroke induction are given in the supplemental material. In Vitro Calpain Cleavage of Recombinant Src—Purified
recombinant Src (100 ng) was digested with different concen-
trations of calpain 1 (Calbiochem) in the digestion buffer (50
mM Tris-HCl, pH 7.4, 2 mM DTT, 30 mM NaCl, 10 mM CaCl2). The reaction mixture was incubated at 30 °C for 45 min. The
digested sample was analyzed by Western blotting with anti-Src
mAb327 and anti-Src mAb(2–17) antibodies. Tyrosine Kinase Activity Assay—Crude lysates of neurons
were diluted with the dilution buffer (25 mM Hepes, pH 7). Aliquots of the diluted lysates were assayed for Src family kinase
activity. For assay of Src kinase activity in the immune complex,
Src and Src mutant were immunoprecipitated from the crude
neuronal lysate with the anti-Src mAb327 antibody or anti-GFP
antibody bound to Protein A-Sepharose beads. The kinase
activity was measured by the rate of phosphorylation of the
Src-optimal peptide (AEEEAYGEAEAKKKK) as described in
the supplemental material. Subcellular Fractionation—Subcellular fractionation of pri-
mary cortical cells was performed based on the method
described by Bernocco et al. (22). First, cells were washed with
ice-cold PBS. Then buffer A (10 mM PIPES, pH 6.8, 100 mM
NaCl, 300 mM sucrose, 3 mM MgCl2, 5 mM EDTA, 0.015%
digitonin, protease and phosphatase inhibitor mixture (Roche
Applied Science)) was added to the attached neuronal cells and
gently shaken for 30 min at 4 °C. Digitonin from the buffer
creates pores in the plasma membrane by solubilizing choles-
terol. Therefore, all of the cytosolic proteins were extracted in
the solution without rupturing the plasma membrane (23). After incubation for 30 min, the supernatant was collected
carefully without detaching neuronal cells. This fraction con-
tains soluble cytosolic proteins. A second extraction was done
using buffer B (10 mM PIPES, pH 7.4, 100 mM NaCl, 300 mM
sucrose, 3 mM MgCl2, 3 mM EDTA, 0.5% Triton X-100). Neurotoxicity Caused by Calpain Cleavage of Src Myr-Akt contains a Src myristoylation signal (Myr) and a
hemagglutinin epitope fused to its N and C termini, respec-
tively (21). For the lentivirus directing the expression of the
transcriptional regulator, it was produced in HEK293FT cells
using the pLVX-Tet-on-Advance plasmid (Clontech). For
transduction, the cultured neurons (0.5 106 cells) at DIV 1
were incubated with a lentivirus generated with PLVX-Tight-
Puro plasmids containing the gene of interest and the lentivirus
directing the expression of the transcriptional regulator at a
multiplicity of infection of 20. The neurobasal medium was
changed after 24 h. At DIV 5, the recombinant Src proteins
were induced by the addition of doxycycline (1 mg/ml). As a
control, cells were also transduced with the lentivirus generated
by the GFP-pLVX-tight-puro plasmid. Experiments to analyze
the effects of expression of the recombinant proteins on the
signaling mechanism and survival of the transduced neurons
were performed at DIV 7. flurane (95% oxygen and 5% isoflurane). A 23-gauge stainless
steel guide cannula was stereotaxically implanted into the piri-
form cortex 2 mm dorsal to the right middle cerebral artery (0.2
mm anterior, 5.2 mm lateral, and 5.9 mm ventral) accord-
ing to the method of Sharkey et al. (24) and as described previ-
ously (25). The cannula was secured using acrylic dental
cement, and the rat was allowed to recover for 5 days prior to
stroke induction. Focal cerebral ischemia was induced in the
conscious rat by constriction of the right middle cerebral artery
with perivascular administration of endothelin-1 (60 pmol in 3
l of saline over 10 min). During stroke induction, counter-
clockwise and/or clockwise circling, clenching, and dragging of
the contralateral forepaw were observed, validating the correct
placement of the cannula. Stroke severity was scored based on
these responses, which have been shown to be a reliable predic-
tion of stroke outcome (25). Thus, this model allows induction
of stroke in a fully conscious rat and allows for accurate predic-
tion of stroke outcome in terms of volume of damage and neu-
rological deficits incurred based on observations made during
stroke induction. Control rats (n 2) underwent cannula
implantation and saline infusion instead of endothelin-1 to
demonstrate that saline injection itself does not induce vaso-
constriction and cerebral infarction, as described previously
(25). Rats were decapitated 3 h after stroke, and forebrains were
removed and frozen over liquid nitrogen until further process-
ing. Neurotoxicity Caused by Calpain Cleavage of Src Cells
were detached and lysed in buffer B by rocking for 30 min at
4 °C. The mixture was centrifuged at 8400 g for 10 min. The
supernatant contained the Triton X-100-soluble plasma mem-
brane proteins. Synthetic Peptides—All peptides were chemically synthe-
sized by Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid
phase peptide synthesis chemistry. Their sequences are listed in
supplemental Table S3. All except the TAT-Scrambled peptide
contain the sequences derived from the segment corresponding
to amino acids 1–79 of Src. For the TAT-Src and TAT-Scram-
bled peptides, they contain a 6-aminohexanoic acid linker con-
necting the TAT protein transduction domain motif (GRK-
KRRQRRRPQ) to the Src(49–79) segment or the scrambled
sequence with the same amino acid composition as the Src(49–
79) segment. Data Analysis—Densitometric analysis for the quantification
of the bands on immunoblots was performed using ImageJ soft-
ware. Statistical analyses were performed with the Statistical
Package for Social Sciences for Windows, version 16 (SPSS,
Inc., Chicago, IL). The data were reported as the means S.D.,
and the statistical significance was determined by Student’s t
test (two-tailed). p 0.05 was considered statistically signifi-
cant for all experiments. EXPERIMENTAL PROCEDURES Transduction of Primary Cortical Neurons with Lentivirus—
The genes encoding GFP fusion proteins of full-length Src
(Src-GFP), (G2A)Src-GFP carrying the myristoylation-defec-
tive G2A mutation, truncated Src lacking residues 1–80
(SrcN-GFP),
a
kinase-dead
version
of
truncated
Src
(SrcN(K/D)-GFP) carrying the K303M mutation, and consti-
tutively active Akt1 mutant (Myr-Akt) were inserted in the
pLVX-Tight-Puro vector (Clontech). They were used to gener-
ate the lentivirus directing expression of the genes of interest in
HEK293FT cells as detailed in the supplemental material. Upon
expression, Src-GFP and its mutants contain a Gly-Ser-Gly-Ser
linker followed by the enhanced green fluorescence protein
fused to the C terminus of Src and mutant Src sequences (20). APRIL 5, 2013•VOLUME 288•NUMBER 14 APRIL 5, 2013•VOLUME 288•NUMBER 14 9697 JOURNAL OF BIOLOGICAL CHEMISTRY Neurotoxicity Caused by Calpain Cleavage of Src 1E
shows that treatment with glutamate leads to degradation of
-fodrin to form the 150-kDa -fodrin fragments, further
confirming activation of calpains in the cultured cortical neu-
rons in response to stimulation by glutamate. Next, we used a combination of Edman sequencing, mass
spectrometry, and biochemical analysis to define the calpain
cleavage site in the unique domain of Src. We first determined
the N-terminal sequence of the truncated Src generated in vitro
by calpain 1-mediated cleavage of recombinant Src (supple-
mental Fig. S3A) by Edman sequencing. Despite several
attempts, we failed to obtain any sequence information, sug-
gesting that the truncated Src does not have a free N terminus. One possible reason for the lack of free N terminus is that the
nascent 52-kDa Src fragment generated by calpain 1-catalyzed
truncation contains a glutamine residue at its N terminus,
which readily undergoes cyclization. To overcome this techni-
cal problem, we employed mass spectrometry to determine the
calpain cleavage site. Briefly, purified intact Src and truncated
Src fragment (supplemental Fig. 3A) were exhaustively digested
with trypsin. The tryptic fragments generated were identified
by mass spectrometry. Supplemental Fig. S3B shows that both
intact and truncated Src proteins gave several detectable tryptic
fragments derived from the region encompassing amino acid
residues 79–461. In addition to these tryptic fragments, mass
spectrometric analysis also detected a tryptic fragment LFG-
GFNSSDTVTSPQR derived from residues 63–78 of the Src
sequence only in the tryptic digest of intact Src (supplemental
Fig. S3, B and C , and Tables S1 and S2), suggesting that the
calpain cleavage site resides in this segment of the unique
domain of Src. p
y g
Concomitant with the decrease in viability and calpain acti-
vation, Src undergoes limited proteolysis to form a truncated
fragment of 52 kDa in neurons as early as 5 min after overstimu-
lation with glutamate (Fig. 1, F and G). The amount of this
truncated Src fragment reaches a maximum level at 2 h of treat-
ment. Calpeptin, a cell-permeable calpain inhibitor effectively
suppresses formation of this truncated Src fragment (Fig. 1, F
and G). Together, these data indicate that the activated calpain
induces proteolysis of Src to form a truncated Src fragment in
neurons. Next, we used pharmacological agents to identify the
type of glutamate receptors involved in excitotoxicity-induced
cleavage of Src in neurons. Neurotoxicity Caused by Calpain Cleavage of Src Src (Fig. 2A). Whereas the mAb327 antibody detects both the
intact and truncated Src, the anti-Src(2–17) antibody only rec-
ognizes full-length Src (Fig. 2B), suggesting that calpain cleaves
Src in the unique domain. To ascertain if Src is a direct sub-
strate of calpain, we incubated purified recombinant Src with
calpain 1 in vitro and performed Western blotting of the pro-
teins in the reaction mixture with the two aforementioned anti-
bodies. Fig. 2C shows that purified recombinant Src is cleaved
by calpain 1 in vitro to produce the 52-kDa proteolytic fragment
recognized by anti-Src mAb327 antibody but not by the anti-
Src mAb(2–17) antibody, indicating that calpain 1 directly
cleaves Src at the unique domain. Further subcellular fraction-
ation studies reveal that the truncated Src resides predomi-
nantly in cytosol (supplemental Fig. S2), suggesting that cal-
pain-mediated cleavage removes the N-terminal myristoyl
moiety that anchors Src to the plasma membrane. system to investigate the role of Src in neuronal death in exci-
totoxicity. After isolation from embryonic day 15–16 mouse
embryos, the cortical neurons were cultured for 7 days prior to
treatment with glutamate. The expression levels of Src and
NMDA receptor subunits GluN1, GluN2A, and GluN2B
undergo time-dependent increase from day 1 to day 7.4 In addi-
tion, at DIV 7, neurite outgrowths of the cultured neurons are
extensive.4 Cortical neurons at DIV7 were used to study the
effects of glutamate overstimulation on neuronal survival. system to investigate the role of Src in neuronal death in exci-
totoxicity. After isolation from embryonic day 15–16 mouse
embryos, the cortical neurons were cultured for 7 days prior to
treatment with glutamate. The expression levels of Src and
NMDA receptor subunits GluN1, GluN2A, and GluN2B
undergo time-dependent increase from day 1 to day 7.4 In addi-
tion, at DIV 7, neurite outgrowths of the cultured neurons are
extensive.4 Cortical neurons at DIV7 were used to study the
effects of glutamate overstimulation on neuronal survival. g
Treatment of cortical neurons with glutamate at DIV 7
induces a time-dependent decrease in viability and increase in
calpain activity (Fig. 1, A–D). Because calpains cleave the cyto-
skeletal protein -fodrin to form the 145- and 150-kDa proteo-
lytic fragments, the appearance of the -fodrin fragments of
150 kDa has been used as a marker of overactivation of cal-
pains in neurons undergoing excitotoxic cell death (26). Fig. 4 M. I. Hossain, D. C. H. Ng, A. F. Hill, and H.-C. Cheng, manuscript in
preparation. RESULTS Induction of Stroke in Rats by Stereotaxic Injection of
Endothelin-1—Male hooded Wistar rats aged 10–12 weeks
(300–350 g) were anesthetized with ketamine/xylazine (75:10
mg/kg intraperitoneally) and maintained by inhalation of iso- A Truncated Src Fragment of 52 kDa Is Formed in Neurons
upon Overstimulation by Glutamate—We used cultured pri-
mary cortical neurons treated with glutamate (100 M) as the 9698 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 14•APRIL 5, 2013 Neurotoxicity Caused by Calpain Cleavage of Src Neurotoxicity Caused by Calpain Cleavage of Src A, MTT assay to monitor neuronal viability at the designated time points. The amount of
med (as measured by absorbance at 570 nm) by the viable neurons in the untreated culture is assigned as 100%. The vi
of treatment is presented as a percentage of the untreated cells (data represented as mean S.D. (error bars), n 5; *, p 0
assay to monitor the extent of neuronal cell damage. The activity of LDH released to the culture medium from the dam
h glutamate at each time point relative to that obtained in the medium of the untreated neurons is presented (data represe
05, Student’s t test). C, the viable neurons were stained with calcein (green), and the damaged neurons were stained with eth
Control, untreated neurons. Glutamate (4 h) and Glutamate (8 h), neurons treated with glutamate for 4 and 8 h, respectiv
cein-stained cells to that of ethidium homodimer-1-stained cells of the control and glutamate-treated neurons (data represe
05,Student’sttest).D,time-dependentchangesofcalpainactivityofprimarycorticalneuronsaftertreatmentwith100Mgl
lysates of the untreated neurons is assigned to be 100% (data represented as mean S.D., n 4; *, p 0.05, Student’s
drin by calpains to form proteolytic fragments of 150 kDa in neurons treated with glutamate and in the presence or abse
n blots (WB) of lysates from neurons treated with either glutamate alone or glutamate and calpeptin for different times and
d
ti t b li
tib d
Th
ti t b li i
ti it
i
l
d
l
di
t
l
G th i
t
icity Caused by Calpain Cleavage of Src Neurotoxicity Caused by Calpain Cleavage of Src Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 1. Overstimulation of glutamate receptors of primary cortical neurons induces cell death, calpain activation, and truncation of Src. Cortical
neurons at DIV 7 were incubated with 100 M glutamate for 8 h. Their viability, their calpain activity, and the structural integrity of neuronal Src and -fodrin
were analyzed at the designated time intervals. A, MTT assay to monitor neuronal viability at the designated time points. The amount of the reaction product
formazan formed (as measured by absorbance at 570 nm) by the viable neurons in the untreated culture is assigned as 100%. The viability of neurons at
different times of treatment is presented as a percentage of the untreated cells (data represented as mean S.D. Neurotoxicity Caused by Calpain Cleavage of Src 6-Cyano-7-nitroquinoxaline-2,3-di-
one, an AMPA/kainate receptor antagonist, fails to abolish cal-
pain-mediated truncation of Src, whereas the NMDA receptor
antagonist MK801 completely blocks the formation of the trun-
cated fragment (supplemental Fig. S1). These results indicate
that NMDA receptor is the major glutamate receptor govern-
ing calpain-mediated truncation of Src in cultured neurons. Stimulation of the GluN2B subunit-containing NMDA
receptors, which are located mostly in extrasynaptic sites,
contributes to brain damage in stroke (27). We therefore
examined the effect of ifenprodil, an antagonist of the
GluN2B subunit-containing NMDA receptor, on neuronal
Src truncation by calpain in response to glutamate over-
stimulation. As shown in supplemental Fig. S1, ifenprodil
suppresses formation of the truncated Src, suggesting
involvement of GluN2B-containing NMDA receptor in cal-
pain-mediated truncation of Src in neurons. Another approach we used to define the calpain cleavage site
in Src is by employing a peptide inhibition assay. We synthe-
sized four peptides (Fig. 3A and supplemental Table S3) derived
from the unique domain of Src and examined their ability to
inhibit calpain 1-catalyzed cleavage of Src in vitro. Among
them, only the synthetic Src(49–79) peptide, which contains
the segment of residues 63–78, could block cleavage of Src by
calpain 1 (Fig. 3B), providing further support to our claim that
the calpain cleavage site resides in this segment (residues
63–78) of the Src unique domain. Indeed, in agreement with the
difference in molecular sizes of intact Src (60 kDa) and the
truncated Src fragment (52 kDa), calpain-mediated cleavage
at this segment is expected to reduce the molecular size of Src
by 8 kDa. Calpain Directly Cleaves Src at the Unique Domain to Gen-
erate the Truncated Src Fragment in Neurons—To define the
cleavage site in Src, we first compared the Western blots of
crude neuronal lysates probed with two site-specific anti-Src
antibodies: (i) the anti-Src mAb327 antibody with epitope at the
SH3 domain, and (ii) the anti-Src mAb(2–17) antibody direct-
ing against the segment corresponding to amino acids 2–17 of Because the sequence of the segment encompassing resi-
dues 63–78 in the Src unique domain shares little homology 9699 APRIL 5, 2013•VOLUME 288•NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY rstimulation of glutamate receptors of primary cortical neurons induces cell death, calpain activation, and trunca
V 7 were incubated with 100 M glutamate for 8 h. Their viability, their calpain activity, and the structural integrity of neuro
at the designated time intervals. Neurotoxicity Caused by Calpain Cleavage of Src (error bars), n 5; *, p 0.05, Student’s t test). B, LDH release assay to monitor the extent of neuronal cell damage. The activity of LDH released to the culture medium from the damaged neurons upon
treatment with glutamate at each time point relative to that obtained in the medium of the untreated neurons is presented (data represented as mean S.D.,
n 5; *, p 0.05, Student’s t test). C, the viable neurons were stained with calcein (green), and the damaged neurons were stained with ethidium homodimer-1
(EthD-1) (red). Control, untreated neurons. Glutamate (4 h) and Glutamate (8 h), neurons treated with glutamate for 4 and 8 h, respectively. Right, ratio of the
number of calcein-stained cells to that of ethidium homodimer-1-stained cells of the control and glutamate-treated neurons (data represented as mean S.D.,
n5;*,p0.05,Student’sttest).D,time-dependentchangesofcalpainactivityofprimarycorticalneuronsaftertreatmentwith100Mglutamate.Thecalpain
activity in the lysates of the untreated neurons is assigned to be 100% (data represented as mean S.D., n 4; *, p 0.05, Student’s t test). E, cleavage of
neuronal -fodrin by calpains to form proteolytic fragments of 150 kDa in neurons treated with glutamate and in the presence or absence of calpeptin (20
M). F, Western blots (WB) of lysates from neurons treated with either glutamate alone or glutamate and calpeptin for different times and probed with anti-Src
mAb327 antibody or anti-tubulin antibody. The anti-tubulin immunoreactivity signals are used as loading controls. G, the immunoreactive signals (in densi-
tometric units) of intact Src (60 kDa) and the truncated Src fragment (52 kDa) (data represented as mean S.D., n 5; *, p 0.05, Student’s t test). in Fig. 2, D and E, although treatment with glutamate
induces a time-dependent increase in the amount of the
52-kDa truncated Src fragment in neurons, no cleavage of with sequences in other SFK members, such as Fyn and Yes,
which are also expressed in neurons, it is logical to predict
that cleavage by calpain is an event unique to Src. As shown VOLUME 288•NUMBER 14•APRIL 5, 2013 C, Western blots of purified
recombinant Src treated with different amounts (in activity units) of calpain in vitro probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. D and
E, Western blots of crude lysates of neurons treated with glutamate for different times (0.5–6 h) and probed with anti-Fyn and anti-Yes antibodies. FIGURE 3. Effects of Src(1–20), Src(20–40), Src(40–49), and Src(49–79) peptides on the calpain 1-mediated cleavage of recombinant Src in vitro. A,
sequenceoftheN-terminalmyristoylationmotifandpartoftheuniquedomainofhumanSrc.ThesegmentsfromwhichSrc(1–20),Src(20–40),Src(40–49),and
Src(49–79) were derived are indicated. B, the four synthetic peptides at concentrations ranging from 2 to 10 M were incubated with the reaction mixture
containingSrcandcalpain1.Asacontrol,calpeptinwasaddedtoinhibitcalpain1activity.FormationofthetruncatedSrcfragmentbycalpain1wasmonitored
by Western blotting (WB) with anti-Src mAb327 antibody. FIGURE 3. Effects of Src(1–20), Src(20–40), Src(40–49), and Src(49–79) peptides on the calpain 1-mediated cleavage of recombinant Src in vitro. A,
sequenceoftheN-terminalmyristoylationmotifandpartoftheuniquedomainofhumanSrc.ThesegmentsfromwhichSrc(1–20),Src(20–40),Src(40–49),and
Src(49–79) were derived are indicated. B, the four synthetic peptides at concentrations ranging from 2 to 10 M were incubated with the reaction mixture
containingSrcandcalpain1.Asacontrol,calpeptinwasaddedtoinhibitcalpain1activity.FormationofthetruncatedSrcfragmentbycalpain1wasmonitored
by Western blotting (WB) with anti-Src mAb327 antibody. Cerebral Ischemia Induces Production of a 52-kDa Truncated
Src Fragment in a Rat Model of Stroke—We aimed to confirm if
excitotoxicity induced by cerebral ischemia can cause trunca-
tion of Src in an animal model. The endothelin-1 (ET-1) model
of stroke in conscious rats was adopted to investigate the
expression of truncated Src following cerebral ischemia with
reperfusion. In this model, a guide cannula was stereotaxically
implanted in the piriform cortex of the rat (Fig. 4A). Following
recovery, ET-1 was then applied locally to the middle cerebral
artery of the conscious rat to induce focal ischemia with reper-
fusion that results in reproducible damage to the ipsilateral ter-
ritory supplied by the middle cerebral artery (Fig. 4B). We
found that following stroke with a 3-h recovery, a 52-kDa trun-
cated Src fragment appeared predominantly in the ipsilateral
side of the cerebral hemisphere (Fig. 4C). The results indicate
that brain injury caused by middle cerebral artery vasoconstric- Fyn and Yes was detected, indicating that cleavage by calpain
is a unique regulatory property of Src in neurons. SFK inhibitors significantly reduce brain damage in rodent
models of stroke and hypoxia (15–17, 28–31), implying that
SFKs are aberrantly activated and that phosphorylation of spe-
cific cellular proteins by the activated SFKs contributes to neu-
ronal death in excitotoxicity. We therefore sought to ascertain
how glutamate overstimulation affects neuronal Src activity. As
shown in supplemental Fig. VOLUME 288•NUMBER 14•APRIL 5, 2013 9700 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 14•APRIL 5, 2013 Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 2. Calpain directly cleaves Src at its unique domain to generate the 52-kDa truncated fragment. A, schematic of Src functional domains. Tyr-416
(Y416) and Tyr-527 (Y527) are the conserved autophosphorylation site in the kinase domain and the C-terminal regulatory tyrosine phosphorylation site,
respectively. The locations of epitopes of the two anti-Src antibodies mAb(2–17) and mAb327 are indicated by arrows. B, Western blots (W.B.) of crude lysates
of neurons treated with glutamate for different times (0.5–6 h) probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. C, Western blots of purified
recombinant Src treated with different amounts (in activity units) of calpain in vitro probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. D and
E, Western blots of crude lysates of neurons treated with glutamate for different times (0.5–6 h) and probed with anti-Fyn and anti-Yes antibodies. y
y
p
g FIGURE 2. Calpain directly cleaves Src at its unique domain to generate the 52-kDa truncated fragment. A, schematic of Src functional domains. Tyr-416
(Y416) and Tyr-527 (Y527) are the conserved autophosphorylation site in the kinase domain and the C-terminal regulatory tyrosine phosphorylation site,
respectively. The locations of epitopes of the two anti-Src antibodies mAb(2–17) and mAb327 are indicated by arrows. B, Western blots (W.B.) of crude lysates
of neurons treated with glutamate for different times (0.5–6 h) probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. C, Western blots of purified
recombinant Src treated with different amounts (in activity units) of calpain in vitro probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. D and
E, Western blots of crude lysates of neurons treated with glutamate for different times (0.5–6 h) and probed with anti-Fyn and anti-Yes antibodies. FIGURE 2. Calpain directly cleaves Src at its unique domain to generate the 52-kDa truncated fragment. A, schematic of Src functional domains. Tyr-416
(Y416) and Tyr-527 (Y527) are the conserved autophosphorylation site in the kinase domain and the C-terminal regulatory tyrosine phosphorylation site,
respectively. The locations of epitopes of the two anti-Src antibodies mAb(2–17) and mAb327 are indicated by arrows. B, Western blots (W.B.) of crude lysates
of neurons treated with glutamate for different times (0.5–6 h) probed with anti-Src mAb327 and anti-Src mAb(2–17) antibodies. Neurotoxicity Caused by Calpain Cleavage of Src Thus, it mimics the action of the truncated Src frag-
ment formed by calpain cleavage in neurons upon overstimu-
lation of glutamate receptors. For the control experiments, we
transduced neurons with lentivirus directing expression of
GFP, GFP fusion proteins of full-length Src (Src-GFP),
(G2A)Src-GFP carrying the myristoylation-defective G2A
mutation, and the kinase-dead SrcN(K/D)-GFP carrying the
K303M mutation (Fig. 6A). FIGURE 4. Endothelin 1 (ET-1)-induced cerebral ischemia induces prote-
olysis of Src kinase in brain cells to form a 52-kDa truncated fragment. A, schematic depicting the ET-1 model of stroke. Vasoconstriction of the mid-
dle cerebral artery by applying ET-1 through the guide cannula generates
ischemic damage in the parietal, insular, and frontal cortex, as well as in the
dorsolateral and corpus striatum. The red arrows indicate some of the regions
adversely affected by the ET-1 treatment. B, hematoxylin and eosin-stained
image of a representative brain section 0.2 mm from Bregma close to the site
of ET-1 infusion during stroke induction. The ischemic infarct (dotted black
line) could be seen evolving as soon as 3 h after stroke induction. C, anti-Src mAb327 blot of lysate of brain tissues collected in the control rat
and three stroke rats. WB, Western blot; Ips, ipsilateral; Con, contralateral; L,
left hemisphere; R, right hemisphere. tion and subsequent reduction in cerebral blood flow in rats is
associated with the formation of a 52-kDa truncated Src frag-
ment similar to that observed in cultured neurons upon over-
stimulation of glutamate receptors. Prevention of Calpain-mediated Truncation of Src Alleviates
Neuronal Death Induced by Overstimulation of Glutamate
Receptors—To investigate if prevention of calpain-mediated
truncation of Src in neurons is cytoprotective, we generated a
cell membrane-permeable fusion peptide containing the pro-
tein transduction domain of the HIV-1 TAT protein at the
N-terminal portion, a 6-aminohexanoic acid linker and the
Src(49–79) segment capable of blocking cleavage of Src in vitro
(Fig. 3 and supplemental Table S3). This peptide (referred to as
the TAT-Src peptide) inhibited calpain cleavage of recombi-
nant Src in vitro (Fig. 5A) and blocked calpain-mediated trun-
cation of Src in glutamate-overstimulated neurons (Fig. 5B). In
contrast, this peptide has no effect on calpain-mediated cleav-
age of -fodrin (Fig. 5C), suggesting that the TAT-Src specifi-
cally blocks calpain-mediated cleavage of Src but not the other
calpain substrates in neurons. More importantly, as shown by
results of the MTT and LDH assays (Fig. Neurotoxicity Caused by Calpain Cleavage of Src Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 4. Endothelin 1 (ET-1)-induced cerebral ischemia induces prote-
olysis of Src kinase in brain cells to form a 52-kDa truncated fragment. A, schematic depicting the ET-1 model of stroke. Vasoconstriction of the mid-
dle cerebral artery by applying ET-1 through the guide cannula generates
ischemic damage in the parietal, insular, and frontal cortex, as well as in the
dorsolateral and corpus striatum. The red arrows indicate some of the regions
adversely affected by the ET-1 treatment. B, hematoxylin and eosin-stained
image of a representative brain section 0.2 mm from Bregma close to the site
of ET-1 infusion during stroke induction. The ischemic infarct (dotted black
line) could be seen evolving as soon as 3 h after stroke induction. C, anti-Src mAb327 blot of lysate of brain tissues collected in the control rat
and three stroke rats. WB, Western blot; Ips, ipsilateral; Con, contralateral; L,
left hemisphere; R, right hemisphere. protect against glutamate-induced neuronal death (Fig. 5),
indicating that the cytoprotective action of the TAT-Src pep-
tide is reliant on the structural integrity of the Src(49–79)
segment. g
Expression of an Active Recombinant N-terminally Trun-
cated Src Mutant Induces Neurons to Undergo Cell Death—Be-
cause the 52-kDa Src fragment generated by calpain-mediated
truncation contains the intact SH3, SH2, and kinase domains
and the C-terminal tail, it can potentially employ these domains
to bind and phosphorylate specific cellular proteins in neurons
to facilitate neuronal death. To examine the validity of this
notion, we first attempted to isolate the truncated Src fragment
in crude neuronal cell lysates and measure its kinase activity
and autophosphorylation status. However, although the anti-
Src mAb327 antibody readily detects both the intact Src and
truncated Src fragment in Western blots, it fails to immunopre-
cipitate the truncated Src fragment from the crude neuronal
lysate (supplemental Fig. S4B). A different approach was there-
fore adopted to monitor the kinase activity and the role of the
truncated Src fragment in excitotoxic neuronal death, we gen-
erated a lentivirus to direct expression of a recombinant GFP
fusion protein of truncated Src mutant (referred to as SrcN-
GFP) under the control of doxycycline in neurons (Fig. 6A). The
SrcN-GFP protein, with the GFP moiety attached to the
C-terminal tail of the truncated Src, lacks the N-terminal seg-
ment encompassing the myristoylation motif and unique
domain. VOLUME 288•NUMBER 14•APRIL 5, 2013 S4, the activity of intact Src in neu-
rons undergoes changes in two phases following glutamate
treatment; an initial phase (0–30 min) of increased activity is
followed by a subsequent phase (30 min or longer) of decreased
activity that is reduced below basal levels. Because intact Src is
involved in maintaining neuronal survival (3, 4, 32), future
investigation may focus on examining if the transient activation
of intact Src is a cytoprotective event in excitotoxicity. APRIL 5, 2013•VOLUME 288•NUMBER 14 9701 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 5, 2013•VOLUME 288•NUMBER 14 Neurotoxicity Caused by Calpain Cleavage of Src Neurotoxicity Caused by Calpain Cleavage of Src 5, D and E) and im-
ages of neurons after staining with calcein-AM/ethidium
homodimer-1 (Fig. 5, F and G), TAT-Src peptide at 20 M pro-
vides significant protection against neuronal death induced by
glutamate overstimulation. In contrast, the TAT-Scrambled
peptide containing the TAT-6-aminohexanoic acid segment
and the scrambled Src(49–79) segment (supplemental Table
S3), however, cannot block calpain-mediated cleavage of Src or Fig. 6A shows that Src-GFP, SrcN-GFP, and its mutant
were expressed at levels comparable with that of endogenous
Src in the transduced neurons. Both Src-GFP and SrcN-GFP
were autophosphorylated at Tyr-416 at levels similar to that of
intact endogenous Src (Fig. 6A). Although the K303M muta-
tion abolishes its kinase activity, the kinase-dead SrcN(K/
D)-GFP mutant was autophosphorylated at Tyr-416, albeit
at a level much lower than those of endogenous Src and
Src-GFP (Fig. 6A). Autophosphorylation of Src is an inter-
molecular event (33, 34). Furthermore, Cooper and Mac-
Auley (33) demonstrated trans-autophosphorylation of a
kinase-dead Src mutant at Tyr-416 by a catalytically active
Src mutant when both mutants were co-expressed in cells. Thus, it is likely that the kinase-dead SrcN(K/D)-GFP
mutant expressed in the transduced neurons was trans-au-
tophosphorylated by the active endogenous Src. To determine the activities of the recombinant Src-GFP and
mutants, we immunoprecipitated them from crude lysates of
the transduced neurons using the anti-GFP antibody and
assayed for the kinase activity associated with the immunopre-
cipitates using the Src-optimal peptide. Fig. 6B shows that the
kinase-dead SrcN(K/D)-GFP exhibited negligible activity,
whereas the Src-GFP and SrcN-GFP exhibit similar kinase
activities. Our data suggest that deletion of the N-terminal myr- 9702 VOLUME 288•NUMBER 14•APRIL 5, 2013 JOURNAL OF BIOLOGICAL CHEMISTRY Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 5. Blockade of calpain-dependent truncation of Src protects against neuronal death in excitotoxicity. Cultured cortical neurons at DIV 7
were treated with glutamate (100 M) for 8 h in the presence or absence of TAT-Src peptide or TAT-Scrambled peptide. The neurons were analyzed for
calpain cleavage of Src and fodrin and cell viability. A, anti-Src Western blot (WB) of purified recombinant Src after treatment with calpain in vitro in the
presence of 0–14 M Tat-Src peptide (C, control). B, anti-Src Western blot of lysates of neurons with and without treatment with 100 M glutamate in the
presence of 0–20 M TAT-Src peptide or 20 M TAT-Scrambled peptide. Neurotoxicity Caused by Calpain Cleavage of Src C, Western blot analysis of the cell lysate to monitor the effect of TAT-Src (20 M)
on calpain cleavage of fodrin in neurons with and without glutamate treatment. D, MTT assay measuring viability of neurons after 8-h treatment under
different conditions. E, LDH release assay to monitor the extent of cellular damage of neurons after 8 h treatment under different conditions. F,
calcein-AM/ethidium homodimer-1 staining of untreated neurons and neurons treated with glutamate (100 M) for 15 h in the presence and absence
of 20 M TAT-Src peptide or 20 M TAT-Scrambled peptide. Green fluorescence, calcein-AM-stained viable neurons. Red fluorescence, ethidium
homodimer-1-stained damaged neurons. G, the ratios of calcein-stained cells versus the ethidium homodimer-1-stained cells in the control sample and
neurons treated with glutamate in the presence or absence of 20 M TAT-Src peptide or 20 M TAT-Scrambled peptide (data represented as mean S.D. (error bars), n 5; *, p 0.05, Student’s t test). y
y
p
g FIGURE 5. Blockade of calpain-dependent truncation of Src protects against neuronal death in excitotoxicity. Cultured cortical neurons at DIV 7
were treated with glutamate (100 M) for 8 h in the presence or absence of TAT-Src peptide or TAT-Scrambled peptide. The neurons were analyzed for
calpain cleavage of Src and fodrin and cell viability. A, anti-Src Western blot (WB) of purified recombinant Src after treatment with calpain in vitro in the
presence of 0–14 M Tat-Src peptide (C, control). B, anti-Src Western blot of lysates of neurons with and without treatment with 100 M glutamate in the
presence of 0–20 M TAT-Src peptide or 20 M TAT-Scrambled peptide. C, Western blot analysis of the cell lysate to monitor the effect of TAT-Src (20 M)
on calpain cleavage of fodrin in neurons with and without glutamate treatment. D, MTT assay measuring viability of neurons after 8-h treatment under
different conditions. E, LDH release assay to monitor the extent of cellular damage of neurons after 8 h treatment under different conditions. F,
calcein-AM/ethidium homodimer-1 staining of untreated neurons and neurons treated with glutamate (100 M) for 15 h in the presence and absence
of 20 M TAT-Src peptide or 20 M TAT-Scrambled peptide. Green fluorescence, calcein-AM-stained viable neurons. Red fluorescence, ethidium
homodimer-1-stained damaged neurons. Neurotoxicity Caused by Calpain Cleavage of Src Expression of Src-GFP and mutants was induced by doxycycline at DIV 5. A, at DIV 7, neurons were collected, and the crude lysates were analyzed by Western
blotting (W.B.) with anti-Src mAb327 antibody and anti-Src-Tyr(P)-416 antibody, which monitors autophosphorylation of Src family kinases at the consensus
site in the activation loop (e.g. Tyr-418 of Src or Tyr-424 of neuronal Src). Control, untransduced neurons. The kinase-dead SrcN(K/D)-GFP mutant was
generated by replacement of the conserved Lys-297 (or Lys-303 of neuronal Src) with methionine. B, the recombinant Src-GFP and mutants were immuno-
precipitated from lysates of the transduced neurons, and the kinase activities associated with the immunoprecipitates were determined. Error bars, S.D. Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 6. Expression, autophosphorylation levels, and kinase activities of Src-GFP and its mutants in transduced primary cortical neurons. Primary
cortical neurons were transduced with lentivirus directing the expression of Src-GFP, (G2A)Src-GFP, SrcN-GFP, and the kinase-dead SrcN(K/D)-GFP at DIV 1. Expression of Src-GFP and mutants was induced by doxycycline at DIV 5. A, at DIV 7, neurons were collected, and the crude lysates were analyzed by Western
blotting (W.B.) with anti-Src mAb327 antibody and anti-Src-Tyr(P)-416 antibody, which monitors autophosphorylation of Src family kinases at the consensus
site in the activation loop (e.g. Tyr-418 of Src or Tyr-424 of neuronal Src). Control, untransduced neurons. The kinase-dead SrcN(K/D)-GFP mutant was
generated by replacement of the conserved Lys-297 (or Lys-303 of neuronal Src) with methionine. B, the recombinant Src-GFP and mutants were immuno-
precipitated from lysates of the transduced neurons, and the kinase activities associated with the immunoprecipitates were determined. Error bars, S.D. FIGURE7.ExpressionofatruncatedSrcmutantlackingthemyristoylationmotifanduniquedomaininducesneuronaldeath.Neuronsweretransduced
with lentivirus directing expression of Src-GFP, SrcN-GFP, the kinase-dead SrcN(K/D)-GFP, and (G2A)Src-GFP mutants at DIV 1. Expression of the recombi-
nant proteins was induced by doxycycline at DIV 5. Effects of their expression were monitored at DIV 7. A, MTT assay to monitor the viability of the cultured
neurons (n 5; *, p 0.05). The mitochondrial reductase activity of the viable neurons in “Control” is arbitrarily assigned as 100%. B, activity of LDH released
from the damaged neurons to the cultured medium was monitored as a measure of the extent of neuronal cell death (n 5; *, p 0.05). The activity in control
is arbitrarily assigned as 100%. Neurotoxicity Caused by Calpain Cleavage of Src G, the ratios of calcein-stained cells versus the ethidium homodimer-1-stained cells in the control sample and
neurons treated with glutamate in the presence or absence of 20 M TAT-Src peptide or 20 M TAT-Scrambled peptide (data represented as mean S.D. (error bars), n 5; *, p 0.05, Student’s t test). and a large portion of the unique domain (Fig. 2 and supple-
mental Fig. S3). Our results strongly suggest that this endoge-
nous 52-kDa Src fragment is a neurotoxic mediator contribut-
ing to neuronal death in excitotoxicity. istoylation motif and the unique domain does not affect the
activity of Src in neurons. Thus, we predict that in neurons
overstimulated by glutamate, intact Src and the 52-kDa trun-
cated Src fragment generated by calpain cleavage exhibit simi-
lar kinase activities. Intriguingly, induced expression of SrcN-
GFP leads to a significant reduction in cell survival (Fig. 7) and
an increase in the number of damaged neurons (Fig. 7C, inset). In contrast, expression of GFP or Src-GFP has no effect on the
viability of the cultured neurons (Fig. 7). These results indicate
that SrcN-GFP is neurotoxic because its expression alone
induces neuronal death. Thus, Src acquires the capability to
induce neuronal death when both the myristoylation motif and
unique domain are deleted. Similar to SrcN-GFP, the 52-kDa
Src fragment generated by calpain cleavage in neurons under-
going excitotoxic cell death also lacks the myristoylation motif Presumably, SrcN-GFP induces neuronal death by phos-
phorylating a specific subset of cellular proteins. In light of this,
we generated the kinase-dead SrcN(K/D)-GFP mutant with
the conserved lysine (equivalent to Lys-303 in the intact neuro-
nal Src) essential for ATP binding mutated to methionine. This
mutation abolishes the neurotoxic ability of the truncated Src
mutant (Fig. 7). The myristoyl group attached to the N terminus of Src is
essential for its targeting to the plasma membrane. Previously,
mutation of the conserved Gly-2 essential for myristoylation
has been shown to abolish the ability of the constitutively active APRIL 5, 2013•VOLUME 288•NUMBER 14 9703 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 5, 2013•VOLUME 288•NUMBER 14 Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 6. Expression, autophosphorylation levels, and kinase activities of Src-GFP and its mutants in transduced primary cortical neurons. Primary
cortical neurons were transduced with lentivirus directing the expression of Src-GFP, (G2A)Src-GFP, SrcN-GFP, and the kinase-dead SrcN(K/D)-GFP at DIV 1. Neurotoxicity Caused by Calpain Cleavage of Src C, the effect of expression of Src-GFP, SrcN-GFP, SrcN(K/D)-GFP, and (G2A)Src-GFP on neuronal survival is monitored by
staining with ethidium homodimer-1 (EthD-1; red fluorescence), which shows the damaged neurons. Inset, numbers of ethidium homodimer-1-positive cells in
the control and transduced neurons (n 5; *, p 0.05). Error bars, S.D. FIGURE7.ExpressionofatruncatedSrcmutantlackingthemyristoylationmotifanduniquedomaininducesneuronaldeath.Neuronsweretransduced
with lentivirus directing expression of Src-GFP, SrcN-GFP, the kinase-dead SrcN(K/D)-GFP, and (G2A)Src-GFP mutants at DIV 1. Expression of the recombi-
nant proteins was induced by doxycycline at DIV 5. Effects of their expression were monitored at DIV 7. A, MTT assay to monitor the viability of the cultured
neurons (n 5; *, p 0.05). The mitochondrial reductase activity of the viable neurons in “Control” is arbitrarily assigned as 100%. B, activity of LDH released
from the damaged neurons to the cultured medium was monitored as a measure of the extent of neuronal cell death (n 5; *, p 0.05). The activity in control
is arbitrarily assigned as 100%. C, the effect of expression of Src-GFP, SrcN-GFP, SrcN(K/D)-GFP, and (G2A)Src-GFP on neuronal survival is monitored by
staining with ethidium homodimer-1 (EthD-1; red fluorescence), which shows the damaged neurons. Inset, numbers of ethidium homodimer-1-positive cells in
the control and transduced neurons (n 5; *, p 0.05). Error bars, S.D. v-Src mutant to induce cellular transformation of cultured
fibroblasts (35, 36). To examine if the cytotoxic activity of the
truncated Src fragment is solely due to the removal the myris-
toyl moiety by calpain, we transduced neurons with the lentivi-
rus that direct the expression of the non-myristoylated
(G2A)Src-GFP mutant. As shown in Fig. 6, recombinant v-Src mutant to induce cellular transformation of cultured
fibroblasts (35, 36). To examine if the cytotoxic activity of the
truncated Src fragment is solely due to the removal the myris-
toyl moiety by calpain, we transduced neurons with the lentivi-
rus that direct the expression of the non-myristoylated
(G2A)Src-GFP mutant. As shown in Fig. 6, recombinant (G2A)Src-GFP mutant was expressed at a level similar to that of
endogenous Src in neurons, and it was autophosphorylated and
exhibited kinase activity. However, in contrast to SrcN-GFP,
(G2A)Src-GFP does not induce neuronal death (Fig. 7), indicat-
ing that removal of just the myristoyl moiety is insufficient to
convert Src to a neurotoxic mediator. VOLUME 288•NUMBER 14•APRIL 5, 2013 VOLUME 288•NUMBER 14•APRIL 5, 2013 9704 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 14•APRIL 5, 2013 FIGURE 8. Glutamate overstimulation or expression of the cytotoxic trun-
cated Src fragment induces a significant decrease in Akt phosphoryla-
tion at serine 473 in neurons. A, time-dependent changes of Akt phosphor-
ylation in neurons treated with 100 M glutamate. B, effects of expression of
Src-GFP, (G2A)Src-GFP mutant, the kinase-dead SrcN(K/D)-GFP mutant, and
SrcN-GFP on neuronal Akt phosphorylation level. Neurons were transduced
with the Src-GFP or its mutants at DIV 1 and then induced with doxycycline
induction at DIV 5 for 48 h. Crude lysates of the untreated neurons (Control)
and the transduced neurons were probed with anti-Akt and anti-Akt-Ser(P)-
473 antibodies. Akt blots are representative of three separate experiments. C, effects of TAT-Src peptide on Akt phosphorylation in glutamate-treated
neurons. Crude lysates were prepared from untreated neurons and neurons
treated with glutamate for 4 and 8 h in the presence and absence of TAT-Src
peptide (12–20 M). The lysates were probed with anti-Akt and anti-Akt-
Ser(P)-473 antibodies. Neurotoxicity Caused by Calpain Cleavage of Src Neurons were transduced
with the Src-GFP or its mutants at DIV 1 and then induced with doxycycline
induction at DIV 5 for 48 h. Crude lysates of the untreated neurons (Control)
and the transduced neurons were probed with anti-Akt and anti-Akt-Ser(P)-
473 antibodies. Akt blots are representative of three separate experiments. C, effects of TAT-Src peptide on Akt phosphorylation in glutamate-treated
neurons. Crude lysates were prepared from untreated neurons and neurons
treated with glutamate for 4 and 8 h in the presence and absence of TAT-Src
peptide (12–20 M). The lysates were probed with anti-Akt and anti-Akt-
Ser(P)-473 antibodies. FIGURE 8. Glutamate overstimulation or expression of the cytotoxic trun-
cated Src fragment induces a significant decrease in Akt phosphoryla-
tion at serine 473 in neurons. A, time-dependent changes of Akt phosphor- FIGURE 8. Glutamate overstimulation or expression of the cytotoxic trun-
cated Src fragment induces a significant decrease in Akt phosphoryla-
tion at serine 473 in neurons. A, time-dependent changes of Akt phosphor-
ylation in neurons treated with 100 M glutamate. B, effects of expression of
Src-GFP, (G2A)Src-GFP mutant, the kinase-dead SrcN(K/D)-GFP mutant, and
SrcN-GFP on neuronal Akt phosphorylation level. Neurons were transduced
with the Src-GFP or its mutants at DIV 1 and then induced with doxycycline
induction at DIV 5 for 48 h. Crude lysates of the untreated neurons (Control)
and the transduced neurons were probed with anti-Akt and anti-Akt-Ser(P)-
473 antibodies. Akt blots are representative of three separate experiments. C, effects of TAT-Src peptide on Akt phosphorylation in glutamate-treated
neurons. Crude lysates were prepared from untreated neurons and neurons
treated with glutamate for 4 and 8 h in the presence and absence of TAT-Src
peptide (12–20 M). The lysates were probed with anti-Akt and anti-Akt-
Ser(P)-473 antibodies. Neurotoxicity Caused by Calpain Cleavage of Src that calpain-induced truncation of Src is a key step in directing
neuronal death and inactivation of Akt kinase. FIGURE 8. Glutamate overstimulation or expression of the cytotoxic trun-
cated Src fragment induces a significant decrease in Akt phosphoryla-
tion at serine 473 in neurons A time dependent changes of Akt phosphor Expression of a Constitutively Active Akt Mutant Reduces the
Neurotoxic Action of the Truncated Src Mutant in Neurons—
Because the recombinant neurotoxic SrcN-GFP inactivates
endogenous Akt, we tested whether expression of the constitu-
tively active Akt mutant protects against neuronal death. Luo et
al. (37) reported that expression of a constitutively active myr-
istoylated mutant of Akt in neurons protects against neuronal
death in excitotoxicity. Thus, we generated a lentivirus to direct
the expression of myristoylated Akt1 (Myr-Akt) with a myris-
toylation motif at the N terminus and a hemagglutinin tag at the
C terminus. After viral transduction, both SrcN-GFP and
Myr-Akt were expressed in transduced neurons (Fig. 9A)
Importantly, Myr-Akt is expressed at a similar level as endoge-
nous Akt. Finally, MTT assay, assay for the activity of LDH
released from the damaged neurons, and staining of damaged
neurons with ethidium homodimer-1 all revealed that the cell
viability of neurons co-expressing Myr-Akt with SrcN-GFP is
significantly higher than that of neurons expressing SrcN-
GFP alone (Fig. 9, B–D). Our data suggest that the constitu-
tively active Myr-Akt attenuates the neurotoxic effect of
SrcN-GFP in neurons, and inactivation of Akt is a key step in
the neurotoxic pathway of SrcN-GFP. The anti-Akt-Ser(P)-473 blot of the neuronal lysates (Fig. 9A) shows that both Myr-Akt and endogenous Akt are phos-
phorylated at Ser-473 to a similar level regardless of the co-ex-
pression of SrcN-GFP. However, in neurons expressing
SrcN-GFP, phosphorylation of endogenous Akt at Ser-473 is
much reduced. This data is in agreement with the findings
shown in Fig. 8 that neuronal death is associated with reduced
phosphorylation of Akt at Ser-473. FIGURE 8. Glutamate overstimulation or expression of the cytotoxic trun-
cated Src fragment induces a significant decrease in Akt phosphoryla-
tion at serine 473 in neurons. A, time-dependent changes of Akt phosphor-
ylation in neurons treated with 100 M glutamate. B, effects of expression of
Src-GFP, (G2A)Src-GFP mutant, the kinase-dead SrcN(K/D)-GFP mutant, and
SrcN-GFP on neuronal Akt phosphorylation level. DISCUSSION Overactivated Calpains Target Multiple Protein Substrates to
Induce Neuronal Death—Overactivation of calpains contrib-
utes to neuronal death in excitotoxicity (38). The overactivated
calpains catalyze proteolysis of many cellular proteins, leading
to their aberrant regulation or premature degradation (39). In
addition to inhibition of calpain cleavage of Src, blocking cal-
pain-mediated cleavage of NCX (sodium-calcium exchanger)
(40), STEP (striatal enriched phosphatase) (41), mGluR-1
(metabotropic glutamate receptor -1) (11), TRPC6 (transient
receptor potential canonical-6) (42), and calcineurin (43, 44)
with cell-permeable peptide inhibitors has been documented to
provide protection against neuronal death in excitotoxicity. These calpain substrates previously proven to be critical to
excitotoxicity can be categorized into two groups. The first
group, including NCX, TRPC6, STEP, and mGluR-1, consists
of key signaling proteins in the neuroprotective signaling path-
ways. Their cleavage by calpain either abolishes their neuropro-
tective functions or leads to their degradation. The second
group contains signaling molecules that acquire the neurotoxic
activities upon cleavage by calpain. An example of this group of
calpain substrates is the protein phosphatase calcineurin (45–
47). Calpain-mediated cleavage irreversibly activates calcineu-
rin by deletion of its autoinhibitory domain. This constitutively
active truncated calcineurin induces neuronal death by dephos- Akt Inactivation Is Associated with Neuronal Death Caused
by Glutamate Treatment and Expression of the Neurotoxic
Truncated Src Mutant—The PI3K/Akt signaling pathway is
known to maintain neuronal survival. We confirmed earlier
findings that glutamate-induced neurotoxicity is associated
with reduced phosphorylation at the essential Akt regulatory
site (Ser-473 in Akt1; Fig. 8A) (37). To test whether calpain-
induced Src truncation also caused Akt inactivation, we trans-
duced neurons with GFP fusion proteins of WT-Src, calpain-
truncated Src (SrcN), and myristoylation signal-mutated Src
((G2A)Src). Fig. 8C shows that SrcN-GFP, but not Src-GFP
and (G2A)Src-GFP, induced a significant reduction of Akt
phosphorylation at Ser-473. In addition, the kinase-dead
mutant of SrcN-GFP (K303M) failed to induce both neuro-
toxic ability (Fig. 7) and Akt inactivation (Fig. 8B). Finally, TAT-
Src peptide treatment prevented glutamate-induced inactiva-
tion of Akt in neurons (Fig. 8C). Together, these data indicate APRIL 5, 2013•VOLUME 288•NUMBER 14 9705 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 5, 2013•VOLUME 288•NUMBER 14 Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 9. The constitutively active Myr-Akt1 mutant antagonizes the cytotoxic action of SrcN-GFP in neurons. DISCUSSION A, Western blots (WB) of crude lysate of
transduced neurons showing the expression of endogenous Src, recombinant SrcN-GFP, endogenous Akt and Myr-Akt, and phosphorylation level of Akt and
Myr-Akt1 at Ser-473. Because Myr-Akt contains a hemagglutinin tag at its C terminus, its expression was detected by anti-HA blotting. B, cell viability of the
transducedneuronsmeasuredbyMTTassay(top)andactivityofLDHreleasedfromthedamagedneuronstotheculturedmediumwasmonitoredasameasure
of the extent of neuronal cell death (bottom). C, phase-contrast and ethidium homodimer-1-stained images of the untreated (Control) and the transduced
neurons. D, number of ethidium homodimer-1-stained cells in the control and transduced neuron (data represented as mean S.D. (error bars), n 5; *, p
0.05, Student’s t test). Neurotoxicity Caused by Calpain Cleavage of Src FIGURE 9. The constitutively active Myr-Akt1 mutant antagonizes the cytotoxic action of SrcN-GFP in neuron r-Akt1 mutant antagonizes the cytotoxic action of SrcN-GFP in neurons. A, Western blots (WB) of crude lysate of FIGURE 9. The constitutively active Myr-Akt1 mutant antagonizes the cytotoxic action of SrcN-GFP in neurons. A, Western blots (WB) of crude lysate of
transduced neurons showing the expression of endogenous Src, recombinant SrcN-GFP, endogenous Akt and Myr-Akt, and phosphorylation level of Akt and
Myr-Akt1 at Ser-473. Because Myr-Akt contains a hemagglutinin tag at its C terminus, its expression was detected by anti-HA blotting. B, cell viability of the
transducedneuronsmeasuredbyMTTassay(top)andactivityofLDHreleasedfromthedamagedneuronstotheculturedmediumwasmonitoredasameasure
of the extent of neuronal cell death (bottom). C, phase-contrast and ethidium homodimer-1-stained images of the untreated (Control) and the transduced
neurons. D, number of ethidium homodimer-1-stained cells in the control and transduced neuron (data represented as mean S.D. (error bars), n 5; *, p
0 05, Student’s t test) FIGURE 10. A model depicting the neurotoxic consequence of calpain-
mediated cleavage of Src in neurons in excitotoxicity. The overstimulated
NMDA receptor allows massive influx of calcium to overactivate calpain in
neurons. The activated calpain cleaves a number of cellular proteins. Calpain-
mediated cleavage removes the N-terminal myristoyl group and a significant
portionoftheuniquedomain,releasingthe52-kDatruncatedSrcfragmentto
the cytosol. This truncated Src fragment facilitates neuronal death in part by
inactivating Akt. By blocking calpain cleavage of Src, the TAT-Src peptide
alleviatesneuronaldeathinducedbyoverstimulationofglutamatereceptors. phorylating BAD, huntingtin, and NFAT (47). Src belongs to
the second group of calpain substrates because calpain-medi-
ated cleavage generates the neurotoxic truncated Src fragment
capable of directing neuronal death. DISCUSSION Future investigation
should focus on understanding how the truncated Src fragment
and the truncated calcineurin fragment generated by calpain
cleavage cooperate to induce neuronal death in excitotoxicity. The Paradox: Src Contributes to Maintenance of Survival and
Death of Neurons—Src plays a key role in maintaining neuronal
survival. It mediates the prosurvival action of the thyroid hor-
mone T3 receptor TR1 and glial cell-derived neurotrophic
factor receptor by activating PI3K to maintain neuronal sur-
vival (3, 4, 32). Furthermore, treatment of cultured cortical neu-
rons with the Src kinase inhibitors PP2 and SU6650 promotes
apoptotic cell death (19), supporting the notion that Src kinase
activity is critical to maintenance of neuronal survival. In a pre-
liminary study, we transduced primary cortical neurons at DIV
1 with the lentivirus directing expression of Src shRNA to
knock down Src expression in neurons. Our data demonstrate
that suppression of Src expression induces cell death of the
cultured neurons,4 further supporting the indispensable role of
intact Src in maintaining neuronal survival. In light of these
findings, our findings presented here therefore reveal a para-
dox; Src is a promoter of cell survival as well as a mediator of cell
death (refer to the model shown in Fig. 10). By acting as the
molecular switch converting Src from a cell survival promoter
to a neurotoxic enzyme, calpain allows Src to perform these FIGURE 10. A model depicting the neurotoxic consequence of calpain-
mediated cleavage of Src in neurons in excitotoxicity. The overstimulated
NMDA receptor allows massive influx of calcium to overactivate calpain in
neurons. The activated calpain cleaves a number of cellular proteins. Calpain-
mediated cleavage removes the N-terminal myristoyl group and a significant
portionoftheuniquedomain,releasingthe52-kDatruncatedSrcfragmentto
the cytosol. This truncated Src fragment facilitates neuronal death in part by
inactivating Akt. By blocking calpain cleavage of Src, the TAT-Src peptide
alleviatesneuronaldeathinducedbyoverstimulationofglutamatereceptors. FIGURE 10. A model depicting the neurotoxic consequence of calpain-
mediated cleavage of Src in neurons in excitotoxicity. The overstimulated
NMDA receptor allows massive influx of calcium to overactivate calpain in
neurons. The activated calpain cleaves a number of cellular proteins. Calpain-
mediated cleavage removes the N-terminal myristoyl group and a significant
portionoftheuniquedomain,releasingthe52-kDatruncatedSrcfragmentto
the cytosol. This truncated Src fragment facilitates neuronal death in part by
inactivating Akt. By blocking calpain cleavage of Src, the TAT-Src peptide
alleviatesneuronaldeathinducedbyoverstimulationofglutamatereceptors. paradoxically conflicting functions in neurons. Neurotoxicity Caused by Calpain Cleavage of Src cultured cortical neurons. Furthermore, Liu et al. (55) reported
that Src plays a key role in stimulating proliferation of the new-
born brain microvascular endothelial cells and perivascular
astrocytes, which are essential for resolution of edema and
repair of damaged blood brain barrier during the recovery
phase of ischemic or hemorrhagic stroke (55). In light of the
roles of intact Src in maintaining neuronal survival and repair-
ing the damaged blood brain barrier following stroke, inhibitors
suppressing Src kinase activity are not suitable for use to mini-
mize brain damage in patients suffering from stroke because
they may adversely affect neuronal survival and the repair of
blood brain barrier during the recovery phase of ischemic
stroke. Because TAT-Src peptide can alleviate neuronal death
by preventing calpain-mediated truncation of Src in neurons
(Fig. 4), prevention of calpain cleavage of Src is a better thera-
peutic strategy than inhibition of Src kinase activity to reduce
brain damage in patients suffering from stroke (refer to the
model shown in Fig. 10). Calpains play conflicting roles in pre-
venting and initiating cell death. In addition to contributing to
neuronal death, calpains are critical for the remodeling process
in neural regeneration following stroke (56). Thus, broad spec-
trum calpain inhibitors are not suitable therapeutics for reduc-
ing brain damage in stroke patients because they can inhibit
calpains in the neural regenerative process. In light of this, small
molecule inhibitors that mimic the TAT-Src peptide to selec-
tively prevent formation of the neurotoxic Src fragment by cal-
pain are better therapeutics for the treatment of stroke patients. Future investigation to elucidate the structural basis of preven-
tion of calpain cleavage of Src by the TAT-Src peptide may
benefit the development of these small molecule calpain inhib-
itors. In light of the failures in recent clinical trials of drugs to
protect against brain damage in stroke patients (57, 58), our
discoveries illustrate how investigation into the molecular basis
of neuronal death in excitotoxicity benefits the search for new
molecular targets with the potential to minimize brain damage
in ischemic stroke and neurodegenerative diseases. exerts its neurotoxic action by phosphorylating specific cellular
proteins in cytosol to induce neuronal death. p
y
PI3K maintains neuronal survival by activating Akt (48–50). Recently, Jo et al. (51) discovered a cell-permeable small mole-
cule capable of activating Akt in cytosol. DISCUSSION As the cleavage
generates the truncated Src fragment residing predominantly
in the cytosol, we propose that the truncated Src fragment 9706 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 14•APRIL 5, 2013 Neurotoxicity Caused by Calpain Cleavage of Src We thank Joan
Brugge for the gift of the hybridoma for generation of the mAb327
anti-Src antibody and Danny Hatters, Terry Mulhern, and Sevgi Irte-
gun for the Src-GFP plasmids from which we generated the PLVX-
tight-puro-Src-GFP and PLVX-tight-puro-SrcN-GFP for the pro-
duction of some of the lentivirus. We are grateful for the technical
assistance provided by Daisy Lio and Ivan Ng in biochemical and
microscopic analyses. It is intriguing that (G2A)Src-GFP, albeit lacking the myris-
toyl group for anchorage to the plasma membrane, is unable to
exhibit neurotoxic action when expressed in the cultured cor-
tical neurons. Because the unique domain in this mutant
remains intact, our results suggest that the unique domain con-
tains the motifs that constrain the neurotoxic action of Src and
that these motifs are removed upon calpain cleavage. 1. Bjorge, J. D., Jakymiw, A., and Fujita, D. J. (2000) Selected glimpses into the
activation and function of Src kinase. Oncogene 19, 5620–5635 Acknowledgments—We thank Harshal Nandurkar, Hong-Jian Zhu,
Marie Bogoyevitch, Isao Matsuura, and Hemant Paudel for advice
and for reviewing the manuscript prior to submission. We thank Joan
Brugge for the gift of the hybridoma for generation of the mAb327
anti-Src antibody and Danny Hatters, Terry Mulhern, and Sevgi Irte-
gun for the Src-GFP plasmids from which we generated the PLVX-
tight-puro-Src-GFP and PLVX-tight-puro-SrcN-GFP for the pro-
duction of some of the lentivirus. We are grateful for the technical
assistance provided by Daisy Lio and Ivan Ng in biochemical and
microscopic analyses. 3. Encinas, M., Crowder, R. J., Milbrandt, J., and Johnson, E. M., Jr. (2004)
Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate
neuronal survival. J. Biol. Chem. 279, 18262–18269 Neurotoxicity Caused by Calpain Cleavage of Src Using this compound,
they demonstrated in cultured cortical neurons and in vivo in a
mouse model of stroke that activation of Akt can suppress exci-
totoxicity and reduces stroke-induced neuronal death (51). In
agreement with their findings, we demonstrated that treatment
with excess glutamate and expression of the recombinant trun-
cated Src (SrcN-GFP) induce a significant decrease in phos-
phorylation of Akt at Ser-473 in cultured cortical neurons (Fig. 8). Furthermore, the neurotoxic effect of SrcN-GFP is signif-
icantly reduced by co-expression of the constitutively active
Myr-Akt1 mutant in neurons (Fig. 9). Taken together, our
results suggest that the truncated Src contributes to neuronal
death at least in part by antagonizing the PI3K/Akt prosurvival
signaling pathway. Relevant to this, Akt employs the PXXP
motif near its C terminus to bind to the SH3 domain of Src in
both Madin-Darby canine kidney and HEK293 cells. The bind-
ing facilitates its phosphorylation by Src and activation by PI3K
(52). Schmid and Bohn (53) demonstrated that Src, Akt, and
-arrestin 2 form stable complexes in mouse frontal cortex and
cultured mouse cortical neurons, and Src in the complex facil-
itates Akt activation in response to stimulation of the serotonin
receptor 5-HT2AR. Given the significant role played by Akt in
maintenance of neuronal survival and our finding that the trun-
cated Src mutant SrcN-GFP induces neuronal death and Akt
inactivation, future investigation to decipher how calpain
cleavage affects the functional interplay between Src and Akt in
neurons will shed light on the neurotoxic mechanism of the
truncated Src fragment. Because the kinase-dead K303M mutation abolishes the neu-
rotoxicity of SrcN-GFP, it is logical to predict that the 52-kDa
truncated Src fragment and SrcN-GFP exert their neurotoxic
action by phosphorylating specific cellular proteins in neurons. Identifying the cellular proteins that are selectively phosphoryl-
ated by the 52-kDa truncated Src fragment and SrcN-GFP is
an avenue to decipher the neurotoxic mechanism of the trun-
cated Src fragment. Given that the kinase-dead SrcN(K/D)-
GFP mutant fails to inactivate neuronal Akt, some of these cel-
lular proteins preferentially phosphorylated by the 52-kDa
truncated Src fragment and SrcN-GFP could be upstream
regulators of neuronal Akt. Acknowledgments—We thank Harshal Nandurkar, Hong-Jian Zhu,
Marie Bogoyevitch, Isao Matsuura, and Hemant Paudel for advice
and for reviewing the manuscript prior to submission. 2. Frame, M. C. (2004) Newest findings on the oldest oncogene. How acti-
vated src does it. J. Cell Sci. 117, 989–998 Neurotoxicity Caused by Calpain Cleavage of Src (2004) Src family kinase inhibitor PP2 reduces focal ischemic
brain injury. Acta Neurol. Scand. 110, 175–179 12. O’Hare, M. J., Kushwaha, N., Zhang, Y., Aleyasin, H., Callaghan, S. M.,
Slack, R. S., Albert, P. R., Vincent, I., and Park, D. S. (2005) Differential
roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic
and excitotoxic neuronal death. J. Neurosci. 25, 8954–8966 j
y
31. Mukaiyama, H., Nishimura, T., Kobayashi, S., Komatsu, Y., Kikuchi, S.,
Ozawa, T., Kamada, N., and Ohnota, H. (2008) Novel pyrazolo[1,5-
a]pyrimidines as c-Src kinase inhibitors that reduce IKr channel blockade. Bioorg. Med. Chem. 16, 909–921 13. Hara, H., Friedlander, R. M., Gagliardini, V., Ayata, C., Fink, K., Huang, Z.,
Shimizu-Sasamata, M., Yuan, J., and Moskowitz, M. A. (1997) Inhibition
of interleukin 1-converting enzyme family proteases reduces ischemic
and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. U.S.A. 94,
2007–2012 32. Cao, X., Kambe, F., Yamauchi, M., and Seo, H. (2009) Thyroid-hormone-
dependent activation of the phosphoinositide 3-kinase/Akt cascade re-
quires Src and enhances neuronal survival. Biochem. J. 424, 201–209 14. Ittner, L. M., Ke, Y. D., Delerue, F., Bi, M., Gladbach, A., van Eersel, J.,
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English
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The many facets of brain aging
|
eLife
| 2,020
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cc-by
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Copyright Nyberg and Wa˚hlin.
This article is distributed under the
terms of the Creative Commons
Attribution License, which permits
unrestricted use and redistribution
provided that the original author and
source are credited. Applying big-data analytic techniques to brain images from 18,707
individuals is shedding light on the influence of aging on the brain. Applying big-data analytic techniques to brain images from 18,707
individuals is shedding light on the influence of aging on the brain. LARS NYBERG AND ANDERS WA˚ HLIN phenotypes represents a different aspect of
brain structure or function, such as how specific
regions are connected or the structure of certain
cortical areas (Figure 1A). phenotypes represents a different aspect of
brain structure or function, such as how specific
regions are connected or the structure of certain
cortical areas (Figure 1A). Related research article Smith SM, Elliott
LT, Alfaro-Almagro F, McCarthy P, Nichols
TE, Douaud G, Miller KL. 2020. Brain aging
comprises many modes of structural and
functional change with distinct genetic and
biophysical associations. eLife 9:e52677. DOI: 10.7554/eLife.52677 Further computational analyses were con-
ducted to combine the phenotypes that vary
together with age across subjects. In total, 62 of
these groups (termed ‘modes’) were estab-
lished, each potentially representing a biological
process affected by aging: for instance, changes
in
white-matter
microstructure
could
reflect
degeneration of axons. These modes were then
weighted to describe how strongly each is pres-
ent in an individual. W W
e are not all equal when it comes to
brain aging: while some people man-
age to maintain well-preserved cog-
nitive function into old age, others do not
(Nyberg et al., 2012). Brain-imaging studies
have
attempted
to
capture
brain
aging
by
exploring age-related changes to specific struc-
tures
and
different
kinds
of
brain
tissue
(Good et al., 2001; Walhovd et al., 2005). But
a more recent approach has been to use one or
more
brain-imaging
techniques
to
define
a
global,
single
brain-age
for
each
individual
(Franke et al., 2010). This estimate is then used
to derive a measure called brain-age delta,
which represents the gap between the age
expected from the brain status and the actual
age of the individual. Now, in eLife, Stephen
Smith (University of Oxford) and colleagues
report a refined brain-age approach that might
better represent how aging affects the biological
processes of the brain (Smith et al., 2020). The next step was to try to use the modes to
assess how an individual would fare in compari-
son to others, and to examine whether these
new measures could correlate with genetic fac-
tors. The modes were first grouped to create an
‘all-in-one’ brain-age delta for each participant,
a measure that turned out not to be associated
with a genetic signature. INSIGHT Applying big-data analytic techniques to brain images from 18,707
individuals is shedding light on the influence of aging on the brain. Refining big-data analytic approaches to reveal the many facets of brain aging. (A) Smith et al. used a
technique called independent component analysis (ICA) to analyze MRI and fMRI data on brain structure,
connectivity or activity from more than 18,000 individuals over the age of 45. This enabled them to identify 62
modes. Most of these modes co-varied with age across the sample, thus potentially reflecting biological processe
affected by aging. (B) Schematic matrix in which each row represents an individual and each column represents a
mode. The color scale represents the brain-age delta, the difference between the actual age of the individual and
what age would have been expected for this person given the value of the mode. (C) The 62 modes can be
grouped into six mode-clusters, such as one which captures the microstructure of brain white-matter. (D) Smith
et al. were able to relate the brain-age deltas for specific modes and the mode-clusters to various phenotypes (for
instance health, genetics and cognition). Modes
ICA
Connectivity
Activity
Structure
A. B C. A. ICA Cognition
Genes
Health
D. D. B. Modes
ICA
B. D
Brain-age delta
-5 years
+5 years
Subjects Modes
Cognition
Genes
Health
B. D. Brain-age delta
-5 years
+5 years
Subjects Figure 1. Refining big-data analytic approaches to reveal the many facets of brain aging. (A) Smith et al. used a
technique called independent component analysis (ICA) to analyze MRI and fMRI data on brain structure,
connectivity or activity from more than 18,000 individuals over the age of 45. This enabled them to identify 62
modes. Most of these modes co-varied with age across the sample, thus potentially reflecting biological processes
affected by aging. (B) Schematic matrix in which each row represents an individual and each column represents a
mode. The color scale represents the brain-age delta, the difference between the actual age of the individual and
what age would have been expected for this person given the value of the mode. (C) The 62 modes can be
grouped into six mode-clusters, such as one which captures the microstructure of brain white-matter. (D) Smith
et al. were able to relate the brain-age deltas for specific modes and the mode-clusters to various phenotypes (for
instance health, genetics and cognition). The mode-cluster that was associated with the
greatest aging effect, for example, linked cogni-
tive processing speed with brain patterns like
ventricular volume and white-matter microstruc-
ture. Applying big-data analytic techniques to brain images from 18,707
individuals is shedding light on the influence of aging on the brain. Next, each specific
mode was used to create a brain-age delta that
assesses the gap between the participant and
the
population
for
this
particular
mode
(Figure 1B). Most of these ‘mode-specific’ brain-
age deltas turned out to be associated with the
genetic makeup of the individuals. This demon-
strates that superimposing distinct brain aging
processes in an all-in-one brain-age may obscure
biological relations. While the individual modes might be related
to specific biological processes, Smith et al. used data-driven optimization to group the 62
modes into coarser ‘mode-clusters’ (Figure 1C). The six clusters that emerged helped to under-
stand
larger
patterns
of
age-related
brain
changes, and how these relate to other ‘non-
imaging’ variables such as health parameters. From a sample of 21,407 participants of the
UK Biobank study, Smith et al. reported the data
of 18,707 individuals over the age of 45 whose
brains have been imaged using the same MRI
and fMRI protocols. The team then generated
3913 imaging-derived phenotypes: each of the Nyberg and Wa˚hlin. eLife 2020;9:e56640. DOI: https://doi.org/10.7554/eLife.56640 1 of 3 Insight Insight In Imaging
The many facets of brain aging Modes
ICA
Connectivity
Cognition
Genes
Health
Activity
Structure
A. B. C. D. Brain-age delta
-5 years
+5 years
Subjects
Mode-clusters
brain white-matter
Figure 1. Refining big-data analytic approaches to reveal the many facets of brain aging. (A) Smith et al. used a
technique called independent component analysis (ICA) to analyze MRI and fMRI data on brain structure,
connectivity or activity from more than 18,000 individuals over the age of 45. This enabled them to identify 62
modes. Most of these modes co-varied with age across the sample, thus potentially reflecting biological processe
affected by aging. (B) Schematic matrix in which each row represents an individual and each column represents a
mode. The color scale represents the brain-age delta, the difference between the actual age of the individual and
what age would have been expected for this person given the value of the mode. (C) The 62 modes can be
grouped into six mode-clusters, such as one which captures the microstructure of brain white-matter. (D) Smith
et al. were able to relate the brain-age deltas for specific modes and the mode-clusters to various phenotypes (fo
instance health, genetics and cognition). Modes
ICA
Connectivity
Cognition
Genes
Health
Activity
Structure
A. B. C. D. Brain-age delta
-5 years
+5 years
Subjects
Mode-clusters
brain white-matter
Figure 1. Applying big-data analytic techniques to brain images from 18,707
individuals is shedding light on the influence of aging on the brain. Diabetes, hypertension, and smoking were
all risk factors distributed across mode-clusters,
suggesting that different aspects of vascular
health influence brain aging through different
biological processes. age delta, deltas defined from modes and
mode-clusters were associated with genetics
(Figure 1D). But what will the optimal ‘unit’ of
brain aging turn out to be in the end? Should it
be six (as the mode-clusters suggest), 62 (from
the analyses of the 3913 imaging-derived phe-
notypes), or another figure altogether? The rele-
vant number will depend on the type of imaging
used in a specific dataset – for example, if it
includes both MRI and PET images or MRI alone. Unique brain-age deltas may be revealing a gap
between chronological and actual brain age –
thus stressing the vast heterogeneity in the older
population – but the work by Smith et al. repre-
sents the next generation of analytic methods This work highlights the challenge in deter-
mining the optimal balance between integration
and diversification in studies of brain aging,
where a single brain-age is at the extreme end
of integration. An argument in favor of diversifi-
cation is the fact that, unlike the all-in-one brain- Nyberg and Wa˚hlin. eLife 2020;9:e56640. DOI: https://doi.org/10.7554/eLife.56640 2 of 3 Insight Insight Published 16 April 2020
Imaging
The many facets of brain aging Published 16 April 2020 that can help to decode the complexity of brain
aging. In the future, genetics and cognition may
even be considered in the early stages of analy-
sis, when distinct modes are initially computed. References Bzdok D, Yeo BTT. 2017. Inference in the age of big
data future perspectives on neuroscience. NeuroImage
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PMID: 22542563 Lars Nyberg is in the Department of Radiation
Sciences, the Umea˚ Center for Functional Brain
Imaging, and the Department of Integrative Medical
Biology, Umea˚ University, Umea˚, Sweden
lars.nyberg@umu.se Smith SM, Elliott LT, Alfaro-Almagro F, McCarthy P,
Nichols TE, Douaud G, Miller KL. 2020. Brain aging
comprises many modes of structural and functional
change with distinct genetic and biophysical
associations. eLife 9:e52677. DOI: https://doi.org/10. Nyberg and Wa˚hlin. eLife 2020;9:e56640. DOI: https://doi.org/10.7554/eLife.56640 Competing interests:
The authors declare that no
competing interests exist. References 7554/eLife.52677 y
g
https://orcid.org/0000-0002-3367-1746
Anders Wa˚hlin is in the Department of Radiation
Sciences and the Umea˚ Center for Functional Brain
Imaging, Umea˚ University, Umea˚, Sweden https://orcid.org/0000-0002-3367-1746
Anders Wa˚hlin is in the Department of Radiation
Sciences and the Umea˚ Center for Functional Brain
Imaging, Umea˚ University, Umea˚, Sweden
https://orcid org/0000-0001-6784-1945 Walhovd KB, Fjell AM, Reinvang I, Lundervold A, Dale
AM, Eilertsen DE, Quinn BT, Salat D, Makris N, Fischl
B. 2005. Effects of age on volumes of cortex, white
matter and subcortical structures. Neurobiology of
Aging 26:1261–1270. DOI: https://doi.org/10.1016/j. Competing interests:
The authors declare that no
competing interests exist. 3 of 3
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Modelling the electricity generation dynamics of Ghana. A Structural VAR Approach
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Modelling the electricity generation dynamics of
Ghana. A Structural VAR Approach Sampson Agyapong Atuahene ( samxin8@gmail.com ) Research Article Keywords: Ghana, VAR, Electricity, Energy, Conventional energy, RES
Posted Date: August 4th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1736061/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License: This work is licensed under a Creative Commons Attribution 4.0 International License. License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Modelling the electricity generation dynamics of Ghana. A Structural VAR Approach
1
2
Sampson Agyapong Atuahene1*, David Alemzero2
3
1&2School of Economics and Finance -Jiangsu University- 301 Xuefu Rd, Jingkou District, Zhenjiang,
4
Jiangsu, China. 5
Email: Samxin8@gmail.com1: awelingazure@gmail.com2
6
Corresponding Author: Sampson Agyapong Atuahene; email address, Samxin8@gmail.com1,
7
Telephone No:18605243710
8
9
Abstract
10
11
This study estimates endogenous parameters to ascertain the dynamics in the electricity generation sector in
12
Ghana. An unrestricted VAR model is utilized to examine the empirical ramifications of the Ghanaian electricity
13
energy sector and power consumption. The data period ranges between 2002 02021. Precisely, the results detected
14
structural long run and short run headwinds to the unrestricted models. The findings depict that the reactions of GDP
15
growth rate and electricity from fossil fuels react directly to headwinds based on their correlation. Also, there is
16
feedback relationship between GDP growth rate rand electricity from fossil fuel sources since they are significant,
17
from the granger causality analysis. Furthermore, the impulse function depicts GDP growth rate reacts to exogenous
18
headwinds and the impacts are enduring to an extended period. Furthermore, the Variance decomposition results
19
backed this analysis via having renewable energy without hydropower explaining less than 1% variance due to shocks
20
and total global greenhouse emissions further explains about 85% variance due to headwinds in the time period. 21
Electricity from fossil fuels sources explains more than 100% of the variance owing to headwinds hitting the system. 22
Implying the nation’s overreliance on conventional energy source. Installed renewable energy will grow to over
23
2500MW by 2036, growing at 57.8% %. 24
Ultimately, this calls for a rapid transformation of energy landscape in the country to a sustainable pathway. 25
This will inform policy discourses regarding diversifying Ghana’s energy mix. 26
KEY Words: Ghana, VAR, Electricity, Energy, Conventional energy, RES. 27
28
29 Modelling the electricity generation dynamics of Ghana. Research Article A Structural VAR Approach
1
2
Sampson Agyapong Atuahene1*, David Alemzero2
3
1&2School of Economics and Finance -Jiangsu University- 301 Xuefu Rd, Jingkou District, Zhenjiang,
4
Jiangsu, China. 5
Email: Samxin8@gmail.com1: awelingazure@gmail.com2
6
Corresponding Author: Sampson Agyapong Atuahene; email address, Samxin8@gmail.com1,
7
Telephone No:18605243710
8
9
Abstract
10
11
This study estimates endogenous parameters to ascertain the dynamics in the electricity generation sector in
12
Ghana. An unrestricted VAR model is utilized to examine the empirical ramifications of the Ghanaian electricity
13
energy sector and power consumption. The data period ranges between 2002 02021. Precisely, the results detected
14
structural long run and short run headwinds to the unrestricted models. The findings depict that the reactions of GDP
15
growth rate and electricity from fossil fuels react directly to headwinds based on their correlation. Also, there is
16
feedback relationship between GDP growth rate rand electricity from fossil fuel sources since they are significant,
17
from the granger causality analysis. Furthermore, the impulse function depicts GDP growth rate reacts to exogenous
18
headwinds and the impacts are enduring to an extended period. Furthermore, the Variance decomposition results
19
backed this analysis via having renewable energy without hydropower explaining less than 1% variance due to shocks
20
and total global greenhouse emissions further explains about 85% variance due to headwinds in the time period. 21
Electricity from fossil fuels sources explains more than 100% of the variance owing to headwinds hitting the system. 22
Implying the nation’s overreliance on conventional energy source. Installed renewable energy will grow to over
23
2500MW by 2036, growing at 57.8% %. 24
Ultimately, this calls for a rapid transformation of energy landscape in the country to a sustainable pathway. 25
This will inform policy discourses regarding diversifying Ghana’s energy mix. 26
KEY Words: Ghana, VAR, Electricity, Energy, Conventional energy, RES. 27
28
29 Modelling the electricity generation dynamics of Ghana. A Structural VAR Approach Sampson Agyapong Atuahene1*, David Alemzero2 Ghana’s total reliable capacity is 4,738.6 MW, that is 13% increase
54
between 2017-2018 installation (Aboagye, et al. 2022). On the other hand, the installed generation capacity
55
connected to the grid is 4,990 Megawatt (MW) in 2019, 4 580 MW is active and running. Additionally, the overall
56
capacity is 4,990 Megawatt (MW) if embedded supply and solar are added (Aboagye, et al. 2022). 57
In addition, Ghana targets to achieve full electrification by 2030 and achieves 10 percent deployment of RES. 58
The country is equally a signatory to the Paris Agreement that seeks to ensure the full access to cleaner energy sources
59
by 2030. Consequently, the country aims to in tandem of the full electrification program, as well as added to the
60
Nationally Determined Contributions (NDCs), seeks to improve and grow access to efficient cooking stoves by 2030,
61
by supplying about two million of those stoves (Assessment, N. et al. 2012). In furtherance to bridging the access gap,
62
the country seeks to decrease the electrification rates between urban and rural centers of 87% and 60%, by taking steps
63
to reduce costs of power connection to the national grid because low income levels of the populace, and due to places
64
that are not economically viable to generate or extend power to (Assessment, N. et al. 2012; Sun, H. et al. 2020). In
65
view of this, other multilateral development institutions, such as the World Bank is working assiduously to scale up
66
off mini grid and mini grid solutions to close the access gap via financial backing (Sun, H. et al. 2020). 67
Furthermore, to address the deficits in the power supply in the late 2000s, the government entered into
68
contract with IPPs to supply power to meet the supply deficit that led to excess supply costing the state a lot of money
69
based on ‘’take or pay it’’ basis. This surplus capacity the government entered into with forty-three power agreements
70 Furthermore, electricity generation increased to 20,170 GW in 2020. This is due to the increased in demand
46
from the about 30.1million people, with yearly power demand increasing at 10%. Additionally, the total gross
47
domestic product (GDP) of the country stands at about 61.6B dollars this size of the GDP can grow extra better when
48
there is adequate energy to turbocharge the growth. Sampson Agyapong Atuahene1*, David Alemzero2 Similarly, RES continues to form an insignificant part of the share
49
of the generation energy mix, increasing at 57% yearly. In addition, the final energy consumption of the country is
50
dominated by the industrial sector as well the residential sector, as the country has per head consumption of 540
51
kWh/capita. The electrification rate of the country is about 87% in 2020. In 2019, the total energy supply was 701.7077
52
TJ, total primary energy supply was 11.19 Mote. As of of 2019, final electricity consumption was 15.5 TWh and
53
cumulative carbon dioxide emissions was 18.5 Mt. Ghana’s total reliable capacity is 4,738.6 MW, that is 13% increase
54
between 2017-2018 installation (Aboagye, et al. 2022). On the other hand, the installed generation capacity
55
connected to the grid is 4,990 Megawatt (MW) in 2019, 4 580 MW is active and running. Additionally, the overall
56
capacity is 4,990 Megawatt (MW) if embedded supply and solar are added (Aboagye, et al. 2022). 57 In addition, Ghana targets to achieve full electrification by 2030 and achieves 10 percent deployment of RES. 58
The country is equally a signatory to the Paris Agreement that seeks to ensure the full access to cleaner energy sources
59
by 2030. Consequently, the country aims to in tandem of the full electrification program, as well as added to the
60
Nationally Determined Contributions (NDCs), seeks to improve and grow access to efficient cooking stoves by 2030,
61
by supplying about two million of those stoves (Assessment, N. et al. 2012). In furtherance to bridging the access gap,
62
the country seeks to decrease the electrification rates between urban and rural centers of 87% and 60%, by taking steps
63
to reduce costs of power connection to the national grid because low income levels of the populace, and due to places
64
that are not economically viable to generate or extend power to (Assessment, N. et al. 2012; Sun, H. et al. 2020). In
65
view of this, other multilateral development institutions, such as the World Bank is working assiduously to scale up
66
off mini grid and mini grid solutions to close the access gap via financial backing (Sun, H. et al. 2020). Sampson Agyapong Atuahene1*, David Alemzero2 Corresponding Author: Sampson Agyapong Atuahene; email address, Samxin8@gmail.com1,
7
Telephone No:18605243710
8 Ultimately, this calls for a rapid transformation of energy landscape in the country to a sustainable pathway. 25
This will inform policy discourses regarding diversifying Ghana’s energy mix. 26 1.0 Introduction
35
Ghana’s electricity generation dynamics is disproportionately in favor of conventional sources generation. 36
Conventional sources makeup about 68.9% of the generation mix, followed by hydropower of 29.1% and the rest of
37
renewable energy sources (RES) 2.1%. respectively [ECG 2020]. Prior, hydropower was the dominating the
38
generation of electricity in Ghana until 2015 when thermal sources overtook the biggest chunk of the generation mix. 39
This comes next after the deregulation of the power sector that sees the formation of independent power producer
40
s(IPPs) alongside government own power plants in the country. The country has institutionalized various reforms in
41
the power sector that attract private sector participation, unbundling the energy sector, even though the utilities are
42
still within the control of the state. Thus, this paper provides an empirical analysis of the energy generation dynamics
43
of the power section in Ghana bearing in mind the global dynamics and to see how they impact on electricity generation
44
of the economy given current shocks prevailing in the energy markets. 45 of the economy given current shocks prevailing in the energy markets. 45
Furthermore, electricity generation increased to 20,170 GW in 2020. This is due to the increased in demand
46
from the about 30.1million people, with yearly power demand increasing at 10%. Additionally, the total gross
47
domestic product (GDP) of the country stands at about 61.6B dollars this size of the GDP can grow extra better when
48
there is adequate energy to turbocharge the growth. Similarly, RES continues to form an insignificant part of the share
49
of the generation energy mix, increasing at 57% yearly. In addition, the final energy consumption of the country is
50
dominated by the industrial sector as well the residential sector, as the country has per head consumption of 540
51
kWh/capita. The electrification rate of the country is about 87% in 2020. In 2019, the total energy supply was 701.7077
52
TJ, total primary energy supply was 11.19 Mote. As of of 2019, final electricity consumption was 15.5 TWh and
53
cumulative carbon dioxide emissions was 18.5 Mt. Sampson Agyapong Atuahene1*, David Alemzero2 67 Furthermore, to address the deficits in the power supply in the late 2000s, the government entered into
68
contract with IPPs to supply power to meet the supply deficit that led to excess supply costing the state a lot of money
69
based on ‘’take or pay it’’ basis. This surplus capacity the government entered into with forty-three power agreements
70 would have costed the nation avoidable $680 million cost yearly in 2017, making the then government to rationalize
71
these emergency power agreements. All these are compounded by high management inefficiencies and operational
72
costs that exacerbate the dire financial situation of the power sector. The energy sector debt will be at $12.5B by 2030
73
(Assessment, N. et al. 2012). The debt is owed to IPPs as well as owing to poor debt collections system, inefficient
74
billing system, state entities’ inability to pay bills, high fuel costs by IPPs, and many more. To help deal with the debt
75
in the sector, the government set up the ESLAC, a special purpose vehicle (SPV) as a limited liability company to
76
tackle the debt issue. The PURC revises tariffs, the average user tariffs stand at 13-15 cents per Kwh. Despite these
77
adjustments, the tariffs rates in Ghana is one of the highest in Africa (Sun, H. et al 2020) yet the sector can’t raise
78
enough finances to meet its operating expenses. 79 would have costed the nation avoidable $680 million cost yearly in 2017, making the then government to rationalize
71
these emergency power agreements. All these are compounded by high management inefficiencies and operational
72
costs that exacerbate the dire financial situation of the power sector. The energy sector debt will be at $12.5B by 2030
73
(Assessment, N. et al. 2012). The debt is owed to IPPs as well as owing to poor debt collections system, inefficient
74
billing system, state entities’ inability to pay bills, high fuel costs by IPPs, and many more. To help deal with the debt
75
in the sector, the government set up the ESLAC, a special purpose vehicle (SPV) as a limited liability company to
76
tackle the debt issue. The PURC revises tariffs, the average user tariffs stand at 13-15 cents per Kwh. Despite these
77
adjustments, the tariffs rates in Ghana is one of the highest in Africa (Sun, H. Sampson Agyapong Atuahene1*, David Alemzero2 et al 2020) yet the sector can’t raise
78
enough finances to meet its operating expenses. 79 Furthermore, the over reliance on fossil fuels on the country makes the nation energy insecure, threating the
80
already energy insecurity nature of the country. Even though the country’s import bill has reduced, the country
81
continues to import gas from the West African Gas Pipeline (WGP) to generate electricity. Ghana spends more than
82
one third of its GDP in importing energy to power its economy( M.A. Nyasapoh, 2022). This is not sustainable. 83 Concerning the empirical analysis, the study deploys an unrestrictive VAR model that ensures innovations
84
of the study by modelling it, applying econometrics analysis to examine its empirical outcomes ( Sack, B. 2020;
85
Calcagnini,et al, 2016). Exactly, the VAR approach breakdowns the endogenous parameters to their unrestrictive
86
functions in a granular. 87 Consequently, a number of results are outstanding, (1) the regressed unrestrictive associations for electricity
88
from fossil fuels sources and GDP growth rate are direct in the medium term to the long-term shocks (2) The impulse
89
response functions depict a continuing increase in Ghana’s total greenhouse emissions levels as well as electricity
90
from fossil fuels sources. (3) The GDP growth rate plus Price at the pump for gasoline per liter, reacts quickly to
91
exogenous shocks than the rest of the parameters. 92 Generally, all the endogenous parameters react positively to shocks in the long term even though few of them
93
reacted negatively and continue on growth trajectory afterwards. And that demand headwinds are rapid and more
94
enduring compared to supply headwinds. Overall, the model explains that the Ghanaian electricity sector and by the
95
extension the economy, the importance of headwinds in shaping the direction of the economy and phase out in the
96
course of time. 97 2. 0 Literature Review The study equally concurs that EVs will overall cut emission levels in the country drastically, up to 33%
108
by a single diesel engine replaced. Furthermore, with RES seen as the panacea to the current energy crisis Aboagye,
109
et al. (2022), seek to determine the degradation of solar PV in varied climate conditions in Ghana. This will ensure
110
that the country adopts the ones most suitable and environmentally benign for the country. With the private sector as
111
the right partner to ensuring the deployment of RES such as solar, Acheampong, et al. (2022) empirical work seeks
112
to back this assertion and advocates the government of Ghana to make their newly craft policy that seeks to attract
113
private sector participation is improved and implemented comprehensively. They opine solar energy can bring about
114
off grid access via PPPs models. 115 Consequently, as Ghana’s emissions levels are on the increase, Acheampong, et al. (2022) utilizes the
116
Stochastic regression to determine the de facto political, economic, social internalization on Ghana’s pollutions
117
levels, and produces a dichotomous results that explain that a negative and positive variation in political
118
internationalization carbon dioxide emissions levels in the long run, whiles a negative and position variation in
119
political internationalization reduces carbon dioxide emissions levels. Mary, et al. (2022), estimated that about 85%
120
of the aggregate landmass in Ghana is appropriate for solar energy deployment. This potential can meet the energy
121
needs of the country sustainable and over yet the country has not explored this potential of solar to the fullest. In
122
justifying the cost competitive nature of solar energy for Ghana, Mary, et al. (2022), estimated the levelized cost of
123
solar energy and arrived at the cost of $0.04/k -$0.15/kWh for utility scale solar, and $0.73/kWh to ∼$2.89/kWh for
124
Concentrated Sola Photovoltaic. Further, Sun, H. et al. (2020) finds the LCOE of diesel thermal plants to be more
125
as high as Gh¢351.44 kWh and Solar thermal plants to be Gh¢4.08 kWh, regarding Ghana. Alemzero and co. 126
(2012), additionally estimated wind energy costs to have fallen by 30% in Africa between 2010-2019.Gyimah, et al. 127
(2021) in their study find renewable energy to promote economic growth in Ghana as well attract foreign direct
128
investment. In the research paper Energy efficiency and electricity expenditure in Ghana Adjei-Mantey et al. 2. 0 Literature Review Within the past years post the deregulation of the electricity sector in Ghana, the power sector has seen
99
significant transformation, with the IPPs playing a prominent role in the power generation. Ghana generates enough
100
electricity to power its economy nonetheless it is coming from mainly from fossil fuel sources. Even though Ghana
101
has adequate energy access now and generates enough electricity, household of electricity for cooking is yet very low
102
at about 20% access to modern cooking solutions (Tabiri, et al. 2022). According to ( M.A. Nyasapoh, 2022) Ghana
103
spends about 27% of its GDP on importing fuels to power its development irrespective of the abundance of local
104 RES.A study by Ayetor,et al (2022), finds that the social cost for owing internal combustion engine vehicle is over
105
164% and that the generation of RES on the energy mix below 20% is harmful to the environment. This calls for
106
scaling up the deployment of EVs as well as the development the necessary infrastructure that will enable the uptake
107
of the sector. The study equally concurs that EVs will overall cut emission levels in the country drastically, up to 33%
108
by a single diesel engine replaced. Furthermore, with RES seen as the panacea to the current energy crisis Aboagye,
109
et al. (2022), seek to determine the degradation of solar PV in varied climate conditions in Ghana. This will ensure
110
that the country adopts the ones most suitable and environmentally benign for the country. With the private sector as
111
the right partner to ensuring the deployment of RES such as solar, Acheampong, et al. (2022) empirical work seeks
112
to back this assertion and advocates the government of Ghana to make their newly craft policy that seeks to attract
113
private sector participation is improved and implemented comprehensively. They opine solar energy can bring about
114
off grid access via PPPs models. 115 RES.A study by Ayetor,et al (2022), finds that the social cost for owing internal combustion engine vehicle is over
105
164% and that the generation of RES on the energy mix below 20% is harmful to the environment. This calls for
106
scaling up the deployment of EVs as well as the development the necessary infrastructure that will enable the uptake
107
of the sector. 2. 0 Literature Review 129
(2021); and Never, et al. (2022), believe that energy efficiency it is very important for environmental sustenance
130
of Ghana. Adjei-Mantey et al. (2021) state that socioeconomic factors in the form of poverty and reduced
131
education levels act as hindrances to the acceptance of light bulbs that are energy efficient in Ghana. 132 This paper takes a different approach to analysis by using an unrestrictive VAR model by evaluation the
133
dynamic relationship among GDP growth rate, electricity from renewable sources excluding hydropower, Ghana’
134
total greenhouse gas emissions, Price at the pump per liter for gasoline, electricity from fossil fuels sources’ VAR
135
makes room for the identification of shocks and to determine their impact on the electricity sector in the long and
136
short periods. The paper makes an empirical contribution by using VAR model to empirical understand shocks,
137
especially energy crisis on the energy generation dynamics and trends. This offers pragmatic importance to policy
138
formulation. 139 The rest of the paper is arranged as follows: Chapter two does an extensive literature review on the energy
140
generation dynamics in Ghana across various lenses. Chapter three presents the methodology, and data in the analysis. 141
Whiles Chapter discusses the results derived in the analysis and Chapter five wraps up the analysis with conclusions. 142
143
3. 0 Method and Data
144
3.1 Model Estimation
145
Vector Autoregression regression (VAR) is a multivariate stochastic process of vector generalization of scalar
146
autoregression. It talks about cointegration that implies casualty among variables. Sometimes it is cumbersome to
147
determine which variables to use as the dependent variable, when estimating the relationship among variables. More
148
so, economic principles at times are unable to determine which variables to be used as the explained parameter and
149
the exploratory variables. Given this background, the VAR is the appropriate model since all the variables are
150
considered endogenous. Similarly, the VAR model was mooted by Sims(Cristiano, 2012; Das, 2019; Sun et al., 2020)
151
for the first time and was made popular by(Robert F. ENGLE and Byung, 1987). 152 The rest of the paper is arranged as follows: Chapter two does an extensive literature review on the energy
140
generation dynamics in Ghana across various lenses. Chapter three presents the methodology, and data in the analysis. 2. 0 Literature Review 141
Whiles Chapter discusses the results derived in the analysis and Chapter five wraps up the analysis with conclusions. 142
143
3. 0 Method and Data
144
3.1 Model Estimation
145
Vector Autoregression regression (VAR) is a multivariate stochastic process of vector generalization of scalar
146
autoregression. It talks about cointegration that implies casualty among variables. Sometimes it is cumbersome to
147
determine which variables to use as the dependent variable, when estimating the relationship among variables. More
148
so, economic principles at times are unable to determine which variables to be used as the explained parameter and
149
the exploratory variables. Given this background, the VAR is the appropriate model since all the variables are
150
considered endogenous. Similarly, the VAR model was mooted by Sims(Cristiano, 2012; Das, 2019; Sun et al., 2020)
151
for the first time and was made popular by(Robert F. ENGLE and Byung, 1987). 152 Vector Autoregression regression (VAR) is a multivariate stochastic process of vector generalization of scalar
146
autoregression. It talks about cointegration that implies casualty among variables. Sometimes it is cumbersome to
147
determine which variables to use as the dependent variable, when estimating the relationship among variables. More
148
so, economic principles at times are unable to determine which variables to be used as the explained parameter and
149
the exploratory variables. Given this background, the VAR is the appropriate model since all the variables are
150
considered endogenous. Similarly, the VAR model was mooted by Sims(Cristiano, 2012; Das, 2019; Sun et al., 2020)
151
for the first time and was made popular by(Robert F. ENGLE and Byung, 1987). atching diminished form is given in an expanded from beneath. 2. 0 Literature Review 152 𝑋1𝑡= 𝛽0 + 𝛽11𝑋2𝑡+ 𝑄11𝑋1𝑡−1 + 𝑄12𝑋2𝑡−1 + 𝜖1𝑡 ( 1)
153
𝑋2𝑡= 𝛽0 + 𝛽22𝑋1𝑡+ 𝑄21𝑋2𝑡−1 + 𝑄22𝑋2𝑡−1 + 𝜖2𝑡
154
(2)
155
𝑋3𝑡= 𝛽0 + 𝛽23𝑋2𝑡+ 𝑄23𝑋3𝑡−1 + 𝑄24𝑋3𝑡−1 + 𝜖3𝑡 (3)
156
𝑋4𝑡= 𝛽0 + 𝛽34𝑋3𝑡+ 𝑄44𝑋4𝑡−1 + 𝑄34𝑋4𝑡−1 + 𝜖4𝑡 (4)
157
𝑋5𝑡= 𝛽0 + 𝛽45𝑋4𝑡+ 𝑄55𝑋5𝑡−1 + 𝑄45𝑋5𝑡−1 + 𝜖5𝑡 (5)
158
159 ( 1) [
=
(
1
−𝛽11
−𝛽12
1
1
−𝛽23
−𝛽34
1
1
−𝛽45
−𝛽55
1 )
+
(
𝑄11
𝑄12
𝑄21
𝑄22
𝑄23
𝑄24
𝑄34
𝑄44
𝑄55
𝑄45
)
+
(
𝑋𝑡−1
𝑋𝑡−3
𝑋𝑡−4
𝑋𝑡−5
)
+
(
𝜖1𝑡
𝜖2𝑡
𝜖3𝑡
𝜖4𝑡
𝜖5𝑡)
]
(6)
161 The above equation can’t be regressed directly because the stochastic term is linked to the 𝜖1𝑡, 𝜖2𝑡𝜖3𝑡, 𝜖4𝑡 is
162
associated to 𝜖5𝑡.Since the regressors in the structural form are linked to the stochastic term, there is likely to be an
163
endogeneity. 164 The above equation can’t be regressed directly because the stochastic term is linked to the 𝜖1𝑡, 𝜖2𝑡𝜖3𝑡, 𝜖4𝑡 is
162
associated to 𝜖5𝑡.Since the regressors in the structural form are linked to the stochastic term, there is likely to be an
163
endogeneity. 164 The matching diminished form is given in an expanded from beneath. 165 𝑋1𝑡= 𝜔01 + 𝜔11𝑋2𝑡+ 𝜔11𝑋1𝑡−1 + 𝜔12𝑋2𝑡−1 + 𝑒1𝑡 (7) (7) 𝑋2𝑡= 𝜔02 + 𝜔12𝑋2𝑡+ 𝜔21𝑋1𝑡−1 + 𝜔22𝑋2𝑡−1 + 𝑒2𝑡 (8)
169
170
𝑋3𝑡= 𝜔03 + 𝜔23 𝑋3𝑡+ 𝜔23𝑋2𝑡−1 + 𝜔22𝑋2𝑡−1 + 𝑒2𝑡 (9)
171
172
𝑋4𝑡= 𝜔0 + 𝜔34𝑋4𝑡+ 𝜔44𝑋4𝑡−1 + 𝜔44𝑋2𝑡−1 + 𝑒4𝑡 (10)
173
174
Order Selection
175
Since the study estimates multivariate of the VAR model with orders from 1 to P. it is proper to estimate. It
176
is interesting to observe the residual of the models. Thus, a more realistically approach is given below. A relevant
177
model order selection criteria for the different variable’s extension for the Akaike and Schwarz information criteria
178
is given below. 179 (8) Order Selection Since the study estimates multivariate of the VAR model with orders from 1 to P. it is proper to estimate. It
176
is interesting to observe the residual of the models. Thus, a more realistically approach is given below. A relevant
177
model order selection criteria for the different variable’s extension for the Akaike and Schwarz information criteria
178
is given below. 179 𝐴𝐼𝐶= 𝐿𝑜𝑔| ∑
𝑃
̂ + 2𝑀2𝑃|𝑇 𝑃= 1, . .2, … . . 𝑃 (11)
80
𝑆𝐼𝐶= 𝐿𝑜𝑔|∑
𝑃
̂ + (𝑙𝑜𝑔)𝑚2𝑃| 𝑇 𝑃= 1, … 2, … … 𝑃 (12)
81
82
Hence, | ∑
𝑃
̂ depicts the casual factor of the residual covariance of the 𝑉𝐴𝑅𝑝 model and 𝑇 portrays the
83
number of effective observations. This model works best even if the explained vector contains unit roots (Paulsen,
84
1984). 85
3 2 Data
86 𝐴𝐼𝐶= 𝐿𝑜𝑔| ∑
𝑃
̂ + 2𝑀2𝑃|𝑇 𝑃= 1, . .2, … . . 𝑃 (11)
𝑆𝐼𝐶= 𝐿𝑜𝑔|∑
𝑃
̂ + (𝑙𝑜𝑔)𝑚2𝑃| 𝑇 𝑃= 1, … 2, … … 𝑃 (12) (12) Hence, | ∑
𝑃
̂ depicts the casual factor of the residual covariance of the 𝑉𝐴𝑅𝑝 model and 𝑇 portrays the
183
number of effective observations. This model works best even if the explained vector contains unit roots (Paulsen,
184
1984). 185
3.2 Data. 186
The study relies on from data from the world Bank development indicators (WDI) between the period of
187
2010-2021, to estimate the electricity generation dynamics in the energy mix of Ghana. The study makes use of an
188
unrestrictive VAR model to determine these endogenous parameters work to impact on the energy mix. The gross
189
domestic product (GDPGRWT) growth rate is one of the endogenous parameters. This will enable the study to
190
comprehend the manner GDP impacts on the electricity generation levels in the country. It is an undeniable fact that
191
no country progress socioeconomically without access to adequate energy. Similarly, EFOSST which is electricity
192
generation from fossil fuel sources is another endogenous parameter. Ghana generates over 70%of electricity from
193
conventional energy sources. Aside, ELRESXHDY depicts electricity from renewable energy sources without
194
hydropower is equally evaluated to determine the contribution of RES sources to electricity generation of the country. 195
Additionally, TGHE further represent the total greenhouse gas emission in Ghana. Order Selection As conspicuous from the table, electricity from fossil fuels source
203
derives the highest mean value. This is so as Ghana generates nearly 70% of electricity from thermal sources. The
204
next variable that has the highest mean value is gross domestic growth rate. Ghana has experienced significant GDP
205
rate in the mid -2000s and was seen as the fastest growing economy in the African continent. More so, electricity from
206
renewable resources excluding hydropower has the least mean value since Ghana does not generate much from RES. 207
Ghana total greenhouse gas is on a growing trajectory since 2010 when the country started exploring and developing
208
hydrocarbons. 209
210
211
212
213
214
215
Table1. Descriptive Statistics
216
EFOSST_
ELRESXHDY
GDPGRWT
TGHE_
PPGPLIT
Mean
5.743783
0.004014
5.704741
139.4096
0.312500
Median
0.000000
0.000000
5.750002
177.9197
0.000000
Maximum
38.84779
0.030857
14.04712
320.6482
1.060000
Minimum
0.000000
0.000000
0.000000
0.000000
0.000000
Std. Dev. 10.62125
0.009880
3.246496
133.4096
0.423256
Skewness
1.914978
2.027200
0.437260
-0.05264
0.714309
Kurtosis
5.930881
5.234799
3.652748
1.135933
1.683751
Jarque-Bera
19.38219
17.86040
0.992389
2.904857
3.144551
Probability
0.000062
0.000132
0.608843
0.234001
0.207572
Sum
114.8757
0.080273
114.0948
2788.192
6.250000
Sum
Sq. Dev. 2143.408
0.001855
200.2550
338164.2
3.403775
Observations
20
20
20
20
20
217
Table 2 Correlation Matrix
218
Furthermore, from table 2. Is the correlation matrix that explains the relationship with the variables. The
219
correlation between electricity from RES excluding hydropower derives the significant relationship to electricity from
220
fossil sources. This direct correlation implies that as there is the need to generate electricity from other renewable
221 From table 1. Is the descriptive statistics of the unrestricted VAR model that analyses the electricity
202
generation dynamics of the Ghana power sector. As conspicuous from the table, electricity from fossil fuels source
203
derives the highest mean value. This is so as Ghana generates nearly 70% of electricity from thermal sources. The
204
next variable that has the highest mean value is gross domestic growth rate. Ghana has experienced significant GDP
205
rate in the mid -2000s and was seen as the fastest growing economy in the African continent. More so, electricity from
206
renewable resources excluding hydropower has the least mean value since Ghana does not generate much from RES. 207
Ghana total greenhouse gas is on a growing trajectory since 2010 when the country started exploring and developing
208
hydrocarbons. 209 Table1. Order Selection This is also assessed to determine
196
the levels of pollution in the country. Ultimately, PPGPLIT explains the price per liter at the pump of gasoline. Ghana
197
consumes a lot of gasoline in the transport sector as well as for farming and the manufacturing sector. Hence the
198
imperative to examine the importance of this variable. Prior to the analysis, the variables were assumed stationary as
199
it is not a necessary pre-condition to the examination process. 200 Hence, | ∑
𝑃
̂ depicts the casual factor of the residual covariance of the 𝑉𝐴𝑅𝑝 model and 𝑇 portrays the
183
number of effective observations. This model works best even if the explained vector contains unit roots (Paulsen,
184
1984). 185 3.2 Data. 186
The study relies on from data from the world Bank development indicators (WDI) between the period of
187
2010-2021, to estimate the electricity generation dynamics in the energy mix of Ghana. The study makes use of an
188
unrestrictive VAR model to determine these endogenous parameters work to impact on the energy mix. The gross
189
domestic product (GDPGRWT) growth rate is one of the endogenous parameters. This will enable the study to
190
comprehend the manner GDP impacts on the electricity generation levels in the country. It is an undeniable fact that
191
no country progress socioeconomically without access to adequate energy. Similarly, EFOSST which is electricity
192
generation from fossil fuel sources is another endogenous parameter. Ghana generates over 70%of electricity from
193
conventional energy sources. Aside, ELRESXHDY depicts electricity from renewable energy sources without
194
hydropower is equally evaluated to determine the contribution of RES sources to electricity generation of the country. 195
Additionally, TGHE further represent the total greenhouse gas emission in Ghana. This is also assessed to determine
196
the levels of pollution in the country. Ultimately, PPGPLIT explains the price per liter at the pump of gasoline. Ghana
197
consumes a lot of gasoline in the transport sector as well as for farming and the manufacturing sector. Hence the
198
imperative to examine the importance of this variable. Prior to the analysis, the variables were assumed stationary as
199
it is not a necessary pre-condition to the examination process. 200 4. Results and Discussion
201
From table 1. Is the descriptive statistics of the unrestricted VAR model that analyses the electricity
202
generation dynamics of the Ghana power sector. Order Selection Implying that as one source increases the other source reduces. This is rightly
247
so, because RES acts as substitutes, not complements in the energy transition drive. Hence, if Ghana is to achieve its
248
10% RES capacity by 2030 and its NDCs, RES will have to be scaled up within the shortest possible time. This fact
249
is backed by De La Pena, et al. (2022) and Haung et al. (2022) regarding China’s evolving energy landscape. The
250
country is behind schedule to achieving 10% of RES on its energy mix by 2030. More so, price per liter at the pump
251 Similarly, from table 3 is the results of the VAR model. The model where total greenhouse gas is the
229
explained(TGHE) parameter, its second lag is significant with a direct correlation. The significant implies the
230
expansion in emissions levels in the country as the economy grows (Shahbaz, et at 2022). The greenhouse gas
231
emissions levels increase from the 2010s onward when the nation discovered oil. Similarly, the electricity from fossil
232
fuels (EFOSS) sources as the dependent variables is significant to price per liter of gasoline. The magnitude of the
233
correlation is direct as well. This is explaining the growing demand for gasoline in the downstream transport in the
234
country and for farming and other industrial purposes in the country. Equally on this model, the lag of total greenhouse
235
gas emissions is significant to the electricity from fossil fuel sources. Also, electricity from renewable sources
236
excluding hydropower is significant as an independent variable to electricity from fossil energy sources. The direction
237
is negative. the magnitude depicts the inverse relationship among the variables. As one increases the other reduces. 238
This is calls for the need to transition to RES electricity generation which is cheaper and sustainable (CBi, 2020). In
239
addition, electricity from fossil fuels sources as the independent variable is meaningful to the gross domestic growth
240
rate. This connotes that electricity consumption impacts on economic growth (CBi, 2020; Yao, et al. 2020))
241
Nonetheless, the coefficient is negative, suggesting the decoupling of the energy consumption from GDP growth. This
242
is quite mind boggling in the sense that Ghana has not decoupled its GDP growth rate to its economic development. 243
Perhaps it depicts future trajectory the economy should take. Order Selection Similarly, the electricity from fossil
232
fuels (EFOSS) sources as the dependent variables is significant to price per liter of gasoline. The magnitude of the
233
correlation is direct as well. This is explaining the growing demand for gasoline in the downstream transport in the
234
country and for farming and other industrial purposes in the country. Equally on this model, the lag of total greenhouse
235
gas emissions is significant to the electricity from fossil fuel sources. Also, electricity from renewable sources
236
excluding hydropower is significant as an independent variable to electricity from fossil energy sources. The direction
237
is negative. the magnitude depicts the inverse relationship among the variables. As one increases the other reduces. 238
This is calls for the need to transition to RES electricity generation which is cheaper and sustainable (CBi, 2020). In
239
addition, electricity from fossil fuels sources as the independent variable is meaningful to the gross domestic growth
240
rate. This connotes that electricity consumption impacts on economic growth (CBi, 2020; Yao, et al. 2020))
241
Nonetheless, the coefficient is negative, suggesting the decoupling of the energy consumption from GDP growth. This
242
is quite mind boggling in the sense that Ghana has not decoupled its GDP growth rate to its economic development. 243
Perhaps it depicts future trajectory the economy should take. Furthermore, the model that has electricity from
244
renewable sources as the explained parameter, the price per liter at the pump of gasoline is meaningful at the lag first
245
and second lags. This confirms that electricity from RES impacts on the price Ghanaians pay at the pump for gasoline. 246
However, the relationship is negative. Implying that as one source increases the other source reduces. This is rightly
247
so, because RES acts as substitutes, not complements in the energy transition drive. Hence, if Ghana is to achieve its
248
10% RES capacity by 2030 and its NDCs, RES will have to be scaled up within the shortest possible time. This fact
249
is backed by De La Pena, et al. (2022) and Haung et al. (2022) regarding China’s evolving energy landscape. The
250
country is behind schedule to achieving 10% of RES on its energy mix by 2030. Order Selection More so, price per liter at the pump
251 EFOSST_
ELRESXHDY
GDPGRWT
TGHE_
PPGPLIT
EFOSST_
1
0.680652906
0.145820915
-0.13120634
0.039199212
ELRESXHDY
0.680652906
1
-0.236258719
-0.446830915
0.095941759
GDPGRWT
0.145820915
-0.23625872
1
0.494582915
0.124347108
TGHE_
-0.13120634
-0.44683091
0.494582915
1
0.206886986
PPGPLIT
0.039199212
0.095941759
0.124347108
0.206886986
1
Source. Author’s estimation. 226
227
Table 3. VAR Results. 228
Similarly, from table 3 is the results of the VAR model. The model where total greenhouse gas is the
229
explained(TGHE) parameter, its second lag is significant with a direct correlation. The significant implies the
230
expansion in emissions levels in the country as the economy grows (Shahbaz, et at 2022). The greenhouse gas
231
emissions levels increase from the 2010s onward when the nation discovered oil. Similarly, the electricity from fossil
232
fuels (EFOSS) sources as the dependent variables is significant to price per liter of gasoline. The magnitude of the
233
correlation is direct as well. This is explaining the growing demand for gasoline in the downstream transport in the
234
country and for farming and other industrial purposes in the country. Equally on this model, the lag of total greenhouse
235
gas emissions is significant to the electricity from fossil fuel sources. Also, electricity from renewable sources
236
excluding hydropower is significant as an independent variable to electricity from fossil energy sources. The direction
237
is negative. the magnitude depicts the inverse relationship among the variables. As one increases the other reduces. 238
This is calls for the need to transition to RES electricity generation which is cheaper and sustainable (CBi, 2020). In
239
addition, electricity from fossil fuels sources as the independent variable is meaningful to the gross domestic growth
240
rate. This connotes that electricity consumption impacts on economic growth (CBi, 2020; Yao, et al. 2020))
241
Nonetheless, the coefficient is negative, suggesting the decoupling of the energy consumption from GDP growth. This
242
is quite mind boggling in the sense that Ghana has not decoupled its GDP growth rate to its economic development. 243
Perhaps it depicts future trajectory the economy should take. Furthermore, the model that has electricity from
244
renewable sources as the explained parameter, the price per liter at the pump of gasoline is meaningful at the lag first
245
and second lags. This confirms that electricity from RES impacts on the price Ghanaians pay at the pump for gasoline. 246
However, the relationship is negative. Order Selection Descriptive Statistics EFOSST_
ELRESXHDY
GDPGRWT
TGHE_
PPGPLIT
Mean
5.743783
0.004014
5.704741
139.4096
0.312500
Median
0.000000
0.000000
5.750002
177.9197
0.000000
Maximum
38.84779
0.030857
14.04712
320.6482
1.060000
Minimum
0.000000
0.000000
0.000000
0.000000
0.000000
Std. Dev. 10.62125
0.009880
3.246496
133.4096
0.423256
Skewness
1.914978
2.027200
0.437260
-0.05264
0.714309
Kurtosis
5.930881
5.234799
3.652748
1.135933
1.683751
Jarque-Bera
19.38219
17.86040
0.992389
2.904857
3.144551
Probability
0.000062
0.000132
0.608843
0.234001
0.207572
Sum
114.8757
0.080273
114.0948
2788.192
6.250000
Sum
Sq. Dev. 2143.408
0.001855
200.2550
338164.2
3.403775
Observations
20
20
20
20
20
217
Table 2 Correlation Matrix
218
Furthermore, from table 2. Is the correlation matrix that explains the relationship with the variables. The
219
correlation between electricity from RES excluding hydropower derives the significant relationship to electricity from
220
fossil sources. This direct correlation implies that as there is the need to generate electricity from other renewable
221 Furthermore, from table 2. Is the correlation matrix that explains the relationship with the variables. The
219
correlation between electricity from RES excluding hydropower derives the significant relationship to electricity from
220
fossil sources. This direct correlation implies that as there is the need to generate electricity from other renewable
221 sources to replace the fossil fuels sources that re expensive and pollute the environment. Another interesting
222
correlation that exists is between electricity from renewable sources excluding hydropower and that total greenhouse
223
gas emissions. The correlation is adverse. This connotes that as RES consumptions increases, emissions levels
224
decrease. Hence RES abates greenhouse gas pollution. 225 EFOSST_
ELRESXHDY
GDPGRWT
TGHE_
PPGPLIT
EFOSST_
1
0.680652906
0.145820915
-0.13120634
0.039199212
ELRESXHDY
0.680652906
1
-0.236258719
-0.446830915
0.095941759
GDPGRWT
0.145820915
-0.23625872
1
0.494582915
0.124347108
TGHE_
-0.13120634
-0.44683091
0.494582915
1
0.206886986
PPGPLIT
0.039199212
0.095941759
0.124347108
0.206886986
1
Source. Author’s estimation. 226 EFOSST_
ELRESXHDY
GDPGRWT
TGHE_
PPGPLIT
EFOSST_
1
0.680652906
0.145820915
-0.13120634
0.039199212
ELRESXHDY
0.680652906
1
-0.236258719
-0.446830915
0.095941759
GDPGRWT
0.145820915
-0.23625872
1
0.494582915
0.124347108
TGHE_
-0.13120634
-0.44683091
0.494582915
1
0.206886986
PPGPLIT
0.039199212
0.095941759
0.124347108
0.206886986
1
Source. Author’s estimation. 226
227
Table 3. VAR Results. 228
Similarly, from table 3 is the results of the VAR model. The model where total greenhouse gas is the
229
explained(TGHE) parameter, its second lag is significant with a direct correlation. The significant implies the
230
expansion in emissions levels in the country as the economy grows (Shahbaz, et at 2022). The greenhouse gas
231
emissions levels increase from the 2010s onward when the nation discovered oil. is significant. Besides, the total greenhouse gas emissions levels are significant in its first and second lags of the
252
model where RES excluding power is the dependent variables. This corroborate the scientific facts that RES abate
253
pollution (De La Pena, et al. (2022). This further was buttressed by the correlation where they are negative. As one
254
variable increases, the variable reduces. Broadly speaking, if Ghana is to reduce its emissions levels, Ghana has to
255
increase renewable electricity generation. Ghana equally bears the brunt of climate change and its worse
256
consequences in Accra in the form of flash floods, changing weather farming seasons in the northern part of Ghana
257
and many others. As result, the country has to hasten to deployment RES in no time. Ultimately, the second lag of
258
renewable energy sources excluding hydropower is significant and but has an inverse relationship. It explains the
259
relevance of RES to encourage economic growth in Ghana (Baarsch, F et al 2020; Mohsin et al. 2021).
260 Order Selection Furthermore, the model that has electricity from
244
renewable sources as the explained parameter, the price per liter at the pump of gasoline is meaningful at the lag first
245
and second lags. This confirms that electricity from RES impacts on the price Ghanaians pay at the pump for gasoline. 246
However, the relationship is negative. Implying that as one source increases the other source reduces. This is rightly
247
so, because RES acts as substitutes, not complements in the energy transition drive. Hence, if Ghana is to achieve its
248
10% RES capacity by 2030 and its NDCs, RES will have to be scaled up within the shortest possible time. This fact
249
is backed by De La Pena, et al. (2022) and Haung et al. (2022) regarding China’s evolving energy landscape. The
250
country is behind schedule to achieving 10% of RES on its energy mix by 2030. More so, price per liter at the pump
251 is significant. Besides, the total greenhouse gas emissions levels are significant in its first and second lags of the
252
model where RES excluding power is the dependent variables. This corroborate the scientific facts that RES abate
253
pollution (De La Pena, et al. (2022). This further was buttressed by the correlation where they are negative. As one
254
variable increases, the variable reduces. Broadly speaking, if Ghana is to reduce its emissions levels, Ghana has to
255
increase renewable electricity generation. Ghana equally bears the brunt of climate change and its worse
256
consequences in Accra in the form of flash floods, changing weather farming seasons in the northern part of Ghana
257
and many others. As result, the country has to hasten to deployment RES in no time. Ultimately, the second lag of
258
renewable energy sources excluding hydropower is significant and but has an inverse relationship. It explains the
259
relevance of RES to encourage economic growth in Ghana (Baarsch, F et al 2020; Mohsin et al. 2021). 260 Table 3. Source. Author’s Calculations Order Selection VAR Results 261 ppgplit
tghe
efosst
gdpgrwt
Elresxhdy
L.ppgplit
-0.295
L.ppgplit
-18.5
L.ppgplit
37.42***
L.ppgplit
-3.035
L.ppgplit
0.0239***
(-1.19)
(-0.30)
-12.88
(-1.92)
-7.72
L2.ppgplit
0.526
L2.ppgplit
-2.635
L2.ppgplit
35.11***
L2.ppgplit
-1.386
L2.ppgplit
0.0184***
-1.81
(-0.04)
-10.34
(-0.75)
-5.06
L.tghe
-0.000271
L.tghe
0.186
L.tghe
-0.0579***
L.tghe
0.00265
L.tghe
-0.0000221*
(-0.31)
-0.86
(-5.74)
-0.48
(-2.05)
L2.tghe
-0.000152
L2.tghe
0.761***
L2.tghe
-0.0553***
L2.tghe
0.00561
L2.tghe
-
0.0000441***
(-0.17)
-3.41
(-5.35)
-1
(-3.98)
L.efosst
-0.00341
L.efosst
1.441
L.efosst
0.392***
L.efosst
0.164**
L.efosst
0.000118
(-0.39)
-0.66
-3.87
-2.97
-1.09
L2.efosst
-0.00731
L2.efosst
-2.713
L2.efosst
-0.157
L2.efosst
0.0409
L2.efosst
0.0000125
(-0.88)
(-1.29)
(-1.61)
-0.77
-0.12
L.gdpgrwt
0.0268
L.gdpgrwt
1.116
L.gdpgrwt
-0.338
L.gdpgrwt
-0.242
L.gdpgrwt
-0.000655*
-1.21
-0.2
(-1.31)
(-1.72)
(-2.37) -1.21 -0.2 (-1.72) (-1.31) L2.gdpgrwt
0.0378
L2.gdpgrwt
2.397
L2.gdpgrwt
0.165
L2.gdpgrwt
-0.158
L2.gdpgrwt
0.000171
-1.61
-0.4
-0.6
(-1.06)
-0.58
L.elresxhdy
20.64
L.elresxhdy
-3084.2
L.elresxhdy
-1167.1***
L.elresxhdy
-350.5***
L.elresxhdy
0.294
-1.34
(-0.79)
(-6.46)
(-3.57)
-1.52
L2.elresxhdy
-3.678
L2.elresxhdy
9784.0***
L2.elresxhdy
81.51
L2.elresxhdy
380.5***
L2.elresxhdy
-0.743***
(-0.32)
-3.38
-0.61
-5.22
(-5.19)
_cons
-0.0607
_cons
-4.719
_cons
2.138
_cons
7.635***
_cons
0.00339
(-0.38)
(-0.12)
-1.14
-7.49
-1.69
N
18
Source Author’s Calculations 262 The figure 1 below depicts the residual graphs, using he lag ‘14’ for endogenous parameters. This depicts
263
that the stochastic term linked to the variance increases reaches maximum and then plunges to the end year for GDP
264
growth rate. The residuals for the electricity from fossil fuels sources is quite steady around the mean diverges away
265
from the stochastic term. All the variables vacillate around the mean thus depicting the steadiness of the stochastic
266
term in the analysis. Generally, the figure that the matching model is a better time series model
267 Source. Author’s calculations. Figure 1. Residual Graphs of the model of the variables. Source. Author’s calculations. Figure 1. Residual Graphs of the model of the variables
269 From figure 2 is the endogenous graphs for the analysis. The endogemosu parameters shpw that GDP growth
271
rate strted off in an increasing path peaked in in the 2010s and then wnet inot a trough and continue on steady
272
pathway.In deed, 2012 and 2013 saw Ghana as one of the fastest growing economies in the workd with 14% GDP
273
grpwth rate, as shown in the diagram.Also electricygeneration peaked in 2006 and decrease in 2009 and 2014.Simialry,
274 Ghana. Order Selection Tottal greenhosue emissions peaked from 2010 and have been on the trajectiry owing to uptake of the upstream
275
petroluem sector in the country, as the graphs depcits.Thia is backed Acheampong, A. O. (2022) where by globalizatio
276
has increased Ghana’s emissions levels. Aside this, Price per litre at the pump is growing at zig zag manner depicting
277
the increaese and decreas pathway. Additonally, RES generation without hydropwer peaked in the midway and has
278
remained flat since then. 279 280
Figure 2. Endogenous Graphs of the variables. 281
282
From table 4 presents eight complex roots of 0.846586 - 0.215968i; 0.846586 + 0.215968i with an eq
283
modulus of 0.873699 0. Also 488625 - 0.671621i; 0.488625 + 0.671621i equally have an equal modulus of 0.8305
284 Figure 2. Endogenous Graphs of the variables. 281 282
From table 4 presents eight complex roots of 0.846586 - 0.215968i; 0.846586 + 0.215968i with an equal
283
modulus of 0.873699 0. Also 488625 - 0.671621i; 0.488625 + 0.671621i equally have an equal modulus of 0.830560
284
as well as 0.033735 - 0.721956i; 0.033735 + 0.721956i attain an equal modulus of 0.722744. Finally, -0.385059 -
285
0.096138i, and -0.385059 + 0.096138i derive an equal modulus of 0.396879. All these confirm that the model is
286
accurate and satisfies the stability condition. 287
288 290 Table 4 LAG STRUCTURE Root
Modulus
-0.915743
0.915743
0.846586 - 0.215968i
0.873699
0.846586 + 0.215968i
0.873699
0.488625 - 0.671621i
0.830560
0.488625 + 0.671621i
0.830560
0.033735 - 0.721956i
0.722744
0.033735 + 0.721956i
0.722744
-0.717079
0.717079
-0.385059 - 0.096138i
0.396879
-0.385059 + 0.096138i
0.396879
No root lies outside the unit circle. VAR satisfies the stability condition. 291
Subsequently, figure 3 presents the actual valued eight roots of the VAR model, which corroborate that the
292
model is stable and graphs the roots using a complex coordinate system. As shown on the graph all the coordinates lie
293
inside the circle signifying the stability condition. Thus, it is appropriate in analysis the electricity dynamic situation
294
in Ghana. 295 Subsequently, figure 3 presents the actual valued eight roots of the VAR model, which corroborate that the
292
model is stable and graphs the roots using a complex coordinate system. As shown on the graph all the coordinates lie
293
inside the circle signifying the stability condition. Thus, it is appropriate in analysis the electricity dynamic situation
294
in Ghana. Order Selection 295 296 -1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
Inverse Roots of AR Characteristic Polynomial
Figure 3. AR ROOT GRAPH. Source. Author’s construct. -1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
Inverse Roots of AR Characteristic Polynomial
297
Figure 3. AR ROOT GRAPH. Source. Author’s construct. 298
299 -1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
Inverse Roots of AR Characteristic Polynomial Inverse Roots of AR Characteristic Polynomial Figure 3. AR ROOT GRAPH. Source. Author’s construct. Table 5. VAR Granger Test/ Wald Test. 300
The granger causality test is done to ascertain the relationship among the variables. To determine which
301
variable granger causes the other. The relationship is based on the null hypothesis that an endogenous variable that
302
doesn’t granger cause the other variables. Given this, the model of GDP growth rate (GDPGRWT) has a chi-sq. of
303
25.00574 and P-value of 0.000 therefore, the null hypothesis is rejected and GDPGRWT does granger cause EFFOSST
304
which has chi-square of 132.3742 and a p-value of 0.000. Furthermore, the model of ELRESXHDY, EFFOSST
305
significantly granger causes total greenhouse gas emissions (THE), as well as price per liter at the pump (PPGPLT). 306
It is therefore concluded that there is a feedback relationship among GDP growth rate, EFOSST and ELRESXHDY. 307
This is apt since GDP growth requires energy to bring about growth. No meaningful development can take place
308
without adequate and sustainable energy. 309
310
311
312 Table 5. VAR Granger Test/ Wald Test. Table 5. VAR Granger Test/ Wald Test. Table 5. VAR Granger Test/ Wald Test. 300
The granger causality test is done to ascertain the relationship among the variables. To determine which
301
variable granger causes the other. The relationship is based on the null hypothesis that an endogenous variable that
302
doesn’t granger cause the other variables. Given this, the model of GDP growth rate (GDPGRWT) has a chi-sq. of
303
25.00574 and P-value of 0.000 therefore, the null hypothesis is rejected and GDPGRWT does granger cause EFFOSST
304
which has chi-square of 132.3742 and a p-value of 0.000. Furthermore, the model of ELRESXHDY, EFFOSST
305
significantly granger causes total greenhouse gas emissions (THE), as well as price per liter at the pump (PPGPLT). 306
It is therefore concluded that there is a feedback relationship among GDP growth rate, EFOSST and ELRESXHDY. 307
This is apt since GDP growth requires energy to bring about growth. No meaningful development can take place
308
without adequate and sustainable energy. 309 312 Dependent variable: GDPGRWT Dependent variable: GDPGRWT
Excluded
Chi-sq
df
Prob. EFOSST_
3.964188
2
0.1378
ELRESXHDY
12.25615
2
0.0022
TGHE_
1.160525
2
0.5598
PPGPLIT
2.896414
2
0.2350
All
25.00574
8
0.0016
Dependent variable: EFOSST_
Excluded
Chi-sq
df
Prob. GDPGRWT
0.757173
2
0.6848
ELRESXHDY
17.05571
2
0.0002
TGHE_
63.48471
2
0.0000
PPGPLIT
66.06707
2
0.0000
All
132.3742
8
0.0000
Dependent variable: ELRESXHDY
Excluded
Chi-sq
df
Prob. GDPGRWT
2.241062
2
0.3261
EFOSST_
0.492105
2
0.7819
TGHE_
19.23845
2
0.0001
PPGPLIT
27.99978
2
0.0000
All
52.51906
8
0.0000
Dependent variable: TGHE_ Excluded
Chi-sq
df
Prob. GDPGRWT
0.085927
2
0.9579
EFOSST_
0.744941
2
0.6890
ELRESXHDY
4.481621
2
0.1064
PPGPLIT
0.117292
2
0.9430
All
9.543200
8
0.2986
Dependent variable: PPGPLIT
Excluded
Chi-sq
df
Prob. GDPGRWT
1.735052
2
0.4200
EFOSST_
0.400585
2
0.8185
ELRESXHDY
0.697107
2
0.7057
TGHE_
0.124586
2
0.9396
All
2.688177
8
0.9524
Source. Author’s calculations
313 Additionally, from table 6. The lag order selection of five lags are adequate which are chosen by the model, which are
314
in tandem with the model aforementioned, according to the SC statistic. Hence the VAR is evaluated with lags interval
315
‘16’. 316 Table 6. Table 5. VAR Granger Test/ Wald Test. Lag Order selection
317
318
319
320
321
322
323
Lag
LogL
LR
FPE
AIC
SC
HQ
0
-174.4750
NA
110.2998
18.89210
19.14064
18.93416
1
-128.6505
62.70721*
13.76872*
16.70005*
18.19127*
16.95242*
* indicates lag order selected by the criterion
LR: sequential modified LR test statistic (each test at 5% level)
FPE: Final prediction error
AIC: Akaike information criterion
SC: Schwarz information criterion 317 From figure 4 presents the correlogram of the VAR model alongside the endogenous variables with the lag
324
interval of ‘16’. It presents five graphs of the endogenous parameter, which depict that the one or two of the matching
325
study unit autocorrelation are meaningful. Their significance confirms the importance of these variables to explaining
326
the dynamics in the electricity generation mix of Ghana. Some of the graphs show that some of the autocorrelations
327
is outside the interval with two standard errors bounds and the other graphs depicts that some of the autocorrelation
328
are outside the interval. Table 5. VAR Granger Test/ Wald Test. 329 -.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,PPGPLIT(-i))
Autocorrelations with 2 Std.Err. Bounds
330
Figure 4.Residual Correlogram of the VAR model. Source. Author’s construct. 331
VAR normality test is done to determine the distribution of the stochastic variables. It employs the Jacque
332
Bera residual normality test testing with the null hypothesis that the residuals are normally distributed and the
333
alternative hypothesis the residuals are not normally distributed. Bearing the residual from the components and their
334
P-Values it is concluded that they are normality distributed since they are generally not significant or more than 0.5%. 335
336
Table 7. Table 5. VAR Granger Test/ Wald Test. VAR Residual Normality test
337
Component
Skewness
Chi-sq
df
Prob. 1
1.053272
3.328143
1
0.0681
2
0.259359
0.201801
1
0.6533
3
0.417071
0.521844
1
0.4701
4
-0.073606
0.016254
1
0.8986
5
-0.501819
0.755466
1
0.3848
Joint
4.823508
5
0.4378
Component
Kurtosis
Chi-sq. df
Prob. 1
3.791337
0.469661
1
0.4931
2
2.319899
0.346903
1
0.5559
3
2.072214
0.645590
1
0.4217
4
2.982980
0.000217
1
0.9882
5
3.935924
0.656966
1
0.4176
Joint
2.119336
5
0.8324 -.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(GDPGRWT,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(EFOSST_,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(ELRESXHDY,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(TGHE_,PPGPLIT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,GDPGRWT(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,EFOSST_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,ELRESXHDY(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,TGHE_(-i))
-.8
-.4
.0
.4
.8
2
4
6
8
10
12
Cor(PPGPLIT,PPGPLIT(-i))
Autocorrelations with 2 Std.Err. Bounds
330
Figure 4.Residual Correlogram of the VAR model. Source. Author’s construct. 331 Figure 4.Residual Correlogram of the VAR model. Source. Author’s construct. Table 5. VAR Granger Test/ Wald Test. Additionally, the reaction of Generally,
349
there is a direct response of the endogenous variables to shocks, which attain a permanent effect that advance to new
350
steady state in the long run. The dynamic reactions of them ost of the endogenous parameters are varied nonetheless
351
they are reassuring, since it leads to increase in the electricity generation mix of Ghana and advocate the reversal of
352
trends of negative reactions that don’t impact on economic development. Endogenous parameters such as total
353
greenhouse emissions, high rising fossil fuels consumptions must be curbed since they are not sustainable generation
354
sources. It is worth mentioning that some these shocks are permeant not transitionary. The reaction of GDP growth
355
rate to shocks from global oil markets, the current energy crisis (the Russia -Ukraine conflict) bears permanently on
356
the economic growth of the economy in the short term to long term. 357 Source. Author’s calculations. Figure 4 shows the impulse response of the endogenous variables to shocks from 𝜖𝑡. These shocks are
340
macroeconomic and could be constructed to make positive contribution to the electricity sector in Ghana. The response
341
of TGHE to shocks from electricity from RES excluding hydropower started from a negative point initially and dipped
342
in the 3rd quarter and went on an increasing trajectory throughout the rest of time. It is projected RES capacity will
343
increase in the future when all projects come online. Also, the reaction of GDP growth rate from shocks from itself
344
leads to an initial expansion in growth and plunges in quarter two and continues in a steady pathway. The response of
345
GDP growth rate to total greenhouse gas emissions started on a positive pathway and peaked around quarter four and
346
continue on an upward trajectory. As the economy grows and relies heavily on fossil fuel sources, emissions levels
347
will rise. A similar situation is noted regarding price at the pump for gasoline per liter where it is reacted to shocks by
348
taking off in a higher trajectory and nosedived and continue in a steady fashion. Additionally, the reaction of Generally,
349
there is a direct response of the endogenous variables to shocks, which attain a permanent effect that advance to new
350
steady state in the long run. Table 5. VAR Granger Test/ Wald Test. VAR normality test is done to determine the distribution of the stochastic variables. It employs the Jacque
Bera residual normality test testing with the null hypothesis that the residuals are normally distributed and the
alternative hypothesis the residuals are not normally distributed. Bearing the residual from the components and their
P-Values it is concluded that they are normality distributed since they are generally not significant or more than 0.5%. Table 7. VAR Residual Normality test
Component
Skewness
Chi-sq
df
Prob. 1
1.053272
3.328143
1
0.0681
2
0.259359
0.201801
1
0.6533
3
0.417071
0.521844
1
0.4701
4
-0.073606
0.016254
1
0.8986
5
-0.501819
0.755466
1
0.3848
Joint
4.823508
5
0.4378
Component
Kurtosis
Chi-sq. df
Prob. 1
3.791337
0.469661
1
0.4931
2
2.319899
0.346903
1
0.5559
3
2.072214
0.645590
1
0.4217
4
2.982980
0.000217
1
0.9882
5
3.935924
0.656966
1
0.4176
Joint
2.119336
5
0.8324 Table 7. VAR Residual Normality test Component
Jarque-Bera
df
Prob. 1
3.797804
2
0.1497
2
0.548704
2
0.7601
3
1.167434
2
0.5578
4
0.016471
2
0.9918
5
1.412432
2
0.4935
Joint
6.942844
10
0.7308
338
Source. Author’s calculations. 339 Joint
6.942844
10
0.7308
338
Source. Author’s calculations. 339
Figure 4 shows the impulse response of the endogenous variables to shocks from 𝜖𝑡. These shocks are
340
macroeconomic and could be constructed to make positive contribution to the electricity sector in Ghana. The response
341
of TGHE to shocks from electricity from RES excluding hydropower started from a negative point initially and dipped
342
in the 3rd quarter and went on an increasing trajectory throughout the rest of time. It is projected RES capacity will
343
increase in the future when all projects come online. Also, the reaction of GDP growth rate from shocks from itself
344
leads to an initial expansion in growth and plunges in quarter two and continues in a steady pathway. The response of
345
GDP growth rate to total greenhouse gas emissions started on a positive pathway and peaked around quarter four and
346
continue on an upward trajectory. As the economy grows and relies heavily on fossil fuel sources, emissions levels
347
will rise. A similar situation is noted regarding price at the pump for gasoline per liter where it is reacted to shocks by
348
taking off in a higher trajectory and nosedived and continue in a steady fashion. Table 5. VAR Granger Test/ Wald Test. VD explains the amount of
361
variance that is ignited by the reaction of a variable to shocks from other variables and itself. The variance of GDP
362
rate to itself from shocks explains a greater percentage rate of all the variance of 100% and declining to around 10%. 363
Thus, the growth rate is very sensitive to shocks since these shocks are exogenous and impact directly on the economy. 364
Similarly, the variance of GDP to shocks from other endogenous parameters started off at less than 1% and increased
365
a bit to about 10% of more. In the same way, the variance explained by EFOSST due to shocks from different
366
endogenous variables is over 20% and some instances more than 70%. This means that EFOSST explains a greater
367
proportion of the variance owing to shocks coming to the electricity generation dynamics. Aside, the variance
368
explained by total greenhouse gas emissions (TGHE) to itself and from different variables varied around less than 1%
369
to nearly 70% in some cases. This equally depicts the greenhouse gas emissions levels of the country. Ghana ‘s THE
370
peaked around 2010 when the country began exploring for oil and gas. Table 5. VAR Granger Test/ Wald Test. Author’s estimation
360 -4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to EFOSST_
Re -4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to TGHE_
Response of EFOSST
to TGHE
S.E. Table 5. VAR Granger Test/ Wald Test. 371 -4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to GDPGRWT
-4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to EFOSST_
-4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to ELRESXHDY
-4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to TGHE_
-4
-2
0
2
4
2
4
6
8
10
Response of GDPGRWT to PPGPLIT
-10
-5
0
5
10
15
2
4
6
8
10
Response of EFOSST_ to GDPGRWT
-10
-5
0
5
10
15
2
4
6
8
10
Response of EFOSST_ to EFOSST_
-10
-5
0
5
10
15
2
4
6
8
10
Response of EFOSST_ to ELRESXHDY
-10
-5
0
5
10
15
2
4
6
8
10
Response of EFOSST_ to TGHE_
-10
-5
0
5
10
15
2
4
6
8
10
Response of EFOSST_ to PPGPLIT
-.01
.00
.01
2
4
6
8
10
Response of ELRESXHDY to GDPGRWT
-.01
.00
.01
2
4
6
8
10
Response of ELRESXHDY to EFOSST_
-.01
.00
.01
2
4
6
8
10
Response of ELRESXHDY to ELRESXHDY
-.01
.00
.01
2
4
6
8
10
Response of ELRESXHDY to TGHE_
-.01
.00
.01
2
4
6
8
10
Response of ELRESXHDY to PPGPLIT
-100
-50
0
50
100
150
2
4
6
8
10
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Response to Cholesky One S.D. Innovations ± 2 S.E. 359
Figure 5. Impulse Response Function(irf). Source. Table 5. VAR Granger Test/ Wald Test. -4
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R
f EFOSST
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59 -100
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ource. Table 5. VAR Granger Test/ Wald Test. The dynamic reactions of them ost of the endogenous parameters are varied nonetheless
351
they are reassuring, since it leads to increase in the electricity generation mix of Ghana and advocate the reversal of
352
trends of negative reactions that don’t impact on economic development. Endogenous parameters such as total
353
greenhouse emissions, high rising fossil fuels consumptions must be curbed since they are not sustainable generation
354
sources. It is worth mentioning that some these shocks are permeant not transitionary. The reaction of GDP growth
355
rate to shocks from global oil markets, the current energy crisis (the Russia -Ukraine conflict) bears permanently on
356
the economic growth of the economy in the short term to long term. Table 5. VAR Granger Test/ Wald Test. 357 358 -4
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Response to Cholesky One S.D. Innovations ± 2 S.E. 359
Figure 5. Impulse Response Function(irf). Source. Author’s estimation
360
From figure 6 is the variance decomposition results for the analysis (VD). Table 5. VAR Granger Test/ Wald Test. Author’s estimation -.4
.0
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ion -.4
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Response of PPGPLIT to ELRESXHDY Response of PPGPLIT to EFOSST_ Figure 5. Impulse Response Function(irf). Source. Author’s estimation From figure 6 is the variance decomposition results for the analysis (VD). VD explains the amount of
361
variance that is ignited by the reaction of a variable to shocks from other variables and itself. The variance of GDP
362
rate to itself from shocks explains a greater percentage rate of all the variance of 100% and declining to around 10%. 363
Thus, the growth rate is very sensitive to shocks since these shocks are exogenous and impact directly on the economy. 364
Similarly, the variance of GDP to shocks from other endogenous parameters started off at less than 1% and increased
365
a bit to about 10% of more. In the same way, the variance explained by EFOSST due to shocks from different
366
endogenous variables is over 20% and some instances more than 70%. This means that EFOSST explains a greater
367
proportion of the variance owing to shocks coming to the electricity generation dynamics. Aside, the variance
368
explained by total greenhouse gas emissions (TGHE) to itself and from different variables varied around less than 1%
369
to nearly 70% in some cases. This equally depicts the greenhouse gas emissions levels of the country. Ghana ‘s THE
370
peaked around 2010 when the country began exploring for oil and gas. 371 The variance connotes the importance of the supply aspect headwinds that account for the greater part of the
372
variance in the electricity generation within the economy. The chart greatly explains the narratives regarding the
373
headwinds and the amounts of variances caused by the variable itself or different variables. Table 5. VAR Granger Test/ Wald Test. 374 0
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0
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0
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Percent PPGPLIT variance due to PPGPLIT
Variance Decomposition
375
Figure 6. Table 5. VAR Granger Test/ Wald Test. Variance Decomposition Graphs. Source. Author’s Construct. 376 Figure 6. Variance Decomposition Graphs. Source. Author’s Construct. From figure 7 below, depicts the electricity generation sources of Ghana. It is obvious that the country has been
377
generation a lot from conventional energy sources such as Natural gas, bioenergy, hydropower and to a lesser extent
378
emerging RES. The data period depicted on the tree map is from 2000-2020. 379 From figure 7 below, depicts the electricity generation sources of Ghana. It is obvious that the country has been
377
generation a lot from conventional energy sources such as Natural gas, bioenergy, hydropower and to a lesser extent
378
emerging RES. The data period depicted on the tree map is from 2000-2020. 379 From figure 7 below, depicts the electricity generation sources of Ghana. It is obvious that the country has been
377
generation a lot from conventional energy sources such as Natural gas, bioenergy, hydropower and to a lesser extent
378
emerging RES. The data period depicted on the tree map is from 2000-2020. 379 From figure 7 below, depicts the electricity generation sources of Ghana. It is obvious that the country has been
377
generation a lot from conventional energy sources such as Natural gas, bioenergy, hydropower and to a lesser extent
378
emerging RES. The data period depicted on the tree map is from 2000-2020. 379 Figure 7. below depicts the cumulative electricity from oil and gas, and coal sources, which is forecasted to increase
380
to 8.8% from 2036 and beyond and installed renewable energy generation capacity for the same period. This implies
381
that the country still relies on the consumption of conventional energy sources. On the other hand, the right handed-
382
sided figure shows Ghana’s RE installed generation capacity will continue to grow despite reaching 6000 MW or
383
more. Hydropower forms a bigger part of the growth, with wind and solar coming next. This forecast is on point since
384
when pipeline projects come on stream. This implies total renewable energy installed capacity would have increased
385
by about 57.8% by 2036. 386 Figure 7. below depicts the cumulative electricity from oil and gas, and coal sources, which is forecasted to increase
380
to 8.8% from 2036 and beyond and installed renewable energy generation capacity for the same period. This implies
381
that the country still relies on the consumption of conventional energy sources. 5. Conclusion 5. Conclusion
397
The study made utilizes data from the WDI for the period of 2002 and 2021 to study the electricity dynamics
398
of Ghana. An unrestrictive VA model was deployed in the analysis. After the analysis it obvious that the Ghana
399
electricity sector is charccte4sied by fossil fuels consumptions that exposed the country to external shocks impacting
400
the macroeconomic stability of the country in the short term to long term eroding the gains made in building the
401
economy .Current shocks that brought untold hardships to the economy were the Covid pandemic as well as the
402
ongoing Russia-Ukraine aggression that has caused acute energy shortages in Ghana reverberating throughout the
403
entire economy. This shock will in no way impact on the GDP growth rate of the country. The variance to GDP growth
404
rate explained over 100% in reactions to shocks, as the analysis revealed. Additionally, Electricity from fossil fuels
405
sources equally explained a significant amount for variance in the variance decomposition as well as was significant
406
in the VAR model. This depicts the importance of fossil fuels to the electricity generation dynamics of the country. 407
This is a supply shock that impact directly on the generation of electricity. Mores, it explained about 96% of the
408
variance in reaction to shocks. This is rightly so as the current energy crisis has brought about queens at filling stations
409
in Ghana. 410 Furthermore, the analysis reveals that renewable energy sources without hydropower is very minimally
411
deployed in Ghana. It very explains very low variance in reactions to shocks depicting the low levels of RES generation
412
in the country, emerging energy sources per se, without traditional hydropower. Also, the granger causality analysis
413
shows a feedback relationship between fossil fuel electricity generation as well as GDP growth rate in the country. 414
Thus, both granger cause another and are significantly. The impulse response (irfs) analysis depicts that the parameters
415
reacted to shocks in a positive manner within the short to long-term keeping a steady trajectory going forward, only
416
one of nosedived to a negative trajectory. Overall, the variables are on an increasing trajectory reassuring their
417
importance to the electricity generation dynamics of the country. Table 5. VAR Granger Test/ Wald Test. On the other hand, the right handed-
382
sided figure shows Ghana’s RE installed generation capacity will continue to grow despite reaching 6000 MW or
383
more. Hydropower forms a bigger part of the growth, with wind and solar coming next. This forecast is on point since
384
when pipeline projects come on stream. This implies total renewable energy installed capacity would have increased
385
by about 57.8% by 2036. 386 388 389
Figure 7. Forecast of EFOSST and RE Installed Generation capacity. Source. Author’s construct. 390
0
10
20
30
40
2000
2010
2020
2030
2040
Year
EFOSST%
EFOSST% (efosst1 f_)
0
10000
20000
30000
2000
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2030
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Year
RE
RE (re f_) 389 Figure 7. Forecast of EFOSST and RE Installed Generation capacity. Source. Author’s construct. 391 Figure 8 Electricity Generation sources of Ghana Figure 8 Electricity Generation sources of Ghana
3 Figure 8 Electricity Generation sources of Ghana
393 396 5. Conclusion The results likewise determine that Ghana is energy
418
secure but that its energy consumption is from fossil fuels sources which is not sustainable. It exports power to its
419
neighbors. The inverse root AR characteristic polynomial analysis confirms the robustness of the model in the analysis
420
since all coordinates lie within the circle, satisfying the stability condition of the analysis. 421 Ultimately, the findings come with policy implications and consequences. They highlight the relevance of
422
wide raging policy toolbox to scaleup RES deployment by diversifying the country’s energy mix in the long run. The
423
energy sector’s indebted to over of $12B owed to IPPs can be avoided if the country deploys RES. RES are cheaper
424
and sustainable means of consumption. Similarly, the use of negative bidding process for conventional power
425
generation will reduce the energy sector. In this process, the bidders pay the state for the generation of electricity. 426
This will avoid the situation of excess capacity. This will result in a deep and sustained reductions in our total
427
greenhouse emissions levels as well as put the country on a pathway to achieving its NDCs. Mores so, there is the
428
need to reforms institutions and breakdown the institutional complex in the public sector, causing red tapeism and
429
stifling private sector initiative, especially in the deployment of RES. Besides, a robust regulatory environment is
430
important to transforming the electricity generation dynamics of Ghana. It is high time the county forms pressure
431 groups such as “The Ghana RES Association” to pressure government to scale up the deployment of RES. That w
432
protect country from exogenous shocks and promotes sustainable economic development. 433
434
Author contribution: All authors contributed to the study conception and design. David Alemzero
435
contributed data and helped wrote the final manuscript. Sampson Agyapong Atuahene aided in drafting
436
the original manuscript and helped in analysis and discussion. All authors read and approved the final
437
manuscript. 438
Funding: No funding was obtained
439
Availability of data and materials: The datasets used and/or analyzed during the current study are
440
available from the corresponding author on reasonable request. 441
Declarations
442
Ethics approval and consent to participate: Not applicable. 443
Consent for publication: Not applicable. 444
Competing interests: The authors declare no competing interests
445
446
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491
growth and carbon emissions: evidence from CIVETS countries. Applied Economics, 52(16),
492
1806-1815. 493
Rezaei, M., Kamal Chaharsooghi, S., & Abbaszadeh, P. (2013). The role of renewable energies in
494
sustainable development: case study Iran. Iranian (Iranica) Journal of Energy &
495
Environment, 4(4). 496
Robert F. ENGLE and Byung Sam YOO University of California, San Diego, CA 92093, U. Co-
497
Integration and Error Correction : Representation , Estimation ,. 55, 251–276 (2012). 498
Sack, B. (2000). Does the Fed act gradually? A VAR analysis. 5. Conclusion Journal of Monetary Economics, 46(1),
499
229-256. 500
Shahbaz, M., Sinha, A., Raghutla, C., & Vo, X. V. (2022). Decomposing scale and technique effects of
501
financial development and foreign direct investment on renewable energy
502
consumption. Energy, 238, 121758. 503
Sun, H., Khan, A. R., Bashir, A., Alemzero, D. A., Abbas, Q., & Abudu, H. (2020). Energy insecurity, pollution
504
mitigation, and renewable energy integration: prospective of wind energy in Ghana. Environmental Scienc
505
and Pollution Research, 27(30), 38259-38275. 506
Sun, H., Khan, A. R., Bashir, A., Alemzero, D. A., Abbas, Q., & Abudu, H. (2020). Energy insecurity, pollution
507
mitigation, and renewable energy integration: prospective of wind energy in Ghana. Environmental Scienc
508
and Pollution Research, 27(30), 38259-38275. 509
Tabiri, K. G., Adusah-Poku, F., & Novignon, J. (2022). Economic inequalities and rural-urban disparities in clean
510
cooking fuel use in Ghana. Energy for Sustainable Development, 68, 480-489. 511
Yao, X., Yi, B., Yu, Y., Fan, Y., & Zhu, L. (2020). Economic analysis of grid integration of variable solar and win
512
power with conventional power system. Applied Energy, 264, 114706. 513 Petrović-Ranđelović, M., Mitić, P., Zdravković, A., Cvetanović, D., & Cvetanović, S. (2020). Economic
491
growth and carbon emissions: evidence from CIVETS countries. Applied Economics, 52(16),
492
1806-1815. 493 Rezaei, M., Kamal Chaharsooghi, S., & Abbaszadeh, P. (2013). The role of renewable energies in
494
sustainable development: case study Iran. Iranian (Iranica) Journal of Energy &
495
Environment, 4(4). 496 Robert F. ENGLE and Byung Sam YOO University of California, San Diego, CA 92093, U. Co-
497
Integration and Error Correction : Representation , Estimation ,. 55, 251–276 (2012). 498 Sack, B. (2000). Does the Fed act gradually? A VAR analysis. Journal of Monetary Economics, 46(1),
499
229-256. 500 Shahbaz, M., Sinha, A., Raghutla, C., & Vo, X. V. (2022). Decomposing scale and technique effects of
501
financial development and foreign direct investment on renewable energy
502
consumption. Energy, 238, 121758. 503 Sun, H., Khan, A. R., Bashir, A., Alemzero, D. A., Abbas, Q., & Abudu, H. (2020). Energy insecurity, pollution
504
mitigation, and renewable energy integration: prospective of wind energy in Ghana. Environmental Science
505
and Pollution Research, 27(30), 38259-38275. 506 Sun, H., Khan, A. R., Bashir, A., Alemzero, D. A., Abbas, Q., & Abudu, H. (2020). Energy insecurity, pollution
507
mitigation, and renewable energy integration: prospective of wind energy in Ghana. 5. Conclusion Environmental Science
508
and Pollution Research, 27(30), 38259-38275. 509 Tabiri, K. G., Adusah-Poku, F., & Novignon, J. (2022). Economic inequalities and rural-urban disparities in clean
510
cooking fuel use in Ghana. Energy for Sustainable Development, 68, 480-489. 511 Yao, X., Yi, B., Yu, Y., Fan, Y., & Zhu, L. (2020). Economic analysis of grid integration of variable solar and wind
512
power with conventional power system. Applied Energy, 264, 114706. 513
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Psychosocial working conditions and the utilization of health care services
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BMC public health
| 2,011
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cc-by
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* Correspondence: s_azagba@live.concordia.ca
† Contributed equally
Department of Economics, Concordia University, 1455 de Maisonneuve Blvd.
West, Montréal, Quebec, H3G 1M8, Canada © 2011 Azagba and Sharaf; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Abstract Background: While there is considerable theoretical and empirical evidence on how job stress affects physical and
mental health, few studies have examined the association between job related stress and health care utilization. Using data from the Canadian National Population Health Survey from 2000 to 2008, this paper examines the
association between stressful working conditions, as measured by the job strain model, and the utilization of
health care services. Methods: A zero inflated negative binomial regression is used to examine the excess health care utilization due to
job strain. Separate regressions are estimated for both males and females since studies have shown gender
differences in health care utilization. Results: Estimates for the whole population show that high or medium job strain has a positive and statistically
significant association with the number of visits to both a general practitioner (GP) and a specialist (SP). On
average, the number of GP visits is up to 26% more (IRR = 1.26, 95% CI = 1.19-1.31) for individuals with high strain
jobs compared to those in the low job strain category. Similarly, SP visits are up to 27% more (IRR = 1.27, 95% CI =
1.14-142) for the high strain category. Results are quantitatively similar for males and females, save for medium
strain. In general, findings are robust to the inclusion of workplace social support, health status, provincial and
occupational-fixed effects. Conclusion: Job strain may be positively associated with the utilization of health care services. This suggests that
improving psychosocial working conditions and educating workers on stress-coping mechanisms could be
beneficial for the physical and mental health of workers. Psychosocial working conditions and the
utilization of health care services Sunday Azagba*† and Mesbah F Sharaf† Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Open Access Background Health care
services
(e.g., outpatient &
inpatient visits) Job strain
(High job demand
&
Low job control) Physical, mental
and emotional
illness Potential health-
risk behaviors
(e.g., smoking,
alcohol abuse,
drug use, physical
inactivity,
excessive eating) Potential health-
risk behaviors
(e.g., smoking,
alcohol abuse,
drug use, physical
inactivity,
excessive eating) Figure 1 A simplified model for the relationship between job stress and health care services. Source: Authors’ compilation. Health care costs account for a considerable portion
of GDP and have shown an upward trend over time in
many countries. For example, in Canada expenditure on
health care utilization as a percentage of GDP increased
from 7% in 1980 to 10.1% in 2007. In the US, total
health care expenditure amount to $2.5 trillion, $8,047
per person and this represents 17.3% of the GDP in
2009 [30]. This rose from 9% of GDP in 1980. linked to back pain, colorectal cancer [14], several infec-
tious disease and cardiovascular problems [15,16] and
can double the risk of heart attack [17]. Stress may also
exacerbate the symptoms of several illnesses including
headaches [18-20], diabetes [21], coronary heart disease
[22] and upper respiratory infections [13]. Third, job
stress may also affect health care usage indirectly by
inducing several health risk behaviors such as smoking,
drug and alcohol abuse [23,24]. Stress may discourage
some healthy behaviors like physical activity, proper diet
and it may reduce the consumption of healthy food like
fruits and vegetables [25] increase the consumption of
fatty and sweet foods [26]. linked to back pain, colorectal cancer [14], several infec-
tious disease and cardiovascular problems [15,16] and
can double the risk of heart attack [17]. Stress may also
exacerbate the symptoms of several illnesses including
headaches [18-20], diabetes [21], coronary heart disease
[22] and upper respiratory infections [13]. Third, job
stress may also affect health care usage indirectly by
inducing several health risk behaviors such as smoking,
drug and alcohol abuse [23,24]. Stress may discourage
some healthy behaviors like physical activity, proper diet
and it may reduce the consumption of healthy food like
fruits and vegetables [25] increase the consumption of
fatty and sweet foods [26]. Given the recent upward trend of health care costs
and the limited empirical research, this study uses
nationally representative data from the Canadian
National Population Health Survey (NPHS) to examine
the association between stressful working conditions
and the utilization of health care services. Background This study is
timely and germane to worldwide efforts aimed at cur-
tailing rising health care expenditures. While the association between job stress and health
care utilization is theoretically clear, the empirical evi-
dence is very limited. Even the few relevant studies have
focused mainly on the effect of major and minor stress-
ful life events and have used small samples that are not
necessarily representative of the whole population [8,27]. For example, Brantley et al. [28] examine the ability of
minor and major stressful life events to predict medical
utilization among 141 low-income, African American
family practice patients. They find that minor stressful
life events are a significant predictor of outpatient visits
but were unrelated to emergency department or inpati-
ent visits. In a related study, Manning et al. [29], using a
sample of 260 individuals from two different industries,
find that health care claims and costs were positively
related to stressful job events and strain. They also find
that environmental, stressor and strain variables
accounted for up to 16 percent of the variance in health
care costs and 21.5 percent of the variance in the num-
ber of health care claims. Background find that the health care expenditures of workers who
report high levels of stress are 46 percent greater than
workers with low levels of stress. There is considerable theoretical and empirical evidence
on how job stress negatively affects physical and mental
health [1]. Surprisingly, the relationship between job
stress and health care utilization has received little
attention. Stress has been widely cited as “the 20th cen-
tury epidemic” and a “worldwide epidemic” according to
the United Nations and the World Health Organization
[2]. In the U.S, 70 percent of employees consider the
work place a significant source of stress, and 51 percent
report that job stress reduces their productivity [3]. Gib-
son [4] estimated that the health care utilization induced
by stress costs U.S. companies $68 billion annually and
reduces their profits by 10 percent. Goetzel et al. [5] According to Karasek’s job strain model, the dominant
job stress theory, the combination of on-the-job high
psychological demands and low decision latitude lead to
physical and mental health problems [6]. Several studies
emphasize the importance of including stress as a deter-
minant in models of health service utilization [7-9]. Stress could be linked to increased usage of health care
services by a number of routes (see Figure 1). First, indi-
viduals may use medical services as a way to cope with
stress [10,11]. Second, job stress may cause physical ill-
ness, mental and emotional problems all of which
increase the demand for health care services. There is
medical evidence that stress can adversely affect an indi-
vidual’s immune system, thereby increasing the risk of
disease [12,13]. For example, excessive stress has been * Correspondence: s_azagba@live.concordia.ca
† Contributed equally
Department of Economics, Concordia University, 1455 de Maisonneuve Blvd. West, Montréal, Quebec, H3G 1M8, Canada Page 2 of 7 Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Job strain
(High job demand
&
Low job control)
Physical, mental
and emotional
illness
Health care
services
(e.g., outpatient &
inpatient visits)
Potential health-
risk behaviors
(e.g., smoking,
alcohol abuse,
drug use, physical
inactivity,
excessive eating)
Figure 1 A simplified model for the relationship between job stress and health care services. Source: Authors’ compilation. Statistical Analysis dependent variable of interest is measured by the num-
ber of visits to (1) a family doctor/general practitioner,
and (2) a specialist (excluding eye specialists) during
the year preceding the survey interview. Job strain, the
main independent variable of interest is an index
(score) that is derived by Statistics Canada from job-
related questions on decision (control) latitude made
up of skill discretion and decision authority, and psy-
chological demands. It is measured as a ratio of psy-
chological demands and decision latitude, where higher
values indicate greater job strain. Individuals are strati-
fied based on the distribution of scores into tertiles to
represent low (reference category), medium, and high
levels of strain. Multivariate analyses are used to investigate the associa-
tion between the intensity of health services utilization
and job related stress. Given that the outcome measures
(GP and SP visits) are positive integer variables (includ-
ing zeros for non users); count data models are more
suitable [31,32]. The benchmark for count data models
is a Poisson regression model, which has some restric-
tive assumptions that are often not satisfied in applied
work. For example, a Poisson regression assumes inde-
pendent count processes, and that the mean and var-
iance are equal (equidispersion). While a negative
binomial can correct for overdispersion, unobserved
individual heterogeneity due to excess zeros are not well
captured. Therefore, a zero-inflated negative binomial
regression may be more appropriate. Since negative
binomial and the zero-inflated negative binomial are not
nested models, the Vuong test is performed to deter-
mine the appropriate model. The test results show zero-
inflated negative binomial as the preferred model, hence
only the zero-inflated negative binomial results are
reported. A number of economic and socio-demographic vari-
ables commonly used in the literature are included in
the analysis. Age is represented in continuous form. Household income is represented by four dummy vari-
ables: low income, middle low income, middle high
income (reference category), and high income. Gender
is captured by a dummy variable (male = 1, female = 0). Four dummy variables represent individual educational
attainment: less than secondary, secondary, some post
secondary (reference category), and post secondary. Marital status is represented by three dummy variables:
married, separated and single (reference category). Smoking status is classified as: never smoker (reference
category), current smoker, and former smoker. Similarly,
never drinker (reference category), current drinker, and
former drinker represent drinking status. Data and Variables Description The data for this study come from the Statistics Canada
NPHS household component. NPHS is a nationally
representative sample of the Canadian population which
collects vital information on health related behavior, as
well as corresponding economic and socio-demographic
variables. This study uses data from cycle four (2000/01)
to cycle eight (2008/09). The sample is restricted to
adults aged 18-65 years since a large fraction of those
above 65 years are not working. Also, the frailty of
health for those over 65 years and unobserved health-
related issues may further complicate the results. After
excluding missing observations (2,445) and those who
are not working (8,237), the final sample includes
29,110 observations. Health services utilization, the Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Page 3 of 7 Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Page 3 of 7 Statistical Analysis Individual phy-
sical activity level is represented by three categories:
active, moderate, and inactive (reference category). Eth-
nicity is captured by a dummy variable (immigrant = 1,
Canadian born = 0). A measure of social support in the
workplace is included since it has been suggested as an
important stress modifier. A higher social support score
indicates lower workplace support. Health status is
represented by the individual health utility index (HUI)
which is a more objective measure than self-rated
health. HUI is a comprehensively-scored system for
measuring individuals’ functional health and a score of 1
indicates perfect health status. It was developed by the
Health Utilities Group, McMaster University. The num-
ber of chronic diseases for each individual is included
and having a regular family doctor is captured by a
dummy variable (reg_doc = 1, no reg_doc = 0). Provin-
cial dummy variables are included with British Colombia
as the reference category. To control for job-specific
effects, seven occupational categories are extracted from
the 2007 North American Industry Classification System
available in NPHS. An individual’s occupation is classi-
fied into one of seven groups: mechanical, trade, profes-
sional, managerial, health, service, and farm (reference
category). Results Table 1 reports the summary statistics of the variables
included in the analysis. About half (48%) of the sample
is female, 63% are married, 47% with post secondary Table 1 Summary statistics
Variables
Mean
S.D
Numerical variables
GP
2.610
4.228
SP
0.745
2.558
Age
40.089
12.076
Social support
4.005
1.915
Health utility index(hui)
0.922
0.127
Chronic conditions
1.228
1.346
Categorical variables
High strain
0.328
Medium strain
0.244
low strain
0.426
Male
0.524
Female
0.476
Single
0.264
Married
0.633
Separated
0.103
Less than secondary education
0.099
Secondary education
0.139
Some post secondary
0.285
Post secondary
0.475
Low income
0.034
Middle low income
0.124
Middle high income
0.345 Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Page 4 of 7 Page 4 of 7 Table 1 Summary statistics (Continued)
High income
0.497
Immigrants
0.150
Non immigrants
0.850
Never smoker
0.331
Current smoker
0.261
Former smoker
0.410
Never drinker
0.036
Current drinker
0.884
Former drinker
0.078
Regular doctor
0.84
No doctor
0.151
Active
0.250
Moderate
0.273
Inactive
0.477
Newfoundland
0.016
Prince Edwards
0.005
Nova Scotia
0.030
New Brunswick
0.024
Quebec
0.250
Ontario
0.370
Manitoba
0.036
Saskatchewan
0.032
Alberta
0.113
British Colombia
0.120
Mechanical
0.191
Trade
0.200
Professional
0.135
Managerial
0.174
Health
0.115
Farm
0.037
Service
0.144
N
29110
The statistics are weighted using the NPHS sampling weights. Numbers
represent percentage and for some variables they do not add up to 100 per
cent because of rounding. Table 1 Summary statistics (Continued) Table 1 Summary statistics (Continued)
High income
0.497
Immigrants
0.150
Non immigrants
0.850
Never smoker
0.331
Current smoker
0.261
Former smoker
0.410
Never drinker
0.036
Current drinker
0.884
Former drinker
0.078
Regular doctor
0.84
No doctor
0.151
Active
0.250
Moderate
0.273
Inactive
0.477
Newfoundland
0.016
Prince Edwards
0.005
Nova Scotia
0.030
New Brunswick
0.024
Quebec
0.250
Ontario
0.370
Manitoba
0.036
Saskatchewan
0.032
Alberta
0.113
British Colombia
0.120
Mechanical
0.191
Trade
0.200
Professional
0.135
Managerial
0.174
Health
0.115
Farm
0.037
Service
0.144
N
29110
The statistics are weighted using the NPHS sampling weights. Numbers
represent percentage and for some variables they do not add up to 100 per
cent because of rounding. analyses (Model 1) is the baseline specification, while
the second model includes an additional confounding
variable: workplace social support. In model 3, covari-
ates representing: individual’s health status, number of
chronic conditions, having a family doctor, province and
occupational fixed effects are included. Heterogeneous results by gender Heterogeneous results by gender
Estimates for the association between high job strain
and GP and SP services are similar for males and
females. For example, for males, GP and SP services uti-
lization increases by 26% (IRR = 1.26) for the high job
strain category compared with the low job strain cate-
gory in model 2. Similarly for females, the excess use of
GP and SP services due to high strain is 23% (IRR =
1.23). The association between medium strain and
health services utilization is statistically significant only
for females. For instance, compared with the low strain
category, GP and SP visits increases by 10% (IRR = 1.10,
95% CI = 1.04-1.17) and 22% (IRR = 1.22, 95% CI =
1.09-1.37, respectively. education, 46% are with high income, 85% are non-
immigrants, 37% are from Ontario, 48% are physically
inactive and 40% are working in mechanical and trade
occupations. The average age of individuals in the sam-
ple is 40 years. On average, individuals in the sample
visit general practitioners and specialists 2.61 and 0.75
times respectively. The average health utility index
(0.92) indicates a high health status for the Canadian
population. The unconditional analysis of health services
utilization according to job strain tertiles are shown in
Figure 2. This indicates that individuals in the high and
medium strain tertiles use more general practitioner and
specialist services than those on the low job strain
tertile. Results In general, the
results are qualitatively similar across the different speci-
fications, namely that job strain has a modest and statis-
tically significant association with the utilization of
health care services. Using the low job strain as the
reference category, the number of GP visits is 26% more
(IRR = 1.26, 95% confidence interval [CI] = 1.19-1.31)
(model 1) for individuals with high strain jobs. When
additional confounding variables are included (see
model 3), individuals in jobs with high strain on average
have 10% more (IRR = 1.10, 95% CI = 1.05-1.14) GP vis-
its than the low job strain category. Also, being in the
medium job strain category increases GP visits by 1.07
(95% CI = 1.02-1.12) and 1.01 (95% CI = 0.97-1.05) in
models 1 and 3, respectively. However, model 3 estimate
is not statistically significant as 1.0 is included in the CI. Expected SP visits increases by 27% (IRR = 1.27, 95% CI
= 1.14-1.42) (model 1) and 14% (IRR = 1.14, 95% CI =
1.04-1.25) (model 3) for the high job strain category
compared with the low job strain category. Furthermore,
for the medium job strain category, the number of SP
visits increases by 15% (IRR = 1.15, 95% CI = 1.04-1.28)
in model 1 and 11% (IRR = 1.11, 95% CI = 1.01-1.22) in
model 3. These results indicate that high/medium job
strain has a statistically significant association with SP
visits. In addition to the whole sample estimation, sepa-
rate analysis is performed for males and females since
studies have shown gender differences in health care uti-
lization, and the results are discussed in the next
session. The statistics are weighted using the NPHS sampling weights. Numbers
represent percentage and for some variables they do not add up to 100 per
cent because of rounding. The statistics are weighted using the NPHS sampling weights. Numbers
represent percentage and for some variables they do not add up to 100 per
cent because of rounding. Discussion The incidence-rate ratios (IRR) from the multivariate
analyses, which are adjusted for potential confounding
variables, are presented in Table 2. The first set of This study uses a nationally representative data from the
Canadian National Population Health Survey to examine Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 Page 5 of 7 0.86
0.77
0.63
3.10
2.52
2.30
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
High strain
Medium strain
Low strain
SP
GP
Figure 2 The unadjusted average health services utilization based on job strain categories. Source: Authors’ calculation based on
Canadian National Population Health Survey. Figure 2 The unadjusted average health services utilization based on job strain categories. Source: Authors’ calculation based on
Canadian National Population Health Survey. the association between job stress and the utilization of
health care services. There is substantial evidence that
job strain negatively affect physical and mental health
whether directly or indirectly. Nonetheless, the literature
on the association between job strain and health care
utilization is sparse. Even the few relevant studies have
focused mainly on the effect of major and minor stress-
ful life events and used small non-generalized samples. the association between job stress and the utilization of
health care services. There is substantial evidence that
job strain negatively affect physical and mental health
whether directly or indirectly. Nonetheless, the literature
on the association between job strain and health care
utilization is sparse. Even the few relevant studies have
focused mainly on the effect of major and minor stress-
ful life events and used small non-generalized samples. and policy makers. Countries are experiencing higher
spending on health care than the growth rate of their
economies. There are concerted efforts aimed at redu-
cing health care cost given the high levels reached in
recent times. Stressful working conditions may be asso-
ciated with higher use of health care services directly by
causing physical illness, mental and emotional problems. Medical evidence suggests that stress suppresses the
immune system, thereby increasing exposure to several
infectious diseases and cardiovascular problems. This table reports estimated coefficients (bi) of the zero-inflated negative binomial regression transformed to incidence-rate ratios (eβi) , *** p < 0.01, ** p <
0.05, * p < 0.1, confidence interval at 95% are in parentheses. Model 1 is the baseline specification, while model 2 includes an additional confounding variable,
workplace social support. In model 3, covariates representing: individual’s health status, number of chronic conditions, having a family doctor, province and
occupational fixed effects are included Discussion It may The increasing growth rate of health care spending in
many countries is of great importance among academics Table 2 Zero inflated negative binomial regression: incidence rate ratio (job strain and the use of health services)
General practitioner visits
Specialist visits
model(1)
model (2)
model (3)
model(1)
model (2)
model (3)
Whole population
High job strain
1.26***
(1.19-1.31)
1.23***
(1.18-1.29)
1.10***
(1.05-1.14)
1.27***
(1.14-1.42)
1.23***
(1.11-1.37)
1.14***
(1.04-1.25)
Medium job strain
1.07***
(1.02-1.12)
1.06***
(1.02-1.11)
1.01
(0.97-1.05)
1.15***
(1.04-1.28)
1.14***
(1.03-1.26)
1.11**
(1.01-1.22)
N
29110
29105
Males
High job strain
1.27***
(1.18-1.38)
1.26***
(1.17-1.36)
1.11***
(1.04-1.19)
1.28***
(1.07-1.54)
1.26***
(1.06-1.50)
1.16*
(1.00-1.34)
Medium job strain
1.03
(0.96-1.10)
1.02
(0.95-1.09)
0.96
(0.90-1.02)
1.04
(0.88-1.23)
1.04
(0.88-1.22)
1.00
(0.86-1.17)
N
14328
14324
Females
High job strain
1.24***
(1.17-1.31)
1.23***
(1.16-1.31)
1.10***
(1.05-1.16)
1.27***
(1.13-1.44)
1.23***
(1.08-1.39)
1.13**
(1.01-1.26)
Medium job strain
1.11***
(1.04-1.18)
1.10***
(1.04-1.17)
1.06**
(1.00-1.12)
1.24***
(1.10-1.39)
1.22***
(1.09-1.37)
1.19***
(1.06-1.33)
N
14782
14781
This table reports estimated coefficients (bi) of the zero-inflated negative binomial regression transformed to incidence-rate ratios (eβi) , *** p < 0.01, ** p <
0.05, * p < 0.1, confidence interval at 95% are in parentheses. Model 1 is the baseline specification, while model 2 includes an additional confounding variable,
workplace social support. In model 3, covariates representing: individual’s health status, number of chronic conditions, having a family doctor, province and
occupational fixed effects are included ative binomial regression: incidence rate ratio (job strain and the use of health services) This table reports estimated coefficients (bi) of the zero-inflated negative binomial regression transformed to incidence-rate ratios (eβi) , *** p < 0.01, ** p <
0.05, * p < 0.1, confidence interval at 95% are in parentheses. Model 1 is the baseline specification, while model 2 includes an additional confounding variable,
workplace social support. In model 3, covariates representing: individual’s health status, number of chronic conditions, having a family doctor, province and
occupational fixed effects are included Page 6 of 7 Azagba and Sharaf BMC Public Health 2011, 11:642
http://www.biomedcentral.com/1471-2458/11/642 also exacerbate symptoms of several illnesses including
headaches [18-20], coronary heart disease [21] and
upper respiratory infections [22]. Moreover, stress may
increase health care usage indirectly by inducing several
health risk behaviors such as smoking [23,24] and dis-
couraging healthy behaviors like physical activity. Indivi-
duals may also use medical services as a way to cope
with stress [10,11]. Received: 6 June 2011 Accepted: 11 August 2011
Published: 11 August 2011 Received: 6 June 2011 Accepted: 11 August 2011
Published: 11 August 2011 Authors’ contributions
B
h
h
ib
d Both authors contributed equally to this work. Both authors read and
approved the final manuscript. It is worth mentioning that the intensity of health care
services may be affected by the source of financing for
these services. In Canada, the health care system is pub-
licly financed, where citizens and permanent residents
are medically covered for inpatient and outpatient visits. Consequently, this may strengthen the association
between job strain and the intensity of using outpatient
visits. Discussion inclusion of the social support index in model 2
reduced the association between job strain and health
service usage. Studies have shown that social support
reduces strains, mitigates perceived stressors, and
moderates the stressor-strain relationship [34]. Pilisuk
et al. [35] found that stress increases utilization of out-
patient services and that social support helps in redu-
cing this effect. Similar results are found by Counte
and Glandon [9]. Results of both the conditional and unconditional ana-
lyses demonstrate that high job strain is associated with
higher health care utilization. On average, individuals in
jobs with high or medium strain use more health care
services than those in jobs with low strain. In particular,
the number of GP visits is up to 26% more for indivi-
duals with high strain jobs compared to those in the
low job strain category. Similarly, SP visits are up to
27% more for the high strain category. In general, the
results are robust to the inclusion of individual’s health
status, number of chronic conditions, having a family
doctor, province and occupational fixed effects. Results
also show that the medium strain has a statistically sig-
nificant association with the utilization of health services
only for females. This could be due to differences in
stress coping abilities between males and females. Research shows that there are gender differences in how
males and females perceive and cope with stressful
events. It has been argued that males usually tend to
use “problem-focused coping” and the “fight-or-flight”
response, while females may use “emotion-focused cop-
ing” and a “tend-and-befriend” response to stress [33]. Acknowledgements
Th
S This paper uses Statistics Canada confidential data, and the opinions
expressed do not represent the views of Statistics Canada. We thank Nikolay
Gospodinov, Ian Irvine, Tatyana Koreshkova and Gordon Fisher. No ethical
approval is needed for this study. Conclusions We find that high job stress is associated with higher
utilization of health care services. The findings of this
paper suggest that improving stressful working condi-
tions and educating workers on stress-coping mechan-
isms may help in reducing health care costs attributable
to psychosocial working conditions. The welfare gains
from these stress management programs are not limited
to reducing health care costs attributable to job stress. Other economic gains, for example, include increased
productivity among workers, reduction in absenteeism
and employee turnover in addition to other costs borne
by employers. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 6 June 2011 Accepted: 11 August 2011
Published: 11 August 2011 Received: 6 June 2011 Accepted: 11 August 2011
Published: 11 August 2011 Pre-publication history p
y
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/642/prepub The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/642/prepub 12. Brosschot JF, Benschop RJ, Godaert G, Olff M, DeSmet M, Heijnen CJ,
Ballieux RE: Influence of life stress on immunological reactivity to mild
psychological stress. Psychosomatic Medicine 1994, 56:216-224. doi:10.1186/1471-2458-11-642
Cite this article as: Azagba and Sharaf: Psychosocial working conditions
and the utilization of health care services. BMC Public Health 2011 11:642. y
g
y
13. Turner-Cobb JM, Steptoe A: Psychosocial stress and susceptibility to
upper respiratory tract illness in an adult population sample. Psychosomatic Medicine 1996, 58:404-412. 14. Health Canada: Best advice on stress risk management in the workplace
Health Canada 2000 [http://www.mtpinnacle.com/pdfs/Best-Advise-on-
Stress-Management.pdf]. 15. Rosengren A, Hawken S, Ounpuu S, Sliwa K, Zubaid M, Almahmeed WA,
Blackett KN, Sitthi-amorn C, Sato H, Yusuf S, INTERHEART investigators:
Association of psychosocial risk factors with risk of acute myocardial
infarction in 11119 cases and 13648 controls from 52 countries (the
INTERHEART study): case-control study. Lancet 2004, 364:953-962. 16. Chandola T, Britton A, Brunner E, Hemingway H, Malik M, Kumari M,
Badrick E, Kivimaki M, Marmot M: Work stress and coronary heart disease:
what are the mechanisms? European Heart Journal 2008, 29:640-648. Heart and Stroke Foundation of Canada: Report of health Stress 17. Heart and Stroke Foundation of Canada: Report of health Stress
threatening Canadians health, Heart and Stroke Foundation warns. 2000
[http://www.heartandstroke.com/site/apps/nlnet/content2.aspx? c=ikIQLcMWJtE&b=4955951&ct=4512825]. 18. Sorbi MJ, Maassen GH, Spierings E: A time-series analysis of daily hassles
and mood changes in the 3 days before the migraine attack. Behavioral
Medicine 1996, 22:102-113. 19. Benedittis GD, Lorenzetti A: The role of stressful life events in the
persistence of primary headache: Major events vs. daily hassles. Pain
1992, 51:35-42. 20. Fernandez E, Sheffield J: Relative contributions of life events versus daily
hassles to the frequency and intensity of headaches. Headache 1996,
36:595-602. 21. Kramer JR, Ledolter J, Manos GN, Bayless ML: Stress and metabolic control
in diabetes mellitus: Methodological issues and an illustrative analysis. Annals of Behavioral Medicine 2000, 22:17-28. 22. Twisk J, Snel J, Kemper H, van Mechelen W: Changes in daily hassles and
life events and the relationship with coronary heart disease risk factors:
A 2-year longitudinal study in 27-29-year-old males and females. Journal
of Psychosomatic Research 1999, 46:229-240. 23. Cohen S, Williamson GM: Perceived stress in a probability sample of the
United States. References . Brantley PJ, Ames SC: Psychobiology of Health and Disease. In 1. Brantley PJ, Ames SC: Psychobiology of Health and Disease. In
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k 1. Brantley PJ, Ames SC: Psychobiology of Health and Disease. In
Comprehensive handbook of psychopathology.. 3 edition. Edited by: Sutker
PB, Adams HE. New York, Plenum publishers; 2001:. This study has some limitations. First, though the
current study controls for potential confounders that
are widely used in the health care utilization literature,
there may be other potential confounders for which
the study did not control. Second, the outcome vari-
ables, SP and GP visits are self reported. However, this
is standard in the health care utilization literature. Third, the findings of the current study may not imply
causality. Hence, future research using prospective data
may be needed to recommend policy changes. Fourth,
the current study did not control for stress coping
ability, as there is no information available about this
in the data set. However, the current study controls
for social support, since it has been suggested that
stress interacts with other factors in influencing medi-
cal utilization. In line with previous studies, the 2. Scott C: Optimal stress. John Wiley and Sons Inc. Hoboken New Jersey;
2010. 3. American Psychological Association: Stress in America. 2009 [http://www. apa.org/news/press/releases/stress-exec-summary.pdf]. 4. Gibson V: Stress in the workplace: A hidden cost factor. HR Focus 1993,
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Wasserman J, The Health Enhancement Research Organization (HERO)
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Medicine 1998, 40(10):843-854. 6. Karasek R, Theorell T: Healthy work: stress, productivity, and the
reconstruction of working life. New York: Basic Books; 1990. 7. Mechanic D: Correlates of physician utilization: Why do major
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http://www.biomedcentral.com/1471-2458/11/642 33. Taylor SE, Klein LC, Lewis BP, Gruenewald TL, Gurung RA, Updegraff JA:
Biobehavioral responses to stress in females: tend-and befriend, not
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services:A time series and cross-sectional analysis. References Journal of Health and
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34. Viswesvaran C, Sanchez JI, Fisher J: The Role of Social Support in the
Process of Job Stress: A Meta-Analysis. Journal of Vocational Behavior
1999, 54(2):314-334. 9. Counte MA, Glandon GL: A panel study of life stress, social support, and
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29:348-361. 35. Pilisuk M, Boylan R, Acredolo C: Social support, life stress, and subsequent
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medical care utilization. Health Psychology 1987, 6:273-288. 10. Mechanic D, Volkart EH: Stress, illness behavior, and the sick role. American Sociological Review 1961, 26:51-58. 35. Pilisuk M, Boylan R, Acredolo C: Social support, life stress, and subsequent
medical care utilization. Health Psychology 1987, 6:273-288. 11. Tessler R, Mechanic D, Dimond M: The effect of psychological distress on
physician utilization: A prospective study. Journal of Health and Social
Behavior 1976, 17:353-364. Pre-publication history In the social psychology of health. Edited by: Spacapan S,
Oscamp S. Newbury Park, CA: Sage; 1988:31-67. Oscamp S. Newbury Park, CA: Sage; 1988:31-67. 24. American institute of stress:[http://www.stress.org/topic-effects.htm]. 25. Oliver G, Wardle J: Perceived effects of stress on food choice. Physiology &
Behavior 1999, 66(3):511-515. 25. Oliver G, Wardle J: Perceived effects of stress on food choice. Physiology &
Behavior 1999, 66(3):511-515. 26. Oliver G, Wardle J, Gibson EL: Stress and food choice: a laboratory study. Psychosomatic Medicine 2000, 62(6):853-865. 26. Oliver G, Wardle J, Gibson EL: Stress and food choice: a laboratory study. Psychosomatic Medicine 2000, 62(6):853-865. 27. Williams R, Zyzanski SJ, Wright AL: Life events and daily hassles and
uplifts as predictors of hospitalization and outpatient visits. Social Science
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and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 28. Brantley PJ, Dutton GR, Grothe KB, Bodenlos JS, Howe J, Jones GN: Minor
Life Events as Predictors of Medical Utilization in Low Income African
American Family Practice Patients. J Behav Med 2005, 28(4):395-401. y
nning MR, Jackson CN, Fusilier MR: Occupational Stress and Health 29. Manning MR, Jackson CN, Fusilier MR: Occupational Stress and Health
Care Use. Journal of Occupational Health Psychology 1996, 1(1):100-109. 30. Fritze J: Medical expenses have very steep rate of growth. USA Today
2010 [http://www.usatoday.com/news/health/2010-02-04-health-care-
costs_N.htm]. 31. Cameron AC, Trivedi PK: Regression Analysis of Count Data. New York:
Cambridge University Press; 1998. 32. Winkelmann R: Econometric Analysis of Count Data. Springer: Heidelberg,
New York;, 5 2008.
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Deliverable 8.1 Deliverable 8.1 Document History Version
Date
Document history/approvals
0.1
15/10/2021
First draft contents
0.2
18/10/2021
Version out for reviewing by the Project coordinator and the STM
0.3
01/11/2021
Version after reviewing
0.4
02/11/2021
Finalized draft ready to be submitted Project Website Project Website Project ref. no. H2020-SU-DS-2020 GA No 101019645
Project title
SECurity And privacy protectioN in Internet of Things devices
Duration of the project
1-09-2021 – 31-08-2024 (36 months)
WP/Task:
WP8/ T8.1, T8.2, T8.3
Dissemination level:
PUBLIC
Document due Date:
M02
Actual date of delivery
02/11/2021
Leader of this deliverable
8BELLS
Author (s)
Alexandros Tavernarakis/Grigoris Katrakazas (8 Bells)
Other Contributors
NΑ
Version
V0.4 D8.1 Project Website
Document History
Version
Date
Document history/approvals
0.1
15/10/2021
First draft contents
0.2
18/10/2021
Version out for reviewing by the Project coordinator and the STM
0.3
01/11/2021
Version after reviewing
0.4
02/11/2021
Finalized draft ready to be submitted D8.1 Project Website Document History DISCLAIMER The authors of this document have taken any available measure for its content to be accurate,
consistent and lawful. However, neither the project consortium as a whole nor the individual
partners that implicitly or explicitly participated in the creation and publication of this document hold
any sort of responsibility that might occur because of using its content. This document reflects only the author's views and the European Community is not responsible for
any use that may be made of the information it contains. Copyright ©SECANT Consortium, 2021 This work is licensed under the Creative Commons License “BY-NC-SA”. Page 2 of 18 GA-No 101019645 Page 2 of 18 D8.1 Project Website
List of acronyms
Item
Description
EC
European Commission
EU
European Union
MySQL
My Structured Query Language
PHP
Hypertext Preprocessor
SSL
Secure Sockets Layer
URL
Uniform Resource Locator
WP
Work Package
WPR
WordPress List of acronyms
Item
Description
EC
European Commission
EU
European Union
MySQL
My Structured Query Language
PHP
Hypertext Preprocessor
SSL
Secure Sockets Layer
URL
Uniform Resource Locator
WP
Work Package
WPR
WordPress D8.1 Project Website GA-No 101019645 Page 3 of 18 Page 3 of 18 D8.1 Project Website Table of contents
DOCUMENT HISTORY.................................................................................................................................................... 2
LIST OF ACRONYMS ...................................................................................................................................................... 3
TABLE OF CONTENTS .................................................................................................................................................... 4
EXECUTIVE SUMMARY .................................................................................................................................................. 5
1. INTRODUCTION .................................................................................................................................................... 6
1.1. PURPOSE ................................................................................................................................................................ 6
1.2. SCOPE AND INTENDED AUDIENCE ................................................................................................................................ 7
1.3. DEVELOPMENT INFORMATION .................................................................................................................................... 7
1.4. STRUCTURE ............................................................................................................................................................. 7
2. POLICY AND IDENTITY .......................................................................................................................................... 8
2.1. SECANT DISSEMINATION FRAMEWORK ....................................................................................................................... 8
2.1.1. Logo visual identity ..................................................................................................................................... 8
2.1.2. Website visual identity................................................................................................................................ 8
3. SECANT WEBSITE .................................................................................................................................................. 9
3.1. WEBSITE TECHNOLOGY & PLUGINS .............................................................................................................................. 9
3.2. DETAILED TECHNOLOGY PROFILE ............................................................................................................................... 10
3.3. WEBSITE STRUCTURE .............................................................................................................................................. 10
3.3.1. Home ........................................................................................................................................................ 11
3.3.2. In Brief ...................................................................................................................................................... 13
3.3.3. The Project ................................................................................................................................................ 13
3.3.4. Use cases .................................................................................................................................................. 14
3.3.5. Partners .................................................................................................................................................... 14
3.3.6. News and Events ....................................................................................................................................... 15
3.3.7. Contact ..................................................................................................................................................... 15
3.3.8. Privacy and Policy ..................................................................................................................................... 16
3.3.9. Accessibility menu..................................................................................................................................... 16
3.3.10. Social Media Links..................................................................................................................................... 16
4. CONCLUSIONS ..................................................................................................................................................... 18 D8.1 Project Website Table of contents 4. GA-No 101019645 Page 4 of 18 Page 4 of 18 D8.1 Project Website 1. Introduction The SECANT platform will enhance the capabilities of organisations’ stakeholders, i •
a collaborative threat intelligence collection, analysis and sharing; •
an innovative risk analysis specifically designed for interconnected nodes of an industria
ecosystem; •
an innovative risk analysis specifically designed for interconnected nodes of an industrial
ecosystem; y
;
•
a cutting-edge trust and accountability mechanisms for data protection and g
g
y
p
•
a security awareness training for more informed security choices. •
a security awareness training for more informed security choices. The proposed solution’s effectiveness and versatility will be validated in four realistic pilot use case
scenarios applied in the healthcare ecosystem. Ultimately, SECANT will contribute decisively
towards improving the readiness and resilience of the organisations against the crippling modern
cyber-threats, increasing the privacy, data protection and accountability across the entire
interconnected ICT ecosystem, and reducing the costs for security training in the European market. The SECANT project is expected to produce results which will be diffused towards the various
audiences using different communication and dissemination channels. The project website will
constitute one of the main channels and its creation is validated by the present report. The “D8.1 Project Website” deliverable report, composed within the scope of “Work Package 8:
Dissemination, Communication and Exploitation of Results”, provides information regarding the
design, specifications, content and delivery of the SECANT project website and its functionalities. Screenshots of the pages and content, that is currently available on the website, are included in this
report. The website will be constantly updated during the project lifecycle. Furthermore, the website
will be monitored with respect to its performance to ensure that it efficiently communicates the
SECANT objectives and disseminates its results to the external stakeholders. 1 More detailed information on how liaisons with external stakeholders will be included into the SECANT
deliverable 8.2 which will be submitted by the end of November 2021. Executive summary The current document presents the website and the social media accounts that were created and
will be used as the main channels to disseminate and communicate the SECANT project news,
results, and achievements. The website is available at the following address: https://secant-project.eu . The website has been
designed to be professional, market facing, crawlable and responsive. It contains information about
the project, its objectives, the challenges, and the use cases. It will be updated throughout the
project lifetime with information about the project, news, events and dissemination material. A twitter account was created using the name @SecantProject. A twitter account was created using the name @SecantProject. Finally, a LinkedIn account was created that will be used to post all project news. The link to the
account is the following: https://www.linkedin.com/in/secant-project/. The purpose of the document is to validate the creation and basic structure of the website and the
creation of the social media accounts. Possible modifications and improvements might be identified
in future to address any needs not identified at this stage of the project. The current report is a working document and will be updated throughout the lifecycle of the project
based on the provided feedback, dissemination needs and activities of the project. A final version
will be submitted at the end of the project. GA-No 101019645 Page 5 of 18 Page 5 of 18 1.3. Development information The website was created by Eight Bells LTD and is on air at the following URL: https://secant-
project.eu. 1.2. Scope and intended Audience A high quality, well-structured website is an essential tool to create awareness of project objectives
and results. Additionally, the dissemination activities can be communicated in a proper manner to
have an increased impact on a variety of audiences including the external stakeholders such as
academia, the general public, SMEs, etc. The scope of website is to define the fundamentals of an effective promotion and communication of
the project and its potential benefits which will lead to possible collaborations with various
organizations mainly in Europe, but also internationally. Furthermore, one of the most significant
goals of the website is to expand the visibility of the project to a wider ranged and diversified
audience as well as to disseminate its plans, actions and results by keeping up to date the relevant
stakeholders. The promotion of the above goals will be enhanced by the presence of SECANT to the social media
networks alongside with an efficient branding strategy2. D8.1 Project Website GA-No 101019645
Page 7
2 More details will be included in the general Dissemination & Communication plan (Deliverable 8.2). GA No 101019645
Page
2 More details will be included in the general Dissemination & Communication plan (Deliverable 8.2). 1.1. Purpose The objective of the website is to become the main source of information regarding the SECANT
project for the general public, the project partners and the EC, as well as to contribute to both its
awareness creation and marketing foundation stage. Moreover, the consortium is responsible to
keep a record of the website visitors as well as to update its content and posts in the SECANT
subsections/pages. This report includes relevant material about the design and structure of the SECANT website whose
purpose is to promote the goals and concepts of the project, along with highlighting its impact in
order to: A. Ensure that achievements and results of the project are promoted to the wider community of
relevant stakeholders and potential new shareholders through various dissemination
activities such as: A. Ensure that achievements and results of the project are promoted to the wider community of
relevant stakeholders and potential new shareholders through various dissemination
activities such as: Webinars and trainings to incubators and associations. •
Webinars and trainings to incubators and associations. •
Webinars and trainings to incubators and associations. •
Publications in conferences, journals, workshops, international events, and relevant
fora. •
Presentations in scientific events and conferences. •
Presentations in scientific events and conferences. •
Quarterly newsletters. •
Quarterly newsletters. B. Engage external stakeholders1 by promoting and communicating events such as:
T i l /Sh •
Exhibitions in industrial and scientific events. 1 More detailed information on how liaisons with external stakeholders will be included into the SECANT
deliverable 8.2 which will be submitted by the end of November 2021. GA-No 101019645 Page 6 of 18 2.1. SECANT Dissemination Framework The dissemination activities that are linked to the completion of the SECANT objectives and results
are highly served by external web-based means that will communicate the actions to the wide and
heterogeneous audience. To this end, a project website has been developed to disseminate the
project outcomes, objectives, significant achievements and public documentation such as
deliverables and scientific publications. The website will support this purpose throughout the whole
duration of the project and beyond as well. Figure 1. Final logo with text Figure 1. Final logo with text 1.4. Structure This report contains the following sections: •
Section 1 - Introduction: In this section, a summary and brief overview are provided to
describe the scope and the objectives of the website. •
Section 1 - Introduction: In this section, a summary and brief overview are provided to
describe the scope and the objectives of the website. •
Section 2 - Policy and Identity: This chapter includes a brief description of the visual identity
of the website. •
Section 3 - SECANT website: This section describes the structure and the logic that this
website has been created. •
Section 4 – Conclusion: The last section of the report summarises the purpose of the
document providing possible future work based on website statistics and feedback from the
external stakeholders. 2 More details will be included in the general Dissemination & Communication plan (Deliverable 8.2). GA-No 101019645 Page 7 of 18 Page 7 of 18 2.1.1. Logo visual identity The logo has been designed on the rationale of a vibrant and unique icon that reflects both its
mission and values. The logo is consisted of two parts: •
Icon part: contains a lock which represents the security and the shapes inside the lock and
means the relationships between different devices (IoT network). •
Icon part: contains a lock which represents the security and the shapes inside the lock and
means the relationships between different devices (IoT network). •
Text part: contains the title of the project. D8.1 Project Website
2. Policy and Identity
2.1. SECANT Dissemination Framework D8.1 Project Website 3. SECANT Website The SECANT website aims to become an effective tool for communicating the SECANT project’s
activities, innovation results and relevant content to different stakeholders. The website has been
designed to provide an easy navigation, accessible and user-friendly experience for its visitors, as
well as to display project information in a fine-looking format to engage stakeholder interest, while
its design is in line with the branding policy of SECANT. Additionally, text and pictures have been chosen with consideration to ensure the proper
communication of the project’s objectives. Furthermore, consideration has been given to link
appearance and feel of the website in conjunction with the project logo and brand to ensure
congruity. This website will be updated and maintained on a regular basis not only during its lifetime, but also
as a static webpage even after the completion of the project. This section of the deliverable presents the technical infrastructure that is used for hosting and
running the website. Furthermore, this section presents the features, functionalities, and content of
the SECANT website. 3.1. Website technology & plugins The project website is built in WordPress (WPR). WPR is a tool that offers loads of possibilities in
order to construct a functional, accessible, responsive and modern website that adequately
promotes the SECANT project. In the following table some important WPR related specifications are described. In the following table some important WPR related specifications are describe the following table some important WPR related specifications are described. Item
Content
Site URL:
Site URL: https://secant-project.eu
WPR Version:
WPR Version: 5.8.1
5.6.1 PHP Version: 7.4.11
PHP Version: 7.4.23
MySQL Version:
MySQL Version: 4.9.7 The website is structured in a way to allow navigation through the major points of the project. On the
one hand, it comprises static pages, with in varying content that give an overview of the project,
describe its objectives and use cases and present the consortium. On the other hand, it comprises
blog pages, dynamically updated with new posts that aim to provide a picture of the project’s current
status, inform the general audience on activities related to the SECANT project. A number of plugins have been installed in order to satisfy the multitude of requirements that
emerge in order to build a functional and attractive website that offers high quality user experience. 2.1.2. Website visual identity The SECANT website URL address is https://secant-project.eu. The choice of the eu-domain is
crucial to enhance their project visibility both within the EU single market and globally and highlight
its impact as well as the funding by the EC. The main colours that have been used on the website are in line with the colours of the logo which
are the HEX: # f36c24 and the HEX: #243c4c. More specifically, dark blue is associated with
expertise and stability and the orange is often used to draw attention. The relevant communication and dissemination activities of SECANT have to comply with the logo
and the colours that have been identified on the visual identity section. GA-No 101019645 Page 8 of 18 Page 8 of 18 D8.1 Project Website 3.2. Detailed Technology profile In order to achieve the functionalities presented in the previous section and fulfil the purposes of the
website, the following technology profile has been used. In order to achieve the functionalities presented in the previous section and fulfil the purposes of the
website, the following technology profile has been used. In order to achieve the functionalities presented in the previous section and fulfil the purposes of the
website, the following technology profile has been used. Item
Name
Link
Plugin
Elementor
https://el.wordpress.org/plugins/elementor/
Plugin
Contact Form 7
https://el.wordpress.org/plugins/contact-form-
7/
Plugin
Essential Addons for
Elementor
https://wordpress.org/plugins/essential-
addons-for-elementor-lite/
Plugin
myStickymenu
https://el.wordpress.org/plugins/mystickymenu/
Plugin
Premium Addons for
Elementor
https://wordpress.org/plugins/premium-
addons-for-elementor/
Security Plugin
Akismet Anti-Spam
https://el.wordpress.org/plugins/akismet/
Theme
OceanWP
https://oceanwp.org/
Content
Management
System
WordPress 5.8, WordPress
https://el.wordpress.org/
JavaScript
Libraries and
Functions
jQuery, PHP
https://jquery.com/, https://www.php.net/
SSL Certificates
SSL by Default
Web Servers
Apache, Apache 2.4 D8.1 Project Website As part of the future development of the website and in order to enhance the website security, it is
planned to add more layers of security. This progress will be reported into the forthcoming
deliverables. As part of the future development of the website and in order to enhance the website security, it is
planned to add more layers of security. This progress will be reported into the forthcoming
deliverables. Finally, the pictures that are included in the project website have been acquired by 8BELLS, royalty
free and licensed by WordPress Consulting and are in line with the dissemination strategy. 3. SECANT Website In the following, we present the functionalities of some of the most important plugins: •
Responsiveness: since nowadays the number of mobile users has undergone a tremendous
rise and so has the variety of devices with internet access (tablets, laptops, PCs, mobiles
etc.), one of the most important features of a modern website is to be responsive; that is to
adapt to the screen size of each possible type of device, through which the user may access
the website. For that reason, a responsive slider was selected, along with several other
WPR settings that ensure the responsiveness of the SECANT website. g
p
•
Anti-Spam: a WPR plugin that protects the website from spam. •
Google Analytics: a WPR plugin that connects Google Analytics with the website, enabling
the administrators to see how visitors find and use the website, discover possible
shortcomings or sources of enthusiasm in the website and manage it in way to increase user
satisfaction. GA-No 101019645 Page 9 of 18 Page 9 of 18 D8.1 Project Website The main menu of the website has the following links: The main menu of the website has the following links: g
•
Home (the house icon)
•
In Brief
•
The project
•
Use cases
•
Partners
•
News & Events
•
Contact •
Home (the house icon)
•
In Brief
•
The project
•
Use cases
•
Partners
•
News & Events
•
Contact rthermore, the website includes four sub-pages to facilitate its navigation to the visitors Furthermore, the website includes four sub-pages to facilitate its navigation to the visitors. The structure of the project website is presented in the following Figure 3. e structure of the project website is presented in the following Figure 3. The structure of the project website is presented in the following Figure 3. Figure 3: Website structure
3.3.1. Home
At the home page each visitor can get useful information about the project such as the main pillars
of it and the involved partners and countries via the SECANT homepage. The project logo is presented on the header and left area while the social media links are located on
the bottom of the homepage. The footer of the home page includes the EU logo, the SECANT project disclaimer, the privacy
policy as well as links for the social media profiles associated to the project Figure 3: Website structure Figure 3: Website structure Figure 3: Website structure 3.3. Website Structure The website is designed to achieve the easy navigation of both the pages and information that the
users request. The SECANT website has one horizontal menu as depicted in Figure 2. The SECANT website has one horizontal menu as depicted in Figure 2. GA-No 101019645 Page 10 of 18 3.3.1. Home At the home page each visitor can get useful information about the project such as the main pillars
of it and the involved partners and countries via the SECANT homepage. At the home page each visitor can get useful information about the project such as the main pillars
of it and the involved partners and countries via the SECANT homepage. The project logo is presented on the header and left area while the social media links are located on
the bottom of the homepage. The footer of the home page includes the EU logo, the SECANT project disclaimer, the privacy
policy as well as links for the social media profiles associated to the project. GA-No 101019645 Page 11 of 18 Page 11 of 18 D8.1 Project Website 3.3.3. The Project This page and the respective sub-pages summarise the motivation, challenges and vision,
objectives and work packages as whole information of SECANT project. This page and the respective sub-pages summarise the motivation, challenges and vision,
objectives and work packages as whole information of SECANT project. Figure 6: ‘’The project’’ page 3.3.2. In Brief The ‘’In Brief’’ section contains a short summary of the objective as well as other useful information
about the project. The ‘’In Brief’’ section contains a short summary of the objective as well as other useful information
about the project. D8.1 Project Website Figure 6: ‘’The project’’ page Figure 4: ‘’Home’’ page Figure 4: ‘’Home’’ page GA-No 101019645 Page 12 of 18 Page 12 of 18 D8.1 Project Website Figure 7: ‘’Use Cases” page Figure 7: ‘’Use Cases” page Figure 6: ‘’The project’’ page Page 13 of 18 GA-No 101019645 3.3.4. Use cases The use cases page includes a brief summary and the demonstration setup for each of the four use
cases The use cases page includes a brief summary and the demonstration setup for each of the four use
cases. p g
y
p
cases. ν
Figure 7: ‘’Use Cases” page
3.3.5. Partners
Using this page, website users have access to the general profiles and the official logos of the
partners involved in the SECANT project, as well as their role and contribution on it. Figure 9: “News & Events” page Figure 9: “News & Events” page D8.1 Project Website 3.3.5. Partners Using this page, website users have access to the general profiles and the official logos of the
partners involved in the SECANT project, as well as their role and contribution on it. Using this page, website users have access to the general profiles and the official logos of the
partners involved in the SECANT project, as well as their role and contribution on it. Figure 8: ‘’Partners’’ page Figure 8: ‘’Partners’’ page GA-No 101019645 Page 14 of 18 Figure 10: ‘’Contact’’ page 3.3.6. News and Events The News & Events page will include any major news or events that are related with the SECANT
project The News & Events page will include any major news or events that are related with the SECANT
project. Figure 9: “News & Events” page Figure 9: “News & Events” page 3.3.8. Privacy and Policy acy and Policy page provides information about the protection of your personal data. The Privacy and Policy page provides information about the protection of your personal data. Figure 11: ‘’Privacy and policy’’ page Figure 11: ‘’Privacy and policy’’ page Figure 11: ‘’Privacy and policy’’ page D8.1 Project Website 3.3.9. Accessibility menu One of the most significant functionalities of the website is the dedicated section with accessibility
features which allows the user to facilitate customising the content representation based on the
special needs of each visitor ensuring that the project does not exclude anyone from accessing its
results and updates. Figure 12: Accessibility website menu Figure 12: Accessibility website menu Figure 12: Accessibility website menu
3.3.10. Social Media Links
Through the website navigation, the user can easily access the social media accounts of SECANT. The respective links for each of the social media are noted below:
•
Twitter: https://twitter.com/SecantProject
•
LinkedIn: https://www.linkedin.com/in/secant-project Figure 12: Accessibility website menu 3.3.7. Contact By navigating to this page, website users can communicate directly to the Project Coordinator either
via email or by using a communication form to provide general feedback or questions regarding the
project. Figure 10: ‘’Contact’’ page Figure 10: ‘’Contact’’ page Figure 10: ‘’Contact’’ page Figure 10: ‘’Contact’’ page Page 15 of 18 Page 15 of 18 GA-No 101019645 3.3.10. 3.3.10. Social Media Links
Through the website navigation, the user can easily access the social media accounts of SECANT. The respective links for each of the social media are noted below: Through the website navigation, the user can easily access the social media accounts of SECANT. The respective links for each of the social media are noted below: The respective links for each of the social media are noted below: •
Twitter: https://twitter.com/SecantProject •
LinkedIn: https://www.linkedin.com/in/secant-project Page 16 of 18 GA-No 101019645 Page 16 of 18 D8.1 Project Website Figure 13: Links to the project’s social media channels (LinkedIn and Twitter) located in the footer of the website. Figure 13: Links to the project’s social media channels (LinkedIn and Twitter) located in the footer of the website. D8.1 Project Website GA-No 101019645 Page 17 of 18 Page 17 of 18 4. Conclusions The main purpose of the current deliverable, ‘’D8.1 Project Website’’ is the creation of a reliable
report that defines: a) the main functionalities, features and content of the SECANT website, a) the main functionalities, features and content of the SECANT website, )
b) its visual identity and the technology that was used for the development of th
alongside with its core functionalities. The SECANT website was created taking under consideration the following tw e SECANT website was created taking under consideration the following two main pilla website was created taking under consideration the following two main pillars: 1) to provide a high-quality user experience to both experts and non-experts by combin
accessibility, attractive graphic design, security, and optimal usability and 2) to provide a comprehensive picture of the achievements and updates of the project. The
SECANT website contains all the relevant information related to the project such as
objectives, consortium contacts, news and use cases details to contribute to the purposes of
WP8. 2) to provide a comprehensive picture of the achievements and updates of the project. The
SECANT website contains all the relevant information related to the project such as
objectives, consortium contacts, news and use cases details to contribute to the purposes of
WP8. Furthermore, it is important to realise that the project website is in a very early stage. Therefore, the
website is expected to keep its capacity to be adapted by adding additional features depending on
the dissemination strategy and project branding. Furthermore, it is important to realise that the project website is in a very early stage. Therefore, the
website is expected to keep its capacity to be adapted by adding additional features depending on
the dissemination strategy and project branding. As one of the most significant dissemination tools, the website will be regularly updated by the
Dissemination Manager with the latest news, information and relevant documents even after the
completion of the project. GA-No 101019645 Page 18 of 18 Page 18 of 18
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Structure of executive functions in young and in older persons
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Abstract a1111111111
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a1111111111 Methods & results Editor: Antonio Verdejo-Garcı´a, Monash University,
AUSTRALIA Editor: Antonio Verdejo-Garcı´a, Monash University,
AUSTRALIA Young and older volunteers completed cognitive tests of the purported executive functions
shifting, updating, inhibition and dual-tasking (two tests per function). Confirmatory and
exploratory factor analyses yielded, for either age group, factorial structures that were within
the range reported in literature. More importantly, when correlations between tests were
sorted in ascending order, and were then fitted them by piecewise linear regression with a
breakpoint, there was no evidence for a distinct breakpoint between low and high correla-
tions in either age group. Correlations between tests were significantly higher in older com-
pared to young participants, and the pattern of test pairs with high and with low correlations
differed between age groups. Received: October 12, 2018
Accepted: April 15, 2019
Published: May 9, 2019 Received: October 12, 2018
Accepted: April 15, 2019
Published: May 9, 2019
Copyright: © 2019 Bock et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Copyright: © 2019 Bock et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. OPEN ACCESS Citation: Bock O, Haeger M, Voelcker-Rehage C
(2019) Structure of executive functions in young
and in older persons. PLoS ONE 14(5): e0216149. https://doi.org/10.1371/journal.pone.0216149 Otmar BockID1,2*, Mathias Haeger1, Claudia Voelcker-Rehage2 Otmar BockID1,2*, Mathias Haeger1, Claudia Voelcker-Rehage2 1 Institute of Physiology and Anatomy, German Sport University, Ko¨ln, Germany, 2 Institute of Human
Movement Science and Health, Technical University Chemnitz, Chemnitz, Germany * bock@dshs-koeln.de RESEARCH ARTICLE Introduction Using factor analysis, several studies reported that higher-order cognitive control involves
separable executive functions. However, the number and definition of the purported func-
tions differed between studies. One possible explanation for this discrepancy is that execu-
tive functions don’t exhibit a clear factorial structure, i.e., there is no clear dichotomy
between executive function tests which are well-correlated (representing a common factor)
and those which are poorly correlated (representing distinct factors). We scrutinize this
explanation separately in data from young and from older persons. Introduction It has been proposed decades ago that human cognition is coordinated and supervised by a
higher-order mechanism, probably residing in the frontal cortex [1,2]. This mechanism has
later been formalized as “supervisory attention system” [3] or “central executive” [4]. Later
authors argued that a monolithic supervisory mechanism is nothing more than a “homuncu-
lus” with little explanatory value [5,6] and instead proposed the existence of multiple supervi-
sory processes, often under the umbrella term ‘executive functions’. As an example, one of
the most influential studies in this field stipulated three executive functions, ‘updating of work-
ing memory contents’, ‘shifting between tasks or mental sets’ and ‘inhibition of prepotent
responses’ [7]. To establish their concept, Miyake et al. [7] asked participants to complete three cognitive
tests which quantified their ability for updating, three which assessed shifting and three which
registered their ability for inhibition. The resultant test scores were submitted to confirmatory
factor analyses (CFAs), a technique which determines the goodness-of-fit between experimen-
tal data and a pre-established factorial model. The authors compared several alternative mod-
els and found the best fit with a model that consisted of three mutually correlated factors, one
associated with tests on updating, one with those on shifting and one with those on inhibition. It is important to note that CFA-based approaches have a propensity for self-fulfilling
prophecy. They can determine which among several pre-established factorial models fits the
available data best, but they are unable to derive a new, even-better-fitting model. Miyake et al. [7] overcame this problem by calculating not only CFAs, but also an exploratory factor analysis
(EFA). This approach is less susceptible to self-fulfilling prophecy since it calculates the best-
fitting factorial model without recourse to any preconceived model. EFA yielded three mutu-
ally correlated factors as well, one closely associated with tests on updating, one with those on
shifting and one with those on inhibition. This outcome therefore seems to support the exis-
tence of the three executive functions stipulated by Miyake et al. [7]. However, the same author
group later presented an alternative factorial model, which replaced ‘inhibition’ by ‘common
executive function’, i.e., by a factor that was associated with all nine cognitive tests [8]. Participants of Miyake et al. [7] also completed a test of dual-tasking. Discussion Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. The absence of a breakpoint indicates that executive function tests don’t segregate into
well-correlated and poorly correlated pairs, and therefore are not well suited for factor analy-
ses. We suggest that executive functions are better described as a partly overlapping rather
than a factorial structure. The increase of correlations in older participants supports the exis-
tence of age-related dedifferentiation, and the dissimilarity of correlations in the two age
groups supports the existence of age-related reorganization. Funding: Funded by OB: BO 649/22-1. CVR: VO
1432/19-1. Both from German Research
Foundation (Special research program SPP 1772). URL: www.dfg.de. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. Competing interests: The authors have declared
that no competing interests exist. 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Introduction The scores on that test
were not associated significantly with any of the three CFA factors; it therefore has been sug-
gested that dual-tasking may represent a distinct executive function [9]. Accordingly, some
recent studies included dual-tasking as a fourth executive function (cf. [10,11]). Yet other stud-
ies stipulated alternative executive functions such as ‘rule detection’, ‘concept formation’, ‘stra-
tegic planning’, ‘estimation’ and ‘emotional control’ (cf. [12]). Summing up, earlier research presented conflicting views about the factorial structure of
executive functions. One possible explanation for this disagreement is that factor analyses, in
general, are not well suited for the data at hand. Simply speaking, factor analyses aggregate
well-correlated variables to a common factor and segregate poorly correlated variables to dis-
tinct factors; this works well if some of the correlations are markedly higher than others, but it
becomes problematic if there is no clear dichotomy between high and low correlations. With-
out such a dichotomy, even small fluctuations of the correlation pattern could substantially
modify the best-fitting factorial model, which could explain the discrepancies in literature. A
glance at published correlations between executive-functions [13–15,7] reveals no easily dis-
cernible dichotomy. One purpose of the present study was, therefore, to scrutinize this obser-
vation in a quantitative fashion. Above research on executive functions dealt with young participants, leaving open how the
structure of those functions changes with advancing age. It is well established that older per-
sons perform less well than young ones on a range of executive function tests (cf. meta- 2 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions analyses by [16–18]([19,20], but poorer performance as such does not necessarily imply a
changed factorial and/or correlational structure. However, that structure could be affected by
two other age-related phenomena. One of them is cognitive dedifferentiation, which manifests
as increased correlation between the performance scores on different cognitive tasks [21,22]. Age-related dedifferentiation probably results from biological decay, cultural influences and
lifelong experience [23–25], and corresponds at the neuronal level to the recruitment of larger
and more overlapping brain areas for a given task [26]. The other phenomenon is cognitive
reorganization, i.e. the engagement of different cognitive processes [27] and brain areas [28]
for the same cognitive task. Introduction Both phenomena are likely to impact the structure of executive
functions: age-related dedifferentiation should reduce the number of factors and/or increase
the correlations between test pairs, while age-related reorganization should decrease the corre-
lations between some test pairs and might increase those between some other test pairs. Several studies used CFA-based approaches similar to Miyake et al. [7] to evaluate the struc-
ture of executive functions in healthy older adults. In one study [29], the best fitting model had
the same tri-factorial structure as that of Miyake et al. [7]. Four other studies yielded best fits
with bi-factorial models which combined updating and shifting [30,15] or updating and inhi-
bition [31] to a single factor, or stipulated the executive functions ‘working memory’ and
‘access to long-term memory’ [13]. Finally, one study reported the best fit with a single-factor
model [14]. Taken together, these studies tell us little about the structure of executive functions
in older compared to young adults. The number and meaning of factors varied between stud-
ies, as they did in research with young participants, and consistent differences between age
groups are therefore difficult to identify. To get a clearer picture about age-related differences,
it might be helpful to administer executive function tests to young and older persons in one
and the same study, thus eliminating differences between the selected tests, instructions, set-
tings and experimenters’ personalities. We are aware of only two factor analytical studies which took such an approach. One of
them used CFA and yielded a model with two factors [13], while the other calculated EFA and
yielded a model with four factors [32]. Importantly, both studies calculated common models
for participants of all ages and treated ‘age’ as a mediating variable. As a consequence, both
studies accounted for the effects of age on factor loadings, but neglected the effects of age on
the factorial and/or correlational structure of executive functions. For example, if young per-
sons were best characterized as having four but older ones as having two executive functions,
the common models calculated by both studies would be oblivious of this fact. The second pur-
pose of the present study was, therefore, to find out whether the structure of executive func-
tions is the same for young and for older persons. To our knowledge, no study has done this
before. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Introduction We expected that in older age, correlations between test pairs will be generally higher
because of age-related dedifferentiation, and the pattern of relatively high and relatively low
correlations will be different because of age-related reorganization. Participants’ performance on executive function tests is often quantified in terms of reac-
tion time. However, this metric may be biased since it doesn’t account for the existence of a
speed-accuracy tradeoff: when responding in a cognitive test, we can be quick at the expense
of accuracy or accurate at the expense of speed [33,34]. This is particularly relevant for studies
which compare young and older individuals, since older persons place a stronger emphasis
on accuracy [35,36]. An age-related increase of reaction time may therefore reflect not only a
deficit of the targeted cognitive function, but also a shift of emphasis from fast to accurate
responses. A second bias may arise because reaction time is sensitive not only to the speed of the tar-
geted cognitive function, but also to the speed of upstream sensory and low-level cognitive
processes [37]. Since processing speed generally decreases in older age [38], an age-related 3 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions increase of reaction time may reflect such generalized slowing rather than a specific deficit of
the targeted cognitive function. It has indeed been shown that the decay of executive functions
in older age is partly attributable to decreasing psychomotor speed [39–41]. increase of reaction time may reflect such generalized slowing rather than a specific deficit of
the targeted cognitive function. It has indeed been shown that the decay of executive functions
in older age is partly attributable to decreasing psychomotor speed [39–41]. It is unknown to what extend the two phenomena, shifts of the speed-accuracy tradeoff and
generalized slowing, are mutually independent. We therefore decided to control for both in
our study. Participants Sixty-three young (age 20–30 years; M = 23.17, SD = 2.83, females = 40) and 61 older adults
(age 65–75 years; M = 69.97, SD = 2.96, females = 22) volunteered to participate in a larger
research project on various aspects of cognitive aging, of which the present study was a smaller
part. They were recruited by paper and electronic postings, and by contacts with local senior
networks in Ko¨ln and in Chemnitz. 39 young and 29 older participants were tested in Ko¨ln
and the remaining ones in Chemnitz, using the same hard- and software and the same formal-
ized instructions. All participants were in good physical and mental health by self-report, had
no history of stroke or brain surgery, and their visual acuity—as assessed by the Freiburg
Vision Test (FrACT version 3.9.0)—was better than 20/60, which is sufficient for safe car driv-
ing [42]. Screening tests ensured that participants didn’t suffer from cognitive impairment
(Mini Mental State Examination > 26 points), from language comprehension deficits (Frei-
burger Sprachversta¨ndlichkeitstest > 50% word recognition at best hearing level) or obesity
(cutoff: BMI 30). The Edinburgh Handedness Inventory [43] revealed that five participants
were left-handed, one was ambidextrous but used the right hand for typing, and all others
were right-handed. Persons who wore contact lenses, prescription glasses or hearing aids in
everyday life did so as well during the tests. This study was carried out in accordance with the
Declaration of Helsinki. All participants signed a written informed-consent statement. The
research protocol was pre-approved by the Ethics Commission of the German Sport Univer-
sity (approval # 27/2015). Participants received a compensation of 60€ in total. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Tests In test switch-semantic, 17 words were sequentially displayed in the center of the
screen for 1500 ms each, with an ISI of 800 to 1200 ms. A central fixation cross was displayed
during each ISI. Participants had to press either the “M” key with their right or the “X” key
with their left index finger, to indicate whether the current word was mono- or bi-syllabic
(subtask A), or whether it denoted an inanimate or a living object (subtask B). The order of
subtasks on each 17-word trial was AABBAABBAABBAABBA, without external cues about
subtask order [20]. The test consisted of six trials, with pauses as in the n-back test. Outcome
measures were the percentage of correct responses and the switching costs across all trials. Switching costs were calculated as the difference between the mean latency of correct
responses after a subtask switch minus that after a subtask repetition, discarding the first stim-
ulus on each trial. Responses outside a time window of 2000 ms after stimulus onset were con-
sidered as ‘incorrect’. Test switch-spatial was similar, except that words were replaced by geometrical shapes and
participants had to discriminate between circular and quadratic shapes (subtask A) or between
big and small shapes (subtask B). Inhibition was registered by a Simon and a Stroop test, both adapted from literature [48–
50]. In the Simon test, 32 left- or rightward pointing arrows were sequentially presented to the
left or right of the screen center for 500 ms each, with an ISI of 800 to 1200 ms. Arrow presen-
tation again alternated with a central fixation cross. On one-half of the trials, position and
direction of the arrow were compatible; on the other half, they were incompatible. Participants
had to depress the “M” key with their right index finger if the arrow pointed to the right, and
the “X” key with their left index finger if the arrow pointed to the left, irrespective of the
arrow’s position. Outcome measures were the percentage of correct responses and the inhibi-
tion costs across all trials. Inhibition costs were calculated as the difference between the mean
latency of correct responses to incompatible stimuli minus that to compatible stimuli. Responses outside a time window of 2000 ms after stimulus onset were considered as
‘incorrect’. Tests We programmed a battery of executive function tests using E-Prime 2.0. Stimuli were pre-
sented on a 24” computer screen and through loudspeakers. Each test was preceded by a stan-
dardized instruction display, and by up to three practice trials. The rationale for selecting those
particular tests was to replicate the three-factor-plus-dual-tasking model of Miyake et al. [7],
since this is one of the most influential models in literature (cf. Introduction). Updating was assessed by a keep-track test and an n-back test, both adapted from literature
[7,44,45]. In the former test, 15 words from six different categories (animals, colors, relatives,
metals, countries, distances) were displayed on the screen in a randomized sequence for 2000
ms each, with an inter-stimulus interval (ISI) of 800 to 1200 ms. Participants were instructed
to attend to three of those categories and, after presentation, to write down the last word from
each of those three categories. Then they pressed the “M” key of a keyboard with their right
index finger to start the next trial, which presented 15 new words from the same six categories. There was a total of six trials, and the number of categories to report changed from trial to trial
in the order 3,3,4,4,5,5. Outcome measure was the percentage of correct responses across all
trials. In the n-back test, a 4x4 grid was displayed continuously on the screen. Black dots were
sequentially presented in the center of different grid cells for 500 ms each, for a total of 19 dot
presentations. ISI was again 800 to 1200 ms. Participants had to press the “M” key within their PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 4 / 19 Structure of executive functions right index finger if the currently displayed dot was in the same position as the second-to-last
dot, and otherwise to press the “X” key with their left index finger. The test consisted of six
19-dot trials, with a pause of five seconds between trials (except after trial 3, where the pause
was 20 s). Outcome measures were the percentage and mean latency of correct responses
across all trials, omitting the first two dots of each trial where no n-back response could be
given. Responses outside a time window of 2000 ms after stimulus onset were considered as
‘incorrect’. Shifting was assessed by two task switching paradigms, again modified from previous work
[46,47]. Tests In the Stroop test, 32 color-denoting words were sequentially presented in the center of the
screen for 500 ms each, with an ISI of 2300 to 2700 ms. Again, stimuli alternated with a central
fixation cross. One-half of the words were displayed in a color that was compatible with the
word’s meaning, red, green, yellow or blue. The other half was displayed in an incompatible
color. Two response words were displayed below each target word for 2000 ms, one somewhat
to the right and the other somewhat to the left. Both response words were white; one named
the color of the target word (i.e. correct response) and the other named one of the other colors. The position (left/right) of the correct response word was randomized across trials. Partici-
pants had to depress the key that was closest to the correct response: either the “M” key with
their right or the “X” key with their left index finger. Outcome measures were as in the Simon
test. Dual-tasking was quantified by a combination of manual tracking and tone monitoring,
again adapted from literature [51]. In the tracking test, a red target square moved across the
screen from left to right along an unpredictable path; vertical position represented the sum of PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 5 / 19 Structure of executive functions six sinewaves, and sweep time was 45 s. Participants tracked the target with a joystick-driven
cross, and their performance was quantified as root mean square error (RMSE). In the tone
monitoring test, ten high-pitched (1086 Hz), ten middle-pitched (652 Hz) and ten low-pitched
(217 Hz) tones were presented in a random sequence from the beginning until the end of each
trial. Participants had to respond to the high-pitched tone only, either by depressing the “M”
key with their right index finger (manual monitoring) or by uttering “yes” (verbal monitoring). Performance was quantified as accuracy and latency of correct responses. Only responses
given before the onset of the subsequent tone were considered for this analysis. Each participant performed the tests one day with manual monitoring, and another day
with verbal monitoring (mixed order). Each day began with a practice trial of tracking with
concurrent monitoring. Then came nine experimental trials: a block of three tracking-only tri-
als, a block of three monitoring-only trials, and a block of thee tracking trials with concurrent
monitoring. Tests The order of blocks was balanced across participants. Outcome measures were
the dual-task costs of tracking ([RMSE(dual)–RMSE(single)] / RMSE(single)), the percentage
of correct monitoring responses, and the dual-task costs of monitoring ([latency(dual)–latency
(single)] / latency (single)). These measures were calculated once for the combination of track-
ing with verbal monitoring (‘tracking-verbal’ for short) and once for the combination of track-
ing with manual monitoring (‘tracking-manual’ for short). Psychomotor speed was assessed by a manual tapping test. Following established procedures
[52], participants placed their non-dominant hand on a table and tapped with their dominant
hand back and forth across the non-dominant hand. The time to complete 25 full tapping
cycles was registered by a stopwatch. The test consisted of three trials, and outcome measure
was the shortest registered time. As already mentioned, this study was part of a larger research project on various aspects of
cognitive aging. Each participant completed four experimental sessions on separate days, with
at least one day off in-between. This took between 8 and 28 days, depending on the partici-
pants’ availability. Before the first session, participants completed at home a set of question-
naires regarding their sex and age, education (highest school degree, years of formal
education), health (overall quality, days sick per year, accidental falls per year, falls efficacy as
per FES), physical activity (hours of moderate and of strenuous physical activity per week), car
use (km driven per year) and social activity (type and frequency). The first session included
screening tests and tapping; the remaining three sessions included the above executive func-
tion tests, in an order that was balanced across participants. In each given session, executive
function tests were administered prior to any other tests. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Data analysis Third, each dual-task yielded two q-scores, one for
tracking and one for task monitoring; in accordance with earlier dual-task research [55,56], we
used the mean of both scores for further analyses. where z(LAT) is the mean latency standardized across participants irrespective of their age,
and z(ACC) is the corresponding standardized mean accuracy. Note that by calculating q-
scores across both age groups, any differences between young and older persons were pre-
served. Note further that higher q-scores indicate poorer performance. Three special cases
emerged when applying Eq (1). First, data from the keep-track test and from the tracking part
of both dual-tasks included no latencies; we therefore decided to calculate the pertinent q-
scores with the numerator of Eq (1) set to 100, i.e., to the mean numerator value of the other
tests. Second, tracking performance on both dual tasks was quantified as error rather than
accuracy; to account for this fact, we entered the error rather than accuracy into Eq (1) and
inverted the sign of the resultant q-scores. Third, each dual-task yielded two q-scores, one for
tracking and one for task monitoring; in accordance with earlier dual-task research [55,56], we
used the mean of both scores for further analyses. Most of our analyses build upon the bivariate correlations between q-scores from different
tests. Since correlations are sensitive to outlying residuals, we identified such outliers by a pro-
cedure adopted from Miyake et al. [7]. We calculated Cook’s D scores for each test pair, and
excluded all individuals with D>1 on any test pair. D>1 is an established criterion for the
identification of outlying residuals [57]. Data from the remaining participants were submitted
to the following analyses. A first set of analyses addressed the predictive value of our home-based tests for partici-
pants’ scores on the executive function tests. To this end, we used multiple stepwise linear
regression analysis with the following regressors: calendric age, sex, years of formal education,
days sick per year, accidental falls per year, falls efficacy, hours of moderate physical activity
per week and hours of strenuous physical activity per week. This analysis was run separately
with the q-scores from each executive function test as dependent variable. The following vari-
ables were not included as regressors since too many scores were zero: number of days sick
and number of falls. Data analysis Outliers were eliminated from latency data using the ±3.29 SD criterion [53], separately for
each participant and variable. Data were then averaged across repetitions, again separately for
each participant and variable. Participants with accuracy <0.60 on any test except keep track
were classified as random performers and were excluded from further analyses. (We did so
because all tests except keep track had two response alternatives, i.e. random responses would
have an accuracy of 0.5; to be on the safe side, we selected 0.6 as rejection criterion. In case of
keep-track, we reasoned that random performers who remember all presented words will be
correct on 1/455 trials, 1/368 trials and 1/286 trials when picking three, four and five words,
respectively, which corresponds to an accuracy of 0.0028. Obviously, participants are not likely
to remember all 15 presented words due to WM capacity limitations, and the words they
remember may include all, some or none of the target words; however, accuracy of random
performance will still be in the same order of magnitude as calculated above. Since the lowest PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 6 / 19 Structure of executive functions keep-track accuracy in our study was 0.25, we decided not to exclude participants because of
their keep-track accuracy.) Data from the other participants were converted into q-scores to
control for speed-accuracy tradeoffs, using [54] q ¼ zðLATÞ 15 þ 100
zðACCÞ 15 þ 100 ;
ð1Þ ð1Þ where z(LAT) is the mean latency standardized across participants irrespective of their age,
and z(ACC) is the corresponding standardized mean accuracy. Note that by calculating q-
scores across both age groups, any differences between young and older persons were pre-
served. Note further that higher q-scores indicate poorer performance. Three special cases
emerged when applying Eq (1). First, data from the keep-track test and from the tracking part
of both dual-tasks included no latencies; we therefore decided to calculate the pertinent q-
scores with the numerator of Eq (1) set to 100, i.e., to the mean numerator value of the other
tests. Second, tracking performance on both dual tasks was quantified as error rather than
accuracy; to account for this fact, we entered the error rather than accuracy into Eq (1) and
inverted the sign of the resultant q-scores. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Data analysis The following variables were not included as regressors since they were
ordinally scaled: overall health quality, car use and social activity. Instead, the latter three vari-
ables served as factors for an analysis of co-variance (ANCoVA), co-variates were the signifi-
cant regressors from above stepwise regression analyses. ANCoVA was run separately with the
q-scores from each executive function test as dependent variable. Subsequent analyses dressed the factorial structure of q-scores. We calculated CFA with
four correlated factors; factor ‘updating’ was linked to n-back and keep-track, factor ‘shifting’
to switching-spatial and switching-semantic, factor ‘inhibition’ to Simon and Stroop, and factor
‘dual-tasking’ to tracking-verbal and tracking-manual. This CFA model is analogous to the best
fitting model of Miyake et al. [7], except that dual-tasking is treated as a factor rather than as
an external variable. Goodness-of-fit was assessed by a χ2-test. CFA was calculated once with
the data from young, and once with those from older participants. We also calculated EFA, using principal component extraction with a minimum eigenvalue
of 1 and with standardized varimax rotation. Again, this was done separately for the data of
young and those of older participants. In accordance with literature [58], factor loadings >0.7
were deemed to be “satisfactory”. 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions The focus of our analyses was not on CFA and EFA, but rather on the underlying bivariate
correlations between executive function tests. Our eight tests could be combined to 28 differ-
ent test pairs, and we therefore yielded 28 test pair correlations for young and 28 for older par-
ticipants. We sorted the correlations from each age group in ascending order, fitted them by
piecewise linear regression with a single breakpoint [59], and used the resultant regression
parameters to calculate the metric ðcpost cpreÞ ðcpre c1Þ:
ð2Þ ð2Þ In Eq (2), c1 is the predicted lowest correlation of the ascending sequence, cpre is the pre-
dicted last correlation before the breakpoint and cpost is the first one after the breakpoint. We
decided to accept the existence of a distinct breakpoint if the above metric returns a positive
value, i.e., if correlations change across the breakpoint more than they do to the left of it. In further analyses, test pair correlations of either age group were tested against zero and
against each other using t-tests. Data analysis Since correlations are not normally distributed in smaller sam-
ples [60], we first transformed them to Fisher’s Z (not to be confused with a z-transformation,
as used for standardizing), performed the t-tests, and then back-transformed the calculated
means and standard deviations for reporting. In a final analysis, we calculated the linear regression of older persons’ correlations on
young persons’ correlations. If the pattern of test pairs with high and with low correlations is
similar in both age groups, the regression slope should be significant. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Results Table 1 lists the number of participants excluded from analysis. By far the most frequent rea-
son for exclusion was random test performance by older persons. Fifty-nine young persons
(23.15 ±2.91 years old, 20 males) and forty-two older persons (69.95 ±2.94 years old, 26 males)
remained for further analyses. Table 2 summarizes the data collected from home-based ques-
tionnaires for those remaining participants: older persons were somewhat less physically
active, less likely to suffer an accidental fall, less often sick, and more likely to drive car over
longer distances. Fig 1 plots the mean q-scores of individuals that remained for further analyses. Older per-
sons had higher q-scores (i.e., poorer performance) on all tests: the age difference ranged from
8.5% in switch-semantic to 36.1% in Stroop. Table 3 summarizes the outcome of stepwise multiple linear regressions. q-scores from all
executive function tests were significantly related to calendric age (higher age ~ poorer perfor-
mance). q-scores from some tests were significantly related to years of education (more years ~
better performance), falls efficacy (more concerns ~ better(!) performance) and hours of mod-
erate physical activity (more activity ~ poorer(!) performance). Notably, q-scores from no test
were significantly related to sex or psychomotor speed. We decided to use only calendric age
as co-variate for the subsequent ANCoVA, since only calendric age had strong and meaningful
associations with all executive function tests. We further decided to proceed with data analyses
without correcting for sex and psychomotor speed. Table 1. Exclusion of participants. exclusion based on:
random performance
outliers
total
young adults
3
1
4
older adults
15
4
19
https://doi.org/10.1371/journal.pone.0216149.t001
one.0216149
May 9, 2019
8 / 19 Table 1. Exclusion of participants. exclusion based on:
random performance
outliers
total
young adults
3
1
4
older adults
15
4
19
https://doi.org/10.1371/journal.pone.0216149.t001 Table 1. Exclusion of participants. exclusion based on:
random performance
outliers
total
young adults
3
1
4
older adults
15
4
19
https://doi.org/10.1371/journal.pone.0216149.t001 8 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions Table 2. Demographics and outcome of home-based questionnaires in young and in older participants. It is interesting to note that correlations
between test pairs which purportedly represent the same executive function (lager symbols in
Fig 3) are not necessarily high. Lines in Fig 3 depict the outcome of piecewise linear regres-
sions, and the pertinent regression parameters are listed in Table 6. According to that table,
Table 4. Outcome of confirmatory factor analyses for young and for older persons. Note: Cell entries are the loadings of a given test (2nd column) on a given factor (1st
column), as well as the pertinent standard errors, values of the asymptotic normal statistic and p-values. Bold p-values indicate statistical significance. The asymptotic nor-
mal statistic is the probability distribution for loading = 0.000. The outcome of CFA with the pre-established four-factor model (see Methods) is presented
in Table 4. Most but not all factor loadings reached statistical significance, and the model as a
whole fitted reasonably well the q-scores of young (χ2(14) = 22.45; p>0.05) as well as to those
of older adults (χ2(14) = 14.59; p>0.05). Correlations between factors ranged from -0.11 to
0.32 in young and from -0.12 to 0.64 in older persons. The outcome of EFA is listed in Table 5. Four factors emerged from the data of young per-
sons; they largely correspond to the four purported executive functions, inhibition, shifting,
dual-tasking and updating, although two of the eight tests had no satisfactory (> 0.7) loadings. Only three factors emerged from the data of older persons, none of them clearly associated
with a presumed executive function. In this age group, four of the eight tests had no satisfac-
tory loadings. Taken together, factors explained 68.4% of total variance in young and 61.2% of
total variance in older persons. Fig 2 shows the frequency distribution of test pair correlations, separately for young and for
older persons. No conspicuous dichotomy between a cluster of lower and a cluster of higher
correlations can be discerned in the distribution for either age group. Fig 3 plots the same cor-
relations individually, sorted in ascending order. It is interesting to note that correlations
between test pairs which purportedly represent the same executive function (lager symbols in
Fig 3) are not necessarily high. Lines in Fig 3 depict the outcome of piecewise linear regres-
sions, and the pertinent regression parameters are listed in Table 6. According to that table, Table 4. Results young persons
older persons
number of females / number of males
39 / 20
16 / 26
age (mean ±SD)
23.15 ±2.91
69.95 ±2.94
school education level
high school diploma
high school diploma
years of formal education (mean ±SD)
15.4 ±2.4
15.6 ±2.6
health quality,
good
good
days sick per year (mean ±SD)
10.9 ±8.4
3.4 ±6.0
accidental falls per year (mean ±SD)
0.102 ±0.443
0.048 ±0.216
falls efficacy (mean ±SD)
16.7 ±1.0
16.7 ±2.4
hrs/week moderate physical activity§
14.3 ±14.9
12.6 ±13.7
hrs/week strenuous physical activity§
6.1 ±6.3
4.5 ±4.2
km/year car use
<6000
9000–12000
number of social activities per month,§§
18
18 Table 2. Demographics and outcome of home-based questionnaires in young and in older participants. Responses were on an ordinal scale; we report the most frequently checked response bin. Five response alternatives, ranging from “poor” to “very good”. Scores range from 16 = no concerns on any item to 64 = strong concerns on all items. $ Includes sports, leisure and work
§§ Indicated on a list of 18 common activities such as concerts, parties, care of dependent persons
https://doi.org/10.1371/journal.pone.0216149.t002 Responses were on an ordinal scale; we report the most frequently checked response bin. ANCoVA yielded no significant effects on any executive function test for the factors health
status (all F < 1.31; all p>0.05) and social activity (all F<2.46; all p>0.05). Car use had a signif-
icant effect on the q-scores from n-back (F(4,64) = 2.96; p <0.05) and from tracking-manual
(F(4,64) = 3.05; p <0.05). Fig 1. Mean value of q-scores in each executive function test. Data from young participants are shown in black, and those from older participants in
gray. Error bars are standard deviations. Fig 1. Mean value of q-scores in each executive function test. Data from young participants are shown in black, and those from older participants in
gray. Error bars are standard deviations. https://doi org/10 1371/journal pone 0216149 g001 Fig 1. Mean value of q-scores in each executive function test. Data from young participants are shown in black, and those from older participants in
gray. Error bars are standard deviations. Fig 1. Mean value of q-scores in each executive function test. Data from young participants are shown in black, and those from older participants in
gray. Error bars are standard deviations. https://doi.org/10.1371/journal.pone.0216149.g001 https://doi.org/10.1371/journal.pone.0216149.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 9 / 19 Structure of executive functions Table 3. Outcome of stepwise multiple linear regression analyses. regressor
test
calendric age
education years
falls efficacy
moder. phys. act. keep-track
0.494
n.s. -0.434
n.s. n-back
0.622
-0.204
n.s. n.s. Simon
0.628
-0.214
-0.225
n.s. Stroop
0.387
n.s. -0.215
0.224
switch-semantic
0.207
n.s. -0.233
n.s. switch-spatial
0.342
n.s. n.s. n.s. tracking-verbal
0.246
n.s. n.s. n.s. tracking-manual
0.350
n.s. n.s. n.s. Note: “moder. phys. act” refers to the time spent on moderate physical activity. Cell entries are partial correlations between regressors and q-scores. Bold font indicates
p<0.001, regular font indicates p<0.05 and n.s. indicates p>0.05. Not shown are regressors which yielded no significant partial correlations with any executive function
test. Table 3. Outcome of stepwise multiple linear regression analyses. Note: “moder. phys. act” refers to the time spent on moderate physical activity. Cell entries are partial correlations between regressors and q-scores. Bold font indicates
p<0.001, regular font indicates p<0.05 and n.s. indicates p>0.05. Not shown are regressors which yielded no significant partial correlations with any executive function
test. The outcome of CFA with the pre-established four-factor model (see Methods) is presented
in Table 4. Most but not all factor loadings reached statistical significance, and the model as a
whole fitted reasonably well the q-scores of young (χ2(14) = 22.45; p>0.05) as well as to those
of older adults (χ2(14) = 14.59; p>0.05). Correlations between factors ranged from -0.11 to
0.32 in young and from -0.12 to 0.64 in older persons. The outcome of EFA is listed in Table 5. Four factors emerged from the data of young per-
sons; they largely correspond to the four purported executive functions, inhibition, shifting,
dual-tasking and updating, although two of the eight tests had no satisfactory (> 0.7) loadings. Only three factors emerged from the data of older persons, none of them clearly associated
with a presumed executive function. In this age group, four of the eight tests had no satisfac-
tory loadings. Taken together, factors explained 68.4% of total variance in young and 61.2% of
total variance in older persons. Fig 2 shows the frequency distribution of test pair correlations, separately for young and for
older persons. No conspicuous dichotomy between a cluster of lower and a cluster of higher
correlations can be discerned in the distribution for either age group. Fig 3 plots the same cor-
relations individually, sorted in ascending order. Outcome of confirmatory factor analyses for young and for older persons. Note: Cell entries are the loadings of a given test (2nd column) on a given factor (1st
column), as well as the pertinent standard errors, values of the asymptotic normal statistic and p-values. Bold p-values indicate statistical significance. The asymptotic nor-
mal statistic is the probability distribution for loading = 0.000. ses for young and for older persons. Note: Cell entries are the loadings of a given test (2nd column) on a given factor (1st
values of the asymptotic normal statistic and p-values. Bold p-values indicate statistical significance. The asymptotic nor-
ing = 0 000 CFA factor
test
loading
S.E. ANS
p
loading
S.E. ANS
p
updating
keep-track
n-back
0.111
0.010
10.77
<0.001
0.185
0.020
9.055
<0.001
0.010
0.017
0.60
>0.05
0.013
0.038
0.338
>0.05
inhibition
Simon
Stroop
0.045
0.017
2.67
<0.01
0.076
0.036
2.126
<0.05
0.145
0.013
10.77
<0.001
0.330
0.095
3.474
<0.01
switching
switch-semantic
switch-spatial
0.181
0.017
10.77
<0.001
0.181
0.020
9.055
<0.001
0.039
0.018
2.16
<0.05
0.047
0.029
1.601
>0.05
dual-tasking
tracking-verbal
tracking-manual
0.110
0.010
10.77
<0.001
0.074
0.033
2.245
<0.05
0.027
0.012
2.30
<0.05
0.261
0.029
9.055
<0.001 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 10 / 19 e: Cell entries are the loadings of a given test (1st column) on a given factor (2nd row). Loadings >0.7 are highlighted in bold Structure of executive functions Fig 3. Bivariate correlations between test pairs, plotted in ascending order. Data from young participants are shown in black, and those from older
participants in gray. Negative correlations imply that good performance on one test was associated with poor performance on the other test. Large
squares denote test pairs which purportedly represent a common executive function. From bottom left to top right, the squares stand for updating,
shifting, dual-tasking and inhibition in young persons, and for updating, shifting, inhibition and dual-tasking in older ones. Solid lines are predictions of
piecewise linear regression with one breakpoint. https://doi.org/10.1371/journal.pone.0216149.g003 Fig 3. Bivariate correlations between test pairs, plotted in ascending order. Data from young participants are shown in black, and those from older
participants in gray. Negative correlations imply that good performance on one test was associated with poor performance on the other test. Large
squares denote test pairs which purportedly represent a common executive function. From bottom left to top right, the squares stand for updating,
shifting, dual-tasking and inhibition in young persons, and for updating, shifting, inhibition and dual-tasking in older ones. Solid lines are predictions of
piecewise linear regression with one breakpoint. https://doi.org/10.1371/journal.pone.0216149.g003 our criterion for the existence of a distinct breakpoint (see above), was not met by the data
from either age group. The mean and standard deviations of test pair correlations were 0.047 ±0.164 in young, and
0.149 ±0.185 in older persons. Young participants’ scores were not significantly different from
zero (t(27) = 1.47; p>0.05), but they were significantly different from older participants’ scores
(t(54) = 2.175; p<0.05). Fig 4 illustrates once more the test pair correlations already shown in
Figs 2 and 3, this time comparing the data of young and older participants for a given test. Lin-
ear regression of the data in Fig 4 yielded a slope of 0.045, which is not statistically significant
(t(26) = 0.244, p>0.05). Table 6. Outcome of piecewise linear regression of test pair correlations in young and older persons. IC1
SL1
IC2
SL2
order
R
cpost −cpre
cpre −c1
DB
young
-0.129
0.018
-0.905
0.052
26
0.994
-0.041
0.228
no
older
-0.229
0.019
-0.444
0.028
16
0.992
0.176
0.447
no
Note: IC1, IC2, SL1 and SL2 are the y-intercepts and slopes of the 1st and 2nd linear segment, respectively. https://doi.org/10.1371/journal.pone.0216149.t006 Structure of executive functions Table 5. Outcome of exploratory factor analyses for young and for older persons. young persons
older persons
test
F1
F2
F3
F4
F1
F2
F3
keep-track
-0.053
-0.042
0.039
0.925
0.815
0.031
0.010
n-back
-0.550
0.194
0.031
0.198
0.155
0.172
-0.815
Simon
0.760
0.141
0.036
-0.013
0.588
0.240
-0.183
Stroop
0.720
-0.036
0.018
0.477
0.424
0.645
0.151
switching-semantic
0.169
0.873
0.218
-0.115
0.815
-0.234
0.227
switching-spatial
-0.323
0.644
-0.274
0.073
0.214
0.249
0.619
tracking-verbal
0.085
0.277
0.769
0.245
-0.127
0.623
0.072
tracking-manual
-0.061
-0.174
0.801
-0.119
0.045
0.868
-0.151
variance expl’d. 0.194
0.168
0.170
0.152
0.243
0.220
0.148
Note: Cell entries are the loadings of a given test (1st column) on a given factor (2nd row) Loadings >0 7 are highlighted in bold Table 5. Outcome of exploratory factor analyses for young and for older persons. manual
-0.061
-0.174
0.801
-0.119
0.045
0.868
-0.151
xpl’d. 0.194
0.168
0.170
0.152
0.243
0.220
0.148
s are the loadings of a given test (1st column) on a given factor (2nd row). Loadings >0.7 are highlighted in bold. 1371/journal.pone.0216149.t005
Fig 2. Frequency distribution of bivariate correlations between test pairs. Data are plotted separately for young (black) and for older participants
(gray). For example, two correlations in older participants were in the range 0.5 to 0.6. https://doi.org/10.1371/journal.pone.0216149.g002 g
g
(
)
g
(
)
g
g
g
1371/journal.pone.0216149.t005 Fig 2. Frequency distribution of bivariate correlations between test pairs. Data are plotted separately for young (black) and for older participants
(gray). For example, two correlations in older participants were in the range 0.5 to 0.6. https://doi.org/10.1371/journal.pone.0216149.g002 Fig 2. Frequency distribution of bivariate correlations between test pairs. Data are plotted separately for young (black) and for older participants
(gray). For example, two correlations in older participants were in the range 0.5 to 0.6. https://doi.org/10.1371/journal.pone.0216149.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 11 / 19 Fig 4. Pattern of test pair correlations in the two age groups. Each symbol represents the correlation for one test pair in older persons, plotted against
the correlation for the same test pair in young persons. Dashed line is the regression line. https://doi org/10 1371/journal pone 0216149 g004 Fig 4. Pattern of test pair correlations in the two age groups. Each symbol represents the correlation for one test pair in older persons, plotted against
the correlation for the same test pair in young persons. Dashed line is the regression line. https://doi.org/10.1371/journal.pone.0216149.g004 https://doi.org/10.1371/journal.pone.0216149.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Order is the serial order of the last test pair correlation before
the breakpoint, and R quantifies the goodness-of-fit of piecewise linear regression. The two subsequent columns list quantities defined in Eq (2) and the rightmost
column indicates whether our criterion for the existence of a distinct breakpoint is met by the data. htt
//d i
/10 1371/j
l
0216149 t006 Table 6. Outcome of piecewise linear regression of test pair correlations in young and older persons. Note: IC1, IC2, SL1 and SL2 are the y-intercepts and slopes of the 1st and 2nd linear segment, respectively. Order is the serial order of the last test pair correlation before
the breakpoint, and R quantifies the goodness-of-fit of piecewise linear regression. The two subsequent columns list quantities defined in Eq (2) and the rightmost
column indicates whether our criterion for the existence of a distinct breakpoint is met by the data. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 12 / 19 Structure of executive functions Fig 4. Pattern of test pair correlations in the two age groups. Each symbol represents the correlation for one test pair in older persons, plotted against
the correlation for the same test pair in young persons. Dashed line is the regression line. https://doi.org/10.1371/journal.pone.0216149.g004 Fig 4. Pattern of test pair correlations in the two age groups. Each symbol represents the correlation for one test pair in older persons, plotted against Discussion The present study examined the structure of executive functions in young and older persons. We found that older participants performed less well than young ones on a range of executive
function tests, that bivariate correlations between test pairs failed to segregate into relatively
high and relatively low correlations in either age group, and that correlations were significantly
higher in older persons than in young ones. We found no strong and consistent effects of sex, education, physical activity and health
on our participants’ executive functions, possibly because our sample was generally healthy PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 13 / 19 Structure of executive functions and fit (cf. Table 2). The observed effect of age is in agreement with numerous earlier studies
(cf. meta-analyses [16,19,17,18,20]. However, earlier work found that this decay is partly
attributable to decreases of psychomotor speed [39–41], which was not the case in our data. This apparent discrepancy is probably due to methodological issues: earlier work quantified
executive performance mainly in terms of response latency, thus disregarding the age-related
shift of priority from speed to accuracy [35,36], while the present study used q-scores, thus
taking speed-accuracy tradeoffs into account. It therefore is conceivable that shifts of priority
and decreases of psychomotor speed in older age are related phenomena. Since we found no
effects of psychomotor speed on q-scores, we disregarded psychomotor speed in subsequent
analyses. Our participants’ q-scores were only in part compatible with a four-factor model, consist-
ing of the three factors postulated by Miyake et al. [7] plus the factor ‘dual-tasking’. In a con-
firmatory factor analysis, data from either age group were not significantly different from
that model’s predictions. However, one test in young and two in older persons achieved only
negligible factor loadings. In an exploratory factor analysis, data from young persons yielded
a four-factor solution reminiscent of the above model while data from older persons yielded
a three-factor solution less clearly related to that model; two tests in young and four in
older persons achieved only unsatisfactory (<0.7) factor loadings. This outcome comple-
ments the discrepant executive-function models available in literature, where the number
and definition of factors differ from study to study, both for young and for older persons
(see Introduction). Discussion The purpose of the present work was not to support any of the executive-function models
in literature, but rather to explore one possible reason for the discrepancies among those mod-
els. As pointed out in the Introduction, the robustness and cogency of factor analytical
approaches may suffer if there is no clear dichotomy between well-correlated and poorly corre-
lated data sets. In absence of such a dichotomy, even small differences in the correlation pat-
tern from different studies could substantially modify the respective best-fitting factorial
models, regarding both the number and the definition of factors. We searched for a clear dichotomy in our data but we found no distinct breakpoint between
relatively low and relatively high test pair correlations. Our data therefore don’t support the
view that the eight tests of the present study can be reduced to a smaller number of distinct
executive functions. Instead, a more likely interpretation of our data is that executive functions
form a partly overlapping structure from which our tests probed eight different regions. The
overlap between the probed regions was generally low, ranging from 0.005% to 11.62% in
young, and from 0.026% to 31.8% in older persons (we calculated overlap as r2). Of course,
studies which use other tests than those in the present work will probe other regions of the pos-
tulated structure, where the overlap may be different and the resultant factorial models may
therefore be different as well. This could explain why some studies found a general executive
function while others did not, why some studies found a common switching-and-updating
function while others did not, and why some studies found two, others three, and yet others
four executive functions. The proposed partial overlap of executive functions could also explain the wide range of
model fits observed in literature. CFA yielded satisfactory loadings (>0.7) for anywhere
between 17% of the administered tests [14] and 78% of those tests [13], with our own data well
within that range (young: 75%; older: 50%). The use of different tests and therefore the probing
of differently overlapping regions could explain this diversity of model fits. Furthermore, the
proposed partial overlap is in accordance with Luria’s notion that higher-level cognition is the
result of integrated activity in a distributed neural network, not of local activities in specialized
modules [61]. PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Discussion In other words, different executive function tests possibly engaged different, PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 14 / 19 Structure of executive functions slightly overlapping regions within such a network. Luria’s notion could also explain the sub-
stantial correlations observed between executive function tests on one side, and tests of fluid
intelligence, memory and attention on the other side [62,63]. To our knowledge, ours is the first study to compare the structure of executive functions in
young and older individuals using the same set of tests, instructions, settings and investigators. We found that correlations were significantly higher in the older group, as predicted by the
concept of age-related dedifferentiation [23,22,21,25]. We further found that the pattern of rel-
atively high and relatively low test pair correlations was not comparable in the two age groups,
which is in accordance with the concept of age-related reorganization [28,27]. Thus with
advancing age, larger, more overlapping, and partly different brain areas may be activated to
accomplish a given task. Several limitations of the present study should be considered. First, as in all experimental
studies, our conclusions are not necessarily generalizable to persons whose education, health
or activity levels differ from those in our participants (cf. Table 2), and to tests other than those
administered in the present study. As a second limitation, psychomotor speed was registered
by means of a stopwatch rather than electronically, and was therefore not as precise and accu-
rate as it could have been. Third, the sample size was quite small: we analyzed data of 101 per-
sons while others examined 100 to 486 [13,14,30,15,7]. As a fourth limitation, the number of
executive function tests was small: we used two tests per putative executive function as did
some earlier authors, but others used three or even four tests per putative executive function. In spite of these limitations, data from the present study are well within the range of earlier
work: neither test pair correlations nor factor analytical outcomes of the present study are con-
spicuously different from earlier research. The unique contribution of our study therefore is
not the data set collected, but rather the analytical treatment of those data. We controlled for the effects of psychomotor speed on test outcomes [39–41] by calculating
q-scores. It might be useful for future work to control for other external influences as well. Supporting information S1 File. Plos One supporting information. (XLSX) Discussion For
example, executive functions are probably influenced by a person’s physical, emotional and
social needs [64] and controlling for those needs might therefore increase test pair correlations
and accentuate age differences. It is interesting to note that the age-related decline of executive functions was substantial in
our work, as it was in earlier studies. Across all tests, q-scores were as much as 19% higher in
our older compared to the young participants (cf. Fig 1). Nevertheless, all our participants
lived independently in the community, arrived for testing at the agreed-upon time in the
agreed-upon place and were able to follow our instructions without noticeable problems. In
other words, older participants functioned well in everyday life, in spite of their deficits on
standardized executive function tests. This casts doubt on the ecological validity [65,66] of
such tests. Possibly, the present study and pertinent earlier work addressed phenomena which
are of theoretical relevance but play only a limited role in normal life. Writing – review & editing: Mathias Haeger, Claudia Voelcker-Rehage. Writing – review & editing: Mathias Haeger, Claudia Voelcker-Rehage. Acknowledgments We thank Dr. Uwe Drescher, Christin Janouch and Konstantin Wechsler for their substantial
contributions to data registration and analysis. 15 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0216149
May 9, 2019 Structure of executive functions Author Contributions Author Contributions
Conceptualization: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Data curation: Mathias Haeger. Formal analysis: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Funding acquisition: Otmar Bock, Claudia Voelcker-Rehage. Investigation: Otmar Bock, Claudia Voelcker-Rehage. Methodology: Otmar Bock, Claudia Voelcker-Rehage. Project administration: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Resources: Otmar Bock, Claudia Voelcker-Rehage. Software: Mathias Haeger. Supervision: Otmar Bock, Claudia Voelcker-Rehage. Validation: Otmar Bock, Claudia Voelcker-Rehage. Visualization: Otmar Bock. Writing – original draft: Otmar Bock. Writing – review & editing: Mathias Haeger, Claudia Voelcker-Rehage. Author Contributions
Conceptualization: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Data curation: Mathias Haeger. Formal analysis: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Funding acquisition: Otmar Bock, Claudia Voelcker-Rehage. Investigation: Otmar Bock, Claudia Voelcker-Rehage. Methodology: Otmar Bock, Claudia Voelcker-Rehage. Project administration: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Resources: Otmar Bock, Claudia Voelcker-Rehage. Software: Mathias Haeger. Supervision: Otmar Bock, Claudia Voelcker-Rehage. Validation: Otmar Bock, Claudia Voelcker-Rehage. Visualization: Otmar Bock. Writing – original draft: Otmar Bock. Writing – review & editing: Mathias Haeger, Claudia Voelcker-Rehage. Conceptualization: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Data curation: Mathias Haeger. Formal analysis: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Funding acquisition: Otmar Bock, Claudia Voelcker-Rehage. Investigation: Otmar Bock, Claudia Voelcker-Rehage. Methodology: Otmar Bock, Claudia Voelcker-Rehage. Project administration: Otmar Bock, Mathias Haeger, Claudia Voelcker-Rehage. Resources: Otmar Bock, Claudia Voelcker-Rehage. Software: Mathias Haeger. Supervision: Otmar Bock, Claudia Voelcker-Rehage. Supervision: Otmar Bock, Claudia Voelcker-Rehage. Validation: Otmar Bock, Claudia Voelcker-Rehage. Validation: Otmar Bock, Claudia Voelcker-Rehage. Visualization: Otmar Bock. Writing – original draft: Otmar Bock. References Neuropsychology 23 (6): 778. https://doi.org/10. 1037/a0016743 PMID: 19899836 15. Hull R, Martin RC, Beier ME, Lane D, Hamilton AC (2008) Executive function in older adults: A structural
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On Some Topological Indices for the Orbit Graph of Dihedral Groups
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* Correspondence: vijaykumarbhat2000@yahoo.com Abstract: Let ΓG be the orbit graph of G, with non-central orbits in the subset of order two com-
muting elements in G, and the vertices of ΓG connected if they are conjugate. The main objective of
this study is to compute several topological indices for the orbit graph of a dihedral group, including
the Wiener index, the Zagreb index, the Schultz index, and others. We also develop a relationship
between the Wiener index and the other indices for the dihedral group’s orbit graph. Furthermore
their polynomial has been computed as well. Keywords: Wiener Index, Zagreb Index, Schultz Index, Somber Index, Randic Index, Dihedral
group, orbit graph Mathematics Subject Classification: 05C10,05C40,05C07,05C25. Mathematics Subject Classification: 05C10,05C40,05C07,05C25. Journal of Combinatorial Mathematics and Combinatorial Computing, 117: 195–208
DOI:10.61091/jcmcc117-18
http://www.combinatorialpress.com/jcmcc
Received 28 November 2023, Accepted 2 December 2023, Published 11 December 2023 Journal of Combinatorial Mathematics and Combinatorial Computing, 117: 195–208
DOI:10.61091/jcmcc117-18
http://www.combinatorialpress.com/jcmcc
Received 28 November 2023, Accepted 2 December 2023, Published 11 December 2023 Vijay Kumar Bhat1,*and Karnika Sharma1 1 School of Mathematics, Shri Mata Vaishno Devi University, Katra-182320, Jammu and Kashmir,
India. Vijay Kumar Bhat and Karnika Sharma 196 The numerical representations of chemical structures with topological indices are significant in
medicinal chemistry and bioinformatics. In the early 1990s, the invention and specification of the
new TIs increased consistently. Topological indices include degree-based, distance-based, counting-
related, and many other topological indices. These indices are used to create quantitative structure-
activity relationships, in which a molecule’s biological activity, as well as other properties like strain
energy, stability, and boiling point, are linked to its structure. To classify these indices, the structural
properties of the graphs are used to calculate the TIs. For example, Zagreb indices are derived using
the degrees of vertices and the wiener index is obtained by using the distance between of vertices in
the given molecular graph. In 1947, [4] the first graph-based molecular structure TI was discovered. Wiener has provided only two important topological indices; the Wiener Index W(G), and the Wiener
polarity index Wp. These indices are used to obtain the chemical and physical properties of chemical
compounds. Some formulas comprising terms of this kind were deduced a long time ago while
studying the dependence of total π-electron energy on molecule structure. That is, M1 =
X
vertices
(di)2
M2 =
X
edges
di.dj
with di standing for the degree of the vertex vi of the molecular graph [4]. In the chemical literature,
M1 and M2 are called the first Zagreb index and the second Zagreb index respectively. M1 =
X
vertices
(di)2
M2 =
X
edges
di.dj with di standing for the degree of the vertex vi of the molecular graph [4]. In the chemical literature,
M1 and M2 are called the first Zagreb index and the second Zagreb index respectively. with di standing for the degree of the vertex vi of the molecular graph [4]. In the chemical literature,
M1 and M2 are called the first Zagreb index and the second Zagreb index respectively. Many studies have been reported regarding the applications of topological indices in the last four
decades. Das et al. [5] introduced several significant characteristics of the second Zagreb index. In
their work, they have declared and verified numerous findings for M2 identities and inequalities. The
M-polynomial for the graph has received a lot of attention since it generates a lot of degree-based
topological indices. Munir et al. [6] used this method to determine M-polynomials of various
nanostar dendrimers and subsequently retrieve a large number of degree-based topological indices. 1. Introduction Graph theory has provided us with several useful tools, one of the most studied and applicable
tools is the topological index. Topological indices (TIs) are the numerical parameters of a graph that
can be employed to characterize its topology. A topological index is also known as a connectedness
index in chemical graph theory, molecular topology, and mathematical chemistry domains. A
molecule can be represented as a graph in the chemical graph theory, with atoms as vertices and
bonds as edges. Topological graph indices have been successfully utilized to predict specific
physicochemical properties as well as to determine the structural properties of chemical compounds. Stankevich et al. [1] examined topological indices as one way of identifying the relationship between
the structure of the chemical compound and its characteristics. Topological indices have also assisted
chemists, physicians, mathematicians, and others in solving chemical and biological problems. Diudea et al. [2] proposed a unified approach to the Wiener topological index and its various
modifications, where they focus on the Schultz, Cluj, Szeged, Harary, and Kirchhoff indices, as
well as their numerous variants and generalizations. They also obtained the relationship between
these indices and their correlations with the physicochemical properties of molecules. Cancan et
al. [3] employed the findings obtained in [2] to investigate the generalized prism network. Further,
they have computed several degree-based indices such as the Airthmetic-Geometric index, modified
Randic, sum connectivity index, the SK index, S K1 index, and S K2 index of generalised prism
network engineering. Journal of Combinatorial Mathematics and Combinatorial Computing On Some Topological Indices for the Orbit Graph of Dihedral Groups 197 n Some Topological Indices for the Orbit Graph of Dihedral Groups The above-mentioned applications motivate us to study topological indices for the orbit graph of
G. This study examines the Wiener index, the first, second, and third Zagreb indices, Hyper first
and second Zagreb indices, Modified first Zagreb indices, Schultz and Modified Schultz indices, the
Forgotten index, Somber index, Randic and Reciprocal Randic indices for the orbit graph of the
dihedral group Dt. Furthermore, we also obtain the relationships between these indices with the
wiener index and the polynomials of indices for the orbit graphs of Dt. Vijay Kumar Bhat and Karnika Sharma Zhou et al. [7] derive several inequalities of three previously investigated descriptors, namely the
Zagreb, Wiener, and Hyper-Wiener indices concerning the given molecular graph. Khalifeh et al. [8]
gave some exact formulae for the first and second Zagreb indices of graph operations containing
the symmetric difference, composition, disjunction, join, and cartesian product of graphs. For the
nanostructure of bridge graphs, Khalaf et al. [9] determine the hyper-Zagreb index, first multiple
Zagreb index, second multiple index, Zagreb polynomials, and M-polynomials. Das et al. [10]
compared the Wiener index and the Zagreb indices, as well as the eccentric connectivity index for
trees. Bello et al. [11] generalized the Wiener index, first and second Zagreb indices of the ordered
product prime graph on dihedral groups. Singh and Bhat [12] investigated the adjacency matrix and
various topological indices of the zero-divisor graph of Zn. Shahistha et al. [13] calculate the Wiener
index of some significant chain graphs. In the recent past, Poojary et al. [14] developed several topological indices and polynomials for
the Issac graph. Filipovski [15] established several relationships between the Sombor index and
degree-based topological indices such as the Zagreb index, Forgotten index, and Randic index. Saleh
et al. [16] introduced the first, second, and forgotten downhill Zagreb indices of graphs. Saeed et
al. [17] investigated degree-based topological indices of Boron B12, such as the Randic index, the
first general Zagreb index, the hyper-Zagreb index, and others. Javaid et al. [18] calculated novel
connection-based Zagreb indices for many wheel-related graphs such as the wheel, gear, helm,
flower, and sunflower graphs. Because of their uses, entire versions of numerous indices have been
introduced and investigated. Naci et al. [19] presented the Wiener index for trees and some graph
families. Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Journal of Combinatorial Mathematics and Combinatorial Computing 2. Preliminaries The following definitions related to the group theory, graph theory and topological indices are
stated as below. nition 1. Dihedral Group: Let G be a dihedral group of order 2t and is represented as Dt = ⟨a, b|at = b2 = e, bab = a−1⟩ Dt = ⟨a, b|at = b2 = e, bab = a−1⟩ with t being a positive integer and t ≥3. with t being a positive integer and t ≥3. Then there are the definitions of several indices that we utilise in this work, which are listed in Table
1. Let ui and u j be two unique vertices with i ≤j and ΓΥ
G a connected graph of t vertices, respectively. Then there are the definitions of several indices that we utilise in this work, which are listed in Table Let ui and u j be two unique vertices with i ≤j and ΓΥ
G a connected graph of t vertices, respectively. Then there are the definitions of several indices that we utilise in this work, which are listed in Table
1. Then there are the definitions of several indices that we utilise in this work, which are listed in Table
1. 1. Table 1. Topological Indices Formulas
Indices
Formulas
W(ΓΥ
G)
1
2
Pt
i=1
Pt
j=1 d(ui, uj)
M1(ΓΥ
G)
P
u∈v(ΓΥ
G)(deg(u))2
M2(ΓΥ
G)
P
u1,u2∈E(ΓΥ
G) deg(u1)deg(u2)
M3(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui)deg(uj))(deg(ui) + deg(uj))
HM1(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui) + deg(uj))2
HM2(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui)deg(uj))2
ModM1(ΓΥ
G)
Pt
i,j=1
1
deg(ui)2
S c(ΓΥ
G)
1
2
P(deg(ui) + deg(uj))d(ui, uj)
S ∗
c(ΓΥ
G)
1
2
P(deg(ui)deg(uj))d(ui, uj)
F(ΓΥ
G)
P t(t−1)
2
i,j=1 deg(ui)2 + deg(uj)2
S o(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui)2 + deg(uj)2)
1
2
R(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui)deg(uj))−1
2
RR(ΓΥ
G)
P t(t−1)
2
i,j=1(deg(ui)deg(u j))
1
2 Table 1. Topological Indices Formulas The orbit graph of all dihedral groups, Dt, can be divided into three parts, according to Omer et
al., [20], as stated in the following theorem. when t and t
2 are even, |Υ| = 5t
2 + 1 and |B| = 1, when t is
even and t
2 is odd, |Υ| = 5t
2 + 1 and |B| = 1 and when t is odd, |Υ| = t and |B| = 0. This fact will be
utilised to support our primary findings. Theorem 1. [20]Let G be a dihedral group of order 2t. If G acts on Υ by conjugation. Then ΓΥ
G =
S5
i=1 K t
2i,
if t is even and t
2 is odd ,
(S4
i=1 K t
2i) S(S2
i=1 K t
4i),
if t and t
2 is odd ,
Kt,
if t is odd . Journal of Combinatorial Mathematics and Combinatorial Computing with t being a positive integer and t ≥3. Definition 2. [20]The Set Υ: The set Υ is the set of all pairs of commuting elements of G and the
least common multiple of the elements having order two and is represented as Definition 3. [20]Orbit:
If a group G operates on a set Υ and v1
∈Υ, then the subset
R(v1) = {gv1 | g ∈G, v1 ∈Υ} is the orbit of v1 and is symbolized by R(v1). In this study, the
conjugation action is considered. Therefore, the orbit is written as R(v1) = {gv1g−1 | g ∈G, v1 ∈Υ}. R(v1) = {gv1g−1 | g ∈G, v1 ∈Υ}. Definition 4. [20]Orbit Graph ΓΥ
G: The orbit graph, ΓΥ
G is a graph whose vertices are non central
orbits under group action on the set Υ that is |V(ΓΥ
G)| = |Υ|−|B|, where Υ is a disjoint union of distinct
orbits and B = {v1 ∈Υ|v1g = gv1, g ∈G}. Two vertices v1, v2 are adjacent if v1, v2 are conjugate that
is v1 = gv2. Figure 1. orbit graph Kn Figure 1. orbit graph Kn Figure 1. orbit graph Kn Figure 1. orbit graph Kn The following are some definitions that are used in computing the topological indices. Definition 5. [21]Degree of a vertex: The number of incident edges |E(u)| on the vertex u is called
degree of a vertex and is denoted by deg(u). Definition 5. [21]Degree of a vertex: The number of incident edges |E(u)| on the vertex u is called
degree of a vertex and is denoted by deg(u). Journal of Combinatorial Mathematics and Combinatorial Computing Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 Volume 117, 195–208 Volume 117, 195–208 198 Vijay Kumar Bhat and Karnika Sharma Vijay Kumar Bhat and Karnika Sharma Definition 6. [21]Distance: The minimum number of edges between two vertices ui and u j of a graph
is said to be a distance of two vertices and is denoted by d(ui, uj), where i, j denotes the number of
vertices. Definition 6. [21]Distance: The minimum number of edges between two vertices ui and u j of a graph
is said to be a distance of two vertices and is denoted by d(ui, uj), where i, j denotes the number of
vertices. Let ui and u j be two unique vertices with i ≤j and ΓΥ
G a connected graph of t vertices, respectively. 3. Indices for orbit graph of Dt According to Theorem 1, only the third case involves a connected graph, while the other two cases
involve disconnected graphs. As a result, we can only think about the case where t is odd. In this
section, the Wiener index, the first, second and third Zagreb indices, Hyper first and second Zagreb
indices, Modified first Zagreb indices, Schultz and Modified Schultz indices, Forgotten index, Somber
index, Randic and Reciprocal Randic indices for the orbit graph of dihedral groups are calculated. Theorem 2. Let G be a dihedral group Dt of order 2t. Then Theorem 2. Let G be a dihedral group Dt of order 2t. Then Theorem 2. Let G be a dihedral group Dt of order 2t. Then Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing On Some Topological Indices for the Orbit Graph of Dihedral Groups On Some Topological Indices for the Orbit Graph of Dihedral Groups On Some Topological Indices for the Orbit Graph of Dihedral Groups 199 1. W(ΓΥ
G) = 1
2t(t −1)
2. M1(ΓΥ
G = t(t −1)2
3. M2(ΓΥ
G) = t(t−1)3
2
4. M3(ΓΥ
G) = t(t −1)4
5. HM1(ΓΥ
G) = 2t(t −1)3
6. HM2(ΓΥ
G) = t(t−1)5
2
7. ModM1(ΓΥ
G) =
t
(t−1)2
8. S c(ΓΥ
G) = t(t −1)2
9. S ∗
c(ΓΥ
G) = t(t−1)3
2
10. F(ΓΥ
G) = t(t −1)3
11. S o(ΓΥ
G) = t(t −1)2
12. R(ΓΥ
G) = t
2
13. RR(ΓΥ
G) = t(t−1)2
2 Proof. In order to calculate the indices of a graph, we have to determine the number of vertices of the
graph. From the Definition 4, if the group action on the set Υ is applied then |Υ| −|B| = t −0 = t and
the number of vertices of the orbit graph is considered to be a non-central orbit. Proof. In order to calculate the indices of a graph, we have to determine the number of vertices of the
graph. From the Definition 4, if the group action on the set Υ is applied then |Υ| −|B| = t −0 = t and
the number of vertices of the orbit graph is considered to be a non-central orbit. 3. Indices for orbit graph of Dt Since, we are considering the third case of the Theorem 1, where the graph is complete then the
number of edges of the orbit graph are t(t−1)
2
and distance and degree of the vertex is (t −1) (All the
indices are determined on the basis of above predefined definitions). Therefore, Since, we are considering the third case of the Theorem 1, where the graph is complete then the
number of edges of the orbit graph are t(t−1)
2
and distance and degree of the vertex is (t −1) (All the
indices are determined on the basis of above predefined definitions). Therefore, W(ΓΥ
G)
=
1
2
tX
i=1
tX
j=1
d(ui, uj)
=
1
2
tX
i=1
[d(ui, u1) + d(ui, u2) + ... + d(ui, ut)]
=
1
2[d(u1, u1) + d(u2, u1) + ... + d(ut, u1) + ... +
d(u1, ut) + d(u2, ut) + ... + d(ut, ut)]
=
1
2[(t −1) + (t −1) + ... + (t −1)]
=
1
2[t(t −1)]
M1(ΓΥ
G)
=
tX
i=1
(deg(ui))2
=
deg(u1)2 + deg(u2)2 + ... + deg(ut)2
=
[(t −1)2 + (t −1)2 + ... + (t −1)2] =
1
2[d(u1, u1) + d(u2, u1) + ... + d(ut, u1) + ... +
d(u1, ut) + d(u2, ut) + ... + d(ut, ut)]
=
1
2[(t −1) + (t −1) + ... + (t −1)]
=
1
2[t(t −1)] Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Journal of Combinatorial Mathematics and Combinatorial Computing
Volum Volume 117, 195–208 Volume 117, 195–208 Vijay Kumar Bhat and Karnika Sharma 200 M2(ΓΥ
G)
=
t(t−1)
2X
i,j=1
deg(ui)deg(uj)
=
(t −1)(t −1) + (t −1)(t −1) + ... + (t −1)(t −1)
=
(t −1)(t −1)(t(t −1)
2
)
=
(t −1)3 t
2 M3(ΓΥ
G)
=
t(t−1)
2X
i,j=1
(deg(ui)deg(uj))(deg(ui) + deg(uj)) G)
X
i,j=1
(
g( i)
g(
j))(
g( i)
g(
j))
=
[((t −1)(t −1))((t −1) + (t −1))] +
[((t −1)(t −1))((t −1) + (t −1))] + ... +
[((t −1)(t −1))((t −1) + (t −1))]
=
(t −1)2[2(t −1)] + (t −1)2[2(t −1)] + ... +
(t −1)2[2(t −1)]
=
2(t −1)3 + 2(t −1)3 + ... Journal of Combinatorial Mathematics and Combinatorial Computing 3. Indices for orbit graph of Dt + 2(t −1)3
=
t(t −1)
2
[2(t −1)3]
=
t(t −1)4
ΓΥ
G)
=
t(t−1)
2X
i j=1
(deg(ui) + deg(uj))2 =
[((t −1)(t −1))((t −1) + (t −1))] +
[((t −1)(t −1))((t −1) + (t −1))] + ... +
[((t −1)(t −1))((t −1) + (t −1))] =
(t −1)2[2(t −1)] + (t −1)2[2(t −1)] + ... +
(t −1)2[2(t −1)] =
(t −1)2[2(t −1)] + (t −1)2[2(t −1)] + ... +
(t −1)2[2(t −1)] =
2(t −1)3 + 2(t −1)3 + ... + 2(t −1)3
(
1) HM1(ΓΥ
G)
=
2X
i,j=1
(deg(ui) + deg(uj))2 j
=
((t −1) + (t −1))2 + ((t −1) + (t −1))2 + ... +
((t −1) + (t −1))2
=
(2(t −1))2 + (2(t −1))2 + ... + (2(t −1))2
=
t(t −1)
2
(2(t −1))2
=
t(t −1)
2
[4(t −1)2]
=
2t(t −1)3 =
((t −1) + (t −1))2 + ((t −1) + (t −1))2 + ... +
((t −1) + (t −1))2 HM2(ΓΥ
G)
=
t(t−1)
2X
i, j=1
(deg(ui)deg(uj))2
=
((t −1)(t −1))2 + ((t −1)(t −1))2 + ... +
((t −1)(t −1))2
=
((t −1)2)2 + ((t −1)2)2 + ... + ((t −1)2)2
=
(t −1)4 + (t −1)4 + ... + (t −1)4
=
t(t −1)
2
(t −1)4
=
t(t −1)5
2 HM2(ΓΥ
G)
=
t(t−1)
2X
i, j=1
(deg(ui)deg(uj))2 Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Journal of Combinatorial Mathematics and Combinatorial Computing
Volum atics and Combinatorial Computing
Volume 117, 195–208 Volume 117, 195–208 Volume 117, 195–208 On Some Topological Indices for the Orbit Graph of Dihedral Groups 201 ModM1(ΓΥ
G)
=
tX
i,j=1
1
deg(ui)2
=
1
(t −1)2 +
1
(t −1)2 + ... +
1
(t −1)2
=
t[
1
(t −1)2]
=
t
(t −1)2 S c(ΓΥ
G)
=
1
2
X
(deg(ui) + deg(uj))d(ui, uj)
=
1
2[((t −1) + (t −1))(t −1) + ((t −1) + (t −1))
(t −1) + ... + ((t −1) + (t −1))(t −1)]
=
1
2[2(t −1)2 + 2(t −1)2 + ... + 2(t −1)2]
=
1
2t(2(t −1)2)
=
t(t −1)2 S c(ΓΥ
G)
=
1
2
X
(deg(ui) + deg(uj))d(ui, uj)
=
1
2[((t −1) + (t −1))(t −1) + ((t −1) + (t −1))
(t −1) + ... 3. Indices for orbit graph of Dt + ((t −1) + (t −1))(t −1)]
=
1
2[2(t −1)2 + 2(t −1)2 + ... + 2(t −1)2]
=
1
2t(2(t −1)2)
=
t(t −1)2 S ∗
c(ΓΥ
G)
=
1
2
X
(deg(ui)deg(uj))d(ui, uj)
=
1
2[((t −1)(t −1))(t −1) + ((t −1)(t −1))(t −1)
+... + ((t −1)(t −1))(t −1)]
=
1
2[(t −1)3 + (t −1)3 + ... + (t −1)3]
=
1
2t(t −1)3 S ∗
c(ΓΥ
G)
=
1
2
X
(deg(ui)deg(uj))d(ui, uj)
=
1
2[((t −1)(t −1))(t −1) + ((t −1)(t −1))(t −1)
+... + ((t −1)(t −1))(t −1)]
=
1
2[(t −1)3 + (t −1)3 + ... + (t −1)3]
=
1
2t(t −1)3 =
1
2[(t −1)3 + (t −1)3 + ... + (t −1)3] F(ΓΥ
G)
=
t(t−1)
2X
i,j=1
deg(ui)2 + deg(uj)2
=
((t −1)2 + (t −1)2) + ((t −1)2 + (t −1)2)
+... + ((t −1)2 + (t −1)2)
=
[2(t −1)2 + 2(t −1)2 + ... + 2(t −1)2]
=
t(t −1)
2
[2(t −1)2]
=
t(t −1)3 F(ΓΥ
G)
=
t(t−1)
2X
i,j=1
deg(ui)2 + deg(uj)2
=
((t −1)2 + (t −1)2) + ((t −1)2 + (t −1)2)
+... + ((t −1)2 + (t −1)2)
=
[2(t −1)2 + 2(t −1)2 + ... + 2(t −1)2]
=
t(t −1)
2
[2(t −1)2]
=
t(t −1)3 F(ΓΥ
G)
=
t(t−1)
2X
i,j=1
deg(ui)2 + deg(uj)2 =
((t −1)2 + (t −1)2) + ((t −1)2 + (t −1)2)
+... + ((t −1)2 + (t −1)2) =
[2(t −1)2 + 2(t −1)2 + ... + 2(t −1)2]
(
1) S o(ΓΥ
G)
=
t(t−1)
2X
i,j=1
(deg(ui)2 + deg(uj)2)
1
2
=
((t −1)2 + (t −1)2)
1
2 + ((t −1)2 + (t −1)2)
1
2
+... + ((t −1)2 + (t −1)2)
1
2
=
(2(t −1)2)
1
2 + (2(t −1)2)
1
2 + ... + (2(t −1)2)
1
2
=
2(t −1) + 2(t −1) + ... + 2(t −1) S o(ΓΥ
G)
=
(
)
2X
i,j=1
(deg(ui)2 + deg(uj)2)
1
2 =
((t −1)2 + (t −1)2)
1
2 + ((t −1)2 + (t −1)2)
1
2
+... + ((t −1)2 + (t −1)2)
1
2
=
(2(t −1)2)
1
2 + (2(t −1)2)
1
2 + ... + (2(t −1)2)
1
2
=
2(t −1) + 2(t −1) + ... 3. Indices for orbit graph of Dt + 2(t −1) Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 202 Vijay Kumar Bhat and Karnika Sharma =
t(t −1)
2
(2(t −1))
=
t(t −1)2
R(ΓΥ
G)
=
t(t−1)
2X
i,j=1
(deg(ui)deg(uj))−1
2
=
t(t−1)
2X
i,j=1
1
pdeg(ui)deg(uj)
=
1
((t −1)(t −1))
1
2 +
1
((t −1)(t −1))
1
2 + ... +
1
((t −1)(t −1))
1
2
=
1
((t −1)2)
1
2 +
1
((t −1)2)
1
2 + ... +
1
((t −1)2)
1
2
=
1
(t −1) +
1
(t −1) + ... +
1
(t −1)
=
t(t −1)
2
(
1
(t −1))
=
t
2 RR(ΓΥ
G)
=
t(t−1)
2X
i,j=1
(deg(ui)deg(uj))
1
2 RR(ΓΥ
G)
=
t(t−1)
2X
i,j=1
(deg(ui)deg(uj))
1
2
=
((t −1)(t −1))
1
2 + ((t −1)(t −1))
1
2 + ... +
((t −1)(t −1))
1
2
=
((t −1)2)
1
2 + ((t −1)2)
1
2 + ... + ((t −1)2)
1
2
=
t(t −1)
2
[((t −1)2)1
2]
=
t(t −1)2
2 ,j
=
((t −1)(t −1))
1
2 + ((t −1)(t −1))
1
2 + ... +
((t −1)(t −1))
1
2
=
((t −1)2)
1
2 + ((t −1)2)
1
2 + ... + ((t −1)2)
1
2
=
t(t −1)
2
[((t −1)2)1
2]
=
t(t −1)2
2 □ □ Example 1: Consider the orbit graph K15 (see Figure 2). Now, the graph K15 have |Υ| −|B| = 15 −0 = 15 vertices and 15(15−1)
2
= 105 edges, respectively. Al
th d
d di t
f
ti
(15
1)
14 Th Example 1: Consider the orbit graph K15 (see Figure 2). Now, the graph K15 have |Υ| −|B| = 15 −0 = 15 vertices and 15(15−1)
2
= 105 edges, respectively. Example 1: Consider the orbit graph K15 (see Figure 2). Now, the graph K15 have |Υ| −|B| = 15 −0 = 15 vertices and 15(15−1)
2
= 105 edges, respectively. 2
Also, the degree and distance of vertices are (15 −1) = 14. Then 2
Also, the degree and distance of vertices are (15 −1) = 14. Journal of Combinatorial Mathematics and Combinatorial Computing 3. Indices for orbit graph of Dt Then W(ΓΥ
G)
=
105
M1(ΓΥ
G)
=
2940
M2(ΓΥ
G)
=
20580
M3(ΓΥ
G)
=
576240
HM1(ΓΥ
G)
=
82320
HM2(ΓΥ
G)
=
4033680
ModM1(ΓΥ
G)
=
0.00510 Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Journal of Combinatorial Mathematics and Combinatorial Computing
Volum On Some Topological Indices for the Orbit Graph of Dihedral Groups 203 On Some Topological Indices for the Orbit Graph of Dihedral Groups On Some Topological Indices for the Orbit Graph of Dihedral Groups
203
Figure 2. Orbit graph K15
S c(ΓΥ
G)
=
2940
S ∗
c(ΓΥ
G)
=
20580
F(ΓΥ
G)
=
41160
S o(ΓΥ
G)
=
2940
R(ΓΥ
G)
=
7.5
RR(ΓΥ
G)
=
1470
4. Relation with wiener index On Some Topological Indices for the Orbit Graph of Dihedral Groups
203
Figure 2. Orbit graph K15
S c(ΓΥ
G)
=
2940
S ∗
c(ΓΥ
G)
=
20580
F(ΓΥ
G)
=
41160
S o(ΓΥ
G)
=
2940
R(ΓΥ
G)
=
7.5
RR(ΓΥ
G)
=
1470
4. Relation with wiener index Figure 2. Orbit graph K15 4. Relation with wiener index In this section, we relate each of the indices with the Wiener index and also their relationship with
each other. In this section, we relate each of the indices with the Wiener index and also their relationship with
each other. Theorem 3. For orbit graph of dihedral group, Theorem 3. For orbit graph of dihedral group,
S ∗
c(ΓΥ
G) = W(ΓΥ
G)(t −1)2 = M2(ΓΥ
G) f
S ∗
c(ΓΥ
G) = W(ΓΥ
G)(t −1)2 = M2(ΓΥ
G) Proof. As we obtain the modified schultz index for the orbit graph of dihedral group in the above
Theorem 2. Then we have Proof. As we obtain the modified schultz index for the orbit graph of dihedral group in the above
Theorem 2. Then we have S ∗
c(ΓΥ
G) = t(t −1)3
2 S ∗
c(ΓΥ
G) = t(t −1)3
2 S ∗
c(ΓΥ
G) = t(t −1)3
2 Since, W(ΓΥ
G) = 1
2t(t −1) then by substituting the value of W(ΓΥ
G) in S ∗
c(ΓΥ
G),we get S ∗
c(ΓΥ
G) =t(t −1)
2
(t −1)2
=W(ΓΥ
G)(t −1)2 S ∗
c(ΓΥ
G) =t(t −1)
2
(t −1)2
=W(ΓΥ
G)(t −1)2 This implies, This implies, This implies,
S ∗
c(ΓΥ
G)=W(ΓΥ
G)(t −1)2. p
S ∗
c(ΓΥ
G)=W(ΓΥ
G)(t −1)2. S c(ΓG)=W(ΓG)(t
1) . and it is obvious from the above prove result that S ∗
c(ΓΥ
G) = M2(ΓΥ
G). □
Theorem 4. M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) c(
G)
(
G)(
)
and it is obvious from the above prove result that S ∗
c(ΓΥ
G) = M2(ΓΥ
G). c(
G)
(
G)(
)
and it is obvious from the above prove result that S ∗
c(ΓΥ
G) = M2(ΓΥ
G). □
Theorem 4. M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) □ c(
G)
(
G)(
)
and it is obvious from the above prove result that S ∗
c(ΓΥ
G) = M2(ΓΥ
G). □ Theorem 4. M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) Theorem 4. M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) Proof. Since, Proof. Since, Proof. 4. Relation with wiener index Since, M1(ΓΥ
G) =t(t −1)2
S c(ΓΥ
G) =t(t −1)2 Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 Volume 117, 195–208 Vijay Kumar Bhat and Karnika Sharma 204 S o(ΓΥ
G) =t(t −1)2 This implies, This implies, W(ΓΥ
G) =1
2t(t −1)
2W(ΓΥ
G) =t(t −1) Therefore, Therefore,
M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) Therefore,
M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G)
□ M1(ΓΥ
G) = S c(ΓΥ
G) = S o(ΓΥ
G) = 2(t −1)W(ΓΥ
G) □ Theorem 5. For orbit graph of dihedral group, the second Zagreb index is the product of Wiener
index and the first Zagreb index. Theorem 5. For orbit graph of dihedral group, the second Zagreb index is the product of Wiener
index and the first Zagreb index. M2(ΓΥ
G) = 1
t W(ΓΥ
G)M1(ΓΥ
G) M2(ΓΥ
G) = 1
t W(ΓΥ
G)M1(ΓΥ
G) M2(ΓΥ
G) = 1
t W(ΓΥ
G)M1(ΓΥ
G) M2(ΓΥ
G) = 1
t W(ΓΥ
G)M1(ΓΥ
G) Proof. we obtain M2(ΓΥ
G) of dihedral group as Proof. we obtain M2(ΓΥ
G) of dihedral group as Proof. we obtain M2(ΓΥ
G) of dihedral group as Proof. we obtain M2(ΓΥ
G) of dihedral group as Proof. we obtain M2(ΓΥ
G) of dihedral group as M2(ΓΥ
G) = t(t −1)3
2
Then,
M2(ΓΥ
G) =t(t −1)2(t −1)
2
=1
2(t −1)M1(ΓΥ
G)
=1
t W(ΓΥ
G)M1(ΓΥ
G) M2(ΓΥ
G) = t(t −1)3
2 M2(ΓΥ
G) = t(t −1)3
2 M2(ΓΥ
G) = t(t −1)3
2
Then, Then, Then, M2(ΓΥ
G) =t(t −1)2(t −1)
2
=1
2(t −1)M1(ΓΥ
G)
=1
t W(ΓΥ
G)M1(ΓΥ
G) M2(ΓΥ
G) =t(t −1)2(t −1)
2
=1
2(t −1)M1(ΓΥ
G)
=1
t W(ΓΥ
G)M1(ΓΥ
G) □ Theorem 6. M3(ΓΥ
G) = (t −1)2M1(ΓΥ
G) = 4
t W(ΓΥ
G)M2(ΓΥ
G) Theorem 6. M3(ΓΥ
G) = (t −1)2M1(ΓΥ
G) = 4
t W(ΓΥ
G)M2(ΓΥ
G) Proof. Proof. Proof. M3(ΓΥ
G) =t(t −1)4
=t(t −1)2(t −1)2
=(t −1)2M1(ΓΥ
G) M3(ΓΥ
G) =t(t −1)4
=t(t −1)2(t −1)2
=(t −1)2M1(ΓΥ
G) Again, Again, By splitting the HM2(ΓΥ
G) = t(t−1)5
2
, we obtain the result Proof. From Theorem 2, we obtain Proof. From Theorem 2, we obtain ModM1(ΓΥ
G) =
t
(t −1)2
and
R(ΓΥ
G) = t
2
This implies,
2R(ΓΥ
G) =t ModM1(ΓΥ
G) =
t
(t −1)2
and
R(ΓΥ
G) = t
2 ModM1(ΓΥ
G) =
t
(t −1)2
and ModM1(ΓΥ
G) =
t
(t −1)2 and R(ΓΥ
G) = t
2 2R(ΓΥ
G) =t Again, M3(ΓΥ
G) =t(t −1)4
=t(t −1)3(t −1)
=2(t −1)M2(ΓΥ
G)
=4
t W(ΓΥ
G)M2(ΓΥ
G) M3(ΓΥ
G) =t(t −1)4
=t(t −1)3(t −1)
=2(t −1)M2(ΓΥ
G)
=4
t W(ΓΥ
G)M2(ΓΥ
G) where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). □
Theorem 7. HM1(ΓΥ
G) = 2(t −1)M1(ΓΥ
G) = 4W(ΓΥ
G)M1(ΓΥ
G) = 4(t −1)2W(ΓΥ
G). where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). □ where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). □ where, (t −1) = 2
t W(ΓΥ
G) and t(t −1)3 = 2M2(ΓΥ
G). □
Theorem 7. HM1(ΓΥ
G) = 2(t −1)M1(ΓΥ
G) = 4
t W(ΓΥ
G)M1(ΓΥ
G) = 4(t −1)2W(ΓΥ
G). 2(t −1)M1(ΓΥ
G) = 4
t W(ΓΥ
G)M1(ΓΥ
G) = 4(t −1)2W(ΓΥ
G). Theorem 7. HM1(ΓΥ
G) = 2(t −1)M1(ΓΥ
G) = 4
t W(ΓΥ
G)M1(ΓΥ
G) = 4(t −1)2W(ΓΥ
G). Proof. Proof. HM1(ΓΥ
G) =2t(t −1)3
=2(t −1)t(t −1)2 HM1(ΓΥ
G) =2t(t −1)3 Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 Volume 117, 195–208 205 On Some Topological Indices for the Orbit Graph of Dihedral Groups n Some Topological Indices for the Orbit Graph of Dihedral Groups =2(t −1)M1(ΓΥ
G)
=4
t W(ΓΥ
G)M1(ΓΥ
G) where, (t −1) = 2
t W(ΓΥ
G). Also, where, (t −1) = 2
t W(ΓΥ
G). Also, here, (t −1) = 2
t W(ΓΥ
G). HM1(ΓΥ
G) =2t(t −1)3
=2t(t −1)(t −1)2
=4(t −1)2W(ΓΥ
G) HM1(ΓΥ
G) =2t(t −1)3
=2t(t −1)(t −1)2
=4(t −1)2W(ΓΥ
G) where, t(t −1) = 2W(ΓΥ
G). □ where, t(t −1) = 2W(ΓΥ
G). □ □ Theorem 8. HM2(ΓΥ
G) = (t −1)4W(ΓΥ
G) = 1
2(t −1)3M1(ΓΥ
G) = (t −1)2M2(ΓΥ
G). Theorem 8. HM2(ΓΥ
G) = (t −1)4W(ΓΥ
G) = 1
2(t −1)3M1(ΓΥ
G) = (t −1)2M2(ΓΥ
G). Proof. By splitting the HM2(ΓΥ
G) = t(t−1)5
2
, we obtain the result
□
Theorem 9. ModM1(ΓΥ
G) =
2
(t−1)2R(ΓΥ
G) Proof. By splitting the HM2(ΓΥ
G) = t(t−1)5
2
, we obtain the result
Theorem 9 ModM (ΓΥ)
2 R(ΓΥ) □ Proof. 2R(ΓΥ
G) =t Substituting the value of t in ModM1(ΓΥ
G), we have
2R(ΓΥ) Substituting the value of t in ModM1(ΓΥ
G), we have Substituting the value of t in ModM1(ΓΥ
G), we have ModM1(ΓΥ
G) =2R(ΓΥ
G)
(t −1)2 ModM1(ΓΥ
G) =2R(ΓΥ
G)
(t −1)2 □ Theorem 10. For the orbit graph of dihedral group, Forgotten index is the twice of second Zagreb
index and modified Schultz index Theorem 10. For the orbit graph of dihedral group, Forgotten index is the twice of second Zagreb
index and modified Schultz index F(ΓΥ
G)
=
2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G)
F(ΓΥ
G)
=
2(t −1)2W(ΓΥ
G) = (t −1)M1(ΓΥ
G) = 2(t −1)RR(ΓΥ
G). Proof. From Theorem 2, F(ΓΥ
G) = 2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G). Now, F(ΓΥ
G)
=
2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G)
F(ΓΥ
G)
=
2(t −1)2W(ΓΥ
G) = (t −1)M1(ΓΥ
G) = 2(t −1)RR(ΓΥ
G). F(ΓΥ
G)
=
2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G)
F(ΓΥ
G)
=
2(t −1)2W(ΓΥ
G) = (t −1)M1(ΓΥ
G) = 2(t −1)RR(ΓΥ
G). orem 2, F(ΓΥ
G) = 2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G). Proof. From Theorem 2, F(ΓΥ
G) = 2M2(ΓΥ
G) = 2S ∗
c(ΓΥ
G). Now, F(ΓΥ
G) = t(t −1)3 by splitting the formula as t(t −1)2(t −1), we have
F(ΓΥ
G) = (t −1)M1(ΓΥ
G) F(ΓΥ
G) = t(t −1)3 by splitting the formula as t(t −1)2(t −1), we have
F(ΓΥ
G) = (t −1)M1(ΓΥ
G) (
G)
(
)
1(
G)
Since, W(ΓΥ
G) = 1
2t(t −1) which gives F(ΓΥ
G) = 2(t −1)2W(ΓΥ
G)
Also, (
G)
(
)
1(
G)
Since, W(ΓΥ
G) = 1
2t(t −1) which gives F(ΓΥ
G) = 2(t −1)2W(ΓΥ
G)
Also, F(ΓΥ
G) =t(t −1)3
=t(t −1)2(t −1)
=2(t −1)RR(ΓΥ
G) F(ΓΥ
G) =t(t −1)3
=t(t −1)2(t −1)
=2(t −1)RR(ΓΥ
G) where, t(t −1)2 = 2RR(ΓΥ
G). □ where, t(t −1)2 = 2RR(ΓΥ
G). □ where, t(t −1)2 = 2RR(ΓΥ
G). where, t(t −1)2 = 2RR(ΓΥ
G). □ Journal of Combinatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 binatorial Mathematics and Combinatorial Computing
Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing
Vo Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 Volume 117, 195–208 Vijay Kumar Bhat and Karnika Sharma Vijay Kumar Bhat and Karnika Sharma 206 5. Polynomial for the Indices of Dt 5. Polynomial for the Indices of Dt In this section, we obtain the polynomial of each index for the orbit graph of the dihedral group. Theorem 11. Let ΓΥ
G be the orbit graph of the dihedral group. Then the polynomial of each index is
1. W(ΓΥ
G) = P x
1
2 t(t−1) In this section, we obtain the polynomial of each index for the orbit graph of the dihedral group. Theorem 11. Let ΓΥ
G be the orbit graph of the dihedral group. Then the polynomial of each index is
1 W(ΓΥ) = P x
1
2 t(t−1) Theorem 11. Let ΓΥ
G be the orbit graph of the dihedral group. Then the polynomial of each index is G
1. W(ΓΥ
G) = P x
1
2 t(t−1)
2. M1(ΓΥ
G) = P xt(t−1)2
3. M2(ΓΥ
G) = P x
t(t−1)3
2
4. M3(ΓΥ
G) = P xt(t−1)4
5. HM1(ΓΥ
G) = P x2t(t−1)3
6. HM2(ΓΥ
G) = P x
t(t−1)5
2
7. ModM1(ΓΥ
G) = P x
t
(t−1)2
8. S c(ΓΥ
G) = P xt(t−1)2
9. S ∗
c(ΓΥ
G) = P x
t(t−1)3
2
10. F(ΓΥ
G) = P xt(t−1)3
11. S o(ΓΥ
G) = P xt(t−1)2
12. R(ΓΥ
G) = P x
t
2
13. RR(ΓΥ
G) = P x
t(t−1)2
2 Proof. From Theorem 2, the result is obvious. □ Proof. From Theorem 2, the result is obvious. Example 2: Consider the orbit graph K9 (see Figure 3). Now, the graph K9 have |Υ| −|B| = 9 −0 = 9 vertices and 9(9−1)
2
= 36 edges, respectively. Figure 3. Orbit graph K9
Also, the degree and distance of vertices are (9 −1) = 8. Then
W(ΓΥ
G) =
P x36 Figure 3. Orbit graph K9 Figure 3. Orbit graph K9 Also, the degree and distance of vertices are (9 −1) = 8. Then
W(ΓΥ
G) =
P x36 Also, the degree and distance of vertices are (9 −1) = 8. 6. Conclusion Because topological indices can only be computed for connected graphs, we only consider the
third case of the dihedral group’s orbit graph. Since the orbit graph of a dihedral group is complete,
we obtain the indices by generalising the Wiener index, the first, second and third Zagreb indices, the
Hyper first and second Zagreb indices, the Schultz and modified Schultz indices, the Somber index,
the Forgotten index, and the Randic and Reciprocal Randic indices of a complete graph. We also learn
how these indices relate to the Wiener index for the orbit graph of the dihedral group. Furthermore,
the polynomial for the indices of the group’s orbit graph has been determined. 5. Polynomial for the Indices of Dt Then
W(ΓΥ
G) =
P x36 Volume 117, 195–208 Journal of Combinatorial Mathematics and Combinatorial Computing Volume 117, 195–208 On Some Topological Indices for the Orbit Graph of Dihedral Groups
207
M1(ΓΥ
G) =
P x576
M2(ΓΥ
G) =
P x2304
M3(ΓΥ
G) =
P x36864
HM1(ΓΥ
G) =
P x9216
HM2(ΓΥ
G) =
P x147456
ModM1(ΓΥ
G) =
P x0.14
S c(ΓΥ
G) =
P x576
S ∗
c(ΓΥ
G) =
P x2304
F(ΓΥ
G) =
P x4608
S o(ΓΥ
G) =
P x576
R(ΓΥ
G) =
P x4.5
RR(ΓΥ
G) =
P x288
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Abstract Background: It is unclear if the rat myocardium undergoes the same rapid reductions in protein content that are classically
observed in fast-twitch skeletal muscle during endotoxaemia. Methodology/Principal Findings: To investigate this further, and to determine if there is any divergence in the response of
skeletal muscle and myocardium in the mechanisms that are thought to be largely responsible for eliciting changes in
protein content, Sprague Dawley rats were implanted with vascular catheters and administered lipopolysaccharide (LPS;
150 mg kg21 h21) intravenously for 2 h, 6 h or 24 h (saline administered control animals were also included), after which the
extensor digitorum longus (EDL) and myocardium were removed under terminal anaesthesia. The protein-to-DNA ratio, a
marker of protein content, was significantly reduced in the EDL following 24 h LPS administration (23%; P,0.05), but was
no different from controls in the myocardium. At the same time point, a significant increase in MAFbx/atrogin-1 and MuRF1
mRNA (3.760.7- and 19.561.9-fold increase vs. controls, respectively; P,0.05), in addition to protein levels of a1-3, 5–7
subunits of the 20S proteasome, were observed in EDL but not myocardium. In contrast, elevations in phosphorylation of
p70 S6K residues Thr421/Ser424, and 4E-BP1 residues Thr37/Thr46 (P,0.05), consistent with an elevation in translation
initiation, were seen exclusively in the myocardium of LPS-treated animals. Conclusions/Significance: In summary, these findings suggest that the myocardium does not undergo the same catabolic
response as skeletal muscle during early endotoxaemia, partly due to the absence of transcriptional and signalling events in
the myocardium typically associated with increased muscle proteolysis and the suppression of protein synthesis. Citation: Murton AJ, Alamdari N, Gardiner SM, Constantin-Teodosiu D, Layfield R, et al. (2009) Effects of Endotoxaemia on Protein Metabolism in Rat Fast-Twitch
Skeletal Muscle and Myocardium. PLoS ONE 4(9): e6945. doi:10.1371/journal.pone.0006945
Editor: Gisela Nogales-Gadea, University Hospital Vall d’Hebron, Spain Citation: Murton AJ, Alamdari N, Gardiner SM, Constantin-Teodosiu D, Layfield R, et al. (2009) Effects of Endotoxaemia on Protein Metabolism in Rat Fast-Twitch
Skeletal Muscle and Myocardium. PLoS ONE 4(9): e6945. doi:10.1371/journal.pone.0006945 Received June 8, 2009; Accepted July 29, 2009; Published September 14, 2009 Copyright: 2009 Murton et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by the UK Medical Research Council. Abstract Capacity Building Studentships were provided by the Medical Research Council to
support Andrew Murton and Nima Alamdari. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andrew.murton@nottingham.ac.uk Andrew J. Murton*, Nima Alamdari, Sheila M. Gardiner, Dumitru Constantin-Teodosiu, Robert Layfield,
Terence Bennett, Paul L. Greenhaff Andrew J. Murton*, Nima Alamdari, Sheila M. Gardiner, Dumitru Constantin-Teodosiu, Robert Layfield,
Terence Bennett, Paul L. Greenhaff Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, United Kingdom PLoS ONE | www.plosone.org Endotoxin and the Myocardium Endotoxin and the Myocardium undergoes the same rapid reduction in protein content classically
observed in rodent fast-twitch skeletal muscle. Conflicting reports
detailing both a decline [15] and the maintenance [16,17] of
myocardial protein content following experimentally induced
endotoxaemia have been described. Thus, we sought to determine
if an i.v., non-lethal dose of bacterial LPS, administered to
conscious rats, resulted in a change in myocardial protein content,
and examined in the same tissue, transcriptional and signalling
events that are typically associated with endotoxaemia-induced
protein loss in fast-twitch skeletal muscle [18]. An attempt was also
made to relate any observed differences between myocardial and
skeletal muscle tissue to localised levels of the two cytokines, tumor
necrosis factor-alpha (TNFa) and interleukin-6 (IL-6), as they
have, in part, been implicated in eliciting reductions in muscle
protein content [19,20]. Here we show that while significant
declines in skeletal muscle protein content are observed in
response to LPS infusion, myocardial protein content is main-
tained at normal levels. Furthermore, we present evidence to
suggest that this is in part due to a differential response to
endotoxin in the processes that govern UP-mediated degradation
and translation initiation in these two tissues. clamped (whilst still beating) with liquid nitrogen-cooled alumin-
ium tongs, and subsequently transferred to liquid nitrogen for
storage. Two 24 h LPS-infused rats and one 6 h saline-infused rat
stopped breathing before removal of tissues was complete, and
were excluded from the study. To examine muscle signalling
events associated with early endotoxaemia, 16 additional animals
underwent the procedures described above and received either
LPS (n = 8) or saline (n = 8) i.v. for a 24 h period. This sole time-
point was selected as previous work emanating from our
laboratory had shown 24 h LPS administration was sufficient to
result in significant suppression of AKT-mediated signalling events
in fast-twitch skeletal muscle [3]. The EDL and myocardium
removed from these animals were used exclusively for the
investigation of changes in protein and phosphorylation levels of
translation initiation signalling events. Cardiovascular recordings Muscle contraction is well known to modulate processes that
control muscle mass [23] and we have previously shown LPS
administration to result in prolonged and persistent tachycardia
[24]. Therefore, to examine any association between the observed
tachycardia seen in LPS-treated animals and myocardial tran-
scriptional and signalling events, recordings of heart rate (HR)
were made. This was accomplished by using a customised,
computer-based system (Haemodynamics Data Acquisition Sys-
tem, University of Limburg, Netherlands) connected to a BP
transducer amplifier (Gould model 13-4615-50; Gould Instrument
Systems Inc., Valley View, USA), providing both HR and BP
recordings. Data were sampled by the Haemodynamics Data
Acquisition System every 2 ms, averaged each cardiac cycle, and
stored to disc every 5 s. Data were analysed off-line (Datview;
University of Maastricht, Netherlands) and measurements were
made under resting conditions and at 1 h intervals for the first 6 h
after LPS administration and again at 24 h. Alkaline soluble protein/DNA ratio The alkaline soluble protein (ASP)/DNA ratio may be used as a
measure of net protein catabolism in skeletal muscle [3,25,26]. Approximately 5 mg of muscle tissue was freeze-dried and
powdered, with care taken to remove any connective tissue
present. ASP and DNA were individually isolated from the
powdered muscle using repeated perchloric acid extractions and
washings, with the ASP being ultimately extracted by the addition
of potassium hydroxide. ASP concentration was determined using
the method of Lowry [27], and DNA was quantified using a
modification of the diphenyl-amine reaction described by Munro
and Fleck [28]. Ethics Statement All procedures involving live animals, as detailed below, were
approved by the University of Nottingham Ethical Review
Committee and were performed under U.K. Home Office Project
and Personal License authority. Animal preparation and experimental protocol Sixty four male Sprague-Dawley rats (380–480 g; Charles River,
Margate, UK) were anesthetised using fentanyl (Janssen-Cilag,
High
Wycombe,
UK) and
medetomidine
(Domitor, Pfizer,
Sandwich, UK; 300 mg?kg21 of each i.p.) and had intravenous
(right jugular vein) catheters implanted for the administration of
substances. In animals designated for 24 h LPS treatment (see
below) catheters were also implanted in the distal abdominal aorta
(via the ventral caudal artery) for monitoring of blood pressure (BP)
and heart rate (HR). We have previously found saline administra-
tion, as used in control animals, to have no significant effect on
baseline cardiovascular status over 24 h [21], and therefore the BP
and HR of control animals were not monitored. Anaesthesia was
reversed with atipamezole (Antisedan, Pfizer, Sandwich, UK) and
nalbuphine (Bristol-Myers Squibb, Uxbridge, UK; 1 mg?kg21 of
each s.c.), with the latter also providing analgesia. Rats were allowed
to recover overnight in their home cages and were given free access
to both food and water. A counter-balanced tether system
connected to a harness and fitted to the rat carried the catheters,
and allowed relatively unrestricted movement within the home
cage. Double channel swivel systems were constructed according to
[22] which enabled the continuous administration of saline or LPS
to the unrestrained animals. PLoS ONE | www.plosone.org Introduction initiation, where increases in AKT activity lead to increases in
both eIF2B guanidine exchange factor activity and eIF4F
assembly, via the phosphorylation and subsequent inhibition of
GSK3b and 4E-BP1, respectively [9], processes that are pivotal to
the correct formation of a functioning ribosomal initiation
complex for translation initiation to occur. Furthermore, when
activated, AKT can phosphorylate the forkhead Foxo family of
transcription factors, leading to their transportation away from the
nucleus and into the cytoplasm where they are inactive [7,8]. However, when present in the nucleus, Foxo1, Foxo3 and Foxo4
can induce the transcription of the two muscle-specific ubiquitin
ligases, MAFbx/atrogin-1 and MuRF1, which are thought
responsible for the specific targeting of proteins for subsequent
degradation by the proteasome [7,8]. A well known consequence of sepsis is the rapid and debilitating
loss of skeletal muscle mass, which appears the result of a
coordinated reduction of muscle protein synthesis together with an
enhancement of proteasomal mediated muscle protein breakdown
[1,2]. However, the effects of sepsis on the mechanisms that
regulate protein turnover in the myocardium are, in comparison,
poorly understood. We have recently shown the induction of lipopolysaccharide
(LPS)-induced endotoxaemia in rodents to result in the reduction
of total AKT protein levels, decreased cytosolic Foxo1 and Foxo3
phosphorylation, and increased levels of MAFbx/atrogin-1 protein
and MuRF1 mRNA in fast-twitch skeletal muscle [3], where
MAFbx/atrogin-1 and MuRF1 are considered important in the
development of skeletal muscle proteolysis during catabolic events
[4–6]. These in vivo findings are important because, when viewed
in conjunction with similar observations in C2C12 cells [7,8], they
suggest AKT as a regulator of both protein translation initiation
and ubiquitin-proteasome (UP) mediated muscle proteolysis. Indeed, AKT is well known as an upstream regulator of translation Interestingly, while numerous studies have focused on skeletal
muscle when considering the endotoxaemia-induced derange-
ments that occur in the pathways that regulate translation
initiation [10–12] and UP-mediated proteolysis [13,14], few have
examined if these pathways are likewise perturbed in the
myocardium. Moreover, from the limited evidence available, it
is currently not possible to discern if the rat myocardium PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e6945 1 September 2009 | Volume 4 | Issue 9 | e6945 Heart rate and mean arterial pressure The LPS infusion resulted in early (1 h) and pronounced
tachycardia which persisted for the length of the LPS infusion
(Fig. 1), in addition to a biphasic fall in mean arterial pressure. The
speed of onset and degree of tachycardia and hypotension were
consistent with previous data obtained under similar conditions [24]. Figure 1. Heart rate and mean arterial pressure during
continuous LPS infusion. Values are mean6SEM; n = 6 animals. * significantly different from baseline (P,0.05). doi:10.1371/journal.pone.0006945.g001 Protein levels of 20S proteasome subunits a1-3 & 5–7 Protein levels of 20S proteasome subunits a1-3 & 5–7
Proteins were extracted by homogenisation in 0.8 ml of pH 7.5
Tris-EDTA buffer, and subsequently quantified and normalised
using the Lowry method [27]. Protein samples (20 mg) were
separated by molecular weight on 5–20% SDS-PAGE gels in 1x
Tris/Glycine/SDS buffer (Biorad, Hemel Hempstead, UK) and
transferred overnight to nitrocellulose membranes (Amersham
Biosciences, Little Chalfont, UK). Equal loading of samples was
confirmed by Ponceau S staining of membranes. Membranes were
blocked by incubation for 1 h with 4% (w/v) milk protein in tris-
buffered saline, and subsequently probed with polyclonal rabbit
anti-a1–3, 5–7 subunits of the proteasome (Biomol, Exeter, UK)
for 1 h, peroxidase-conjugated donkey anti-rabbit (Amersham
Biosciences) for 1 h, and visualised by enhanced chemilumines-
cence plus (Amersham Biosciences). Thus, the primary antibody
cross reacts with the a1 subunit examined by RT-PCR, in addition
to other core proteasome subunits. Real-time quantitative PCR The expression of HMBS was previously found to be unaffected by
LPS-infusion within myocardium and skeletal muscle (data not shown). Real-time PCR was performed using PCR Universal Master Mix
(Applied Biosystems) in a MicroAmp 96-well reaction plate. Each well
contained 2 ml cDNA template, 12.5 ml PCR Master Mix and 1.25 ml
of Assay on Demand primer/probe mix in a reaction volume of 25 ml. Data from the LPS-treatment group was normalised to the average of
the saline (control) group for each muscle tissue type and time point. phosphorylation of Thr37 and Thr46 by mTOR does not prevent
the binding of 4E-BP1 to eIF4E, it is thought to be a necessary
prerequisite for subsequent phosphorylation at Ser65 and Thr70; as
such, the phosphorylation state of 4E-BP1 is considered a good
surrogate measure of translation initiation. This was followed by
incubation with peroxidase-conjugated donkey anti-rabbit for 1 h
(Amersham Biosciences), and subsequently visualised by enhanced
chemiluminescence plus (Amersham Biosciences). Developed films
were digitised, and band densities determined using GeneTools
software (Syngene, Cambridge, UK). Real-time quantitative PCR Total RNA was extracted from approximately 30 mg of frozen
muscle using a TRIzolH (Invitrogen) based method that has been
described in detail elsewhere [29]. After subsequent extraction,
RNA samples were quantified by measuring their absorbance at
260 nm and 280 nm. Reverse transcription was carried out using
1.0 mg of total RNA in a total volume of 30 ml, containing MMLV
reverse transcriptase, random hexamer primers, RNase inhibitor
N2511, and the four nucleotides dATP, dGTP, dCTP and dUTP
(all sourced from Promega, Southampton, UK). After incubation
at 42uC for 1 h, freshly synthesised cDNA was diluted four-fold
with RNase-free H2O. Prepared rats were divided into 6 groups; 3 groups were infused
with sterile saline (0.4 ml h21) for either 2 h (n = 8), 6 h (n = 7) or
24 h (n = 8), and the remaining 3 groups received a continuous
infusion of LPS (E. coli, serotype 0127:B8, Sigma, Poole, UK;
dissolved in sterile, isotonic saline; 150 mg kg21 h21) for either 2 h
(n = 8), 6 h (n = 8) or 24 h (n = 6). At the specified time after onset
of saline or LPS infusion, animals were terminally anesthetised (i.v. sodium pentobarbital, Sagatal, Rhoˆne Me´rieux, Harlow, UK). The EDL muscle was removed from the left hind-limb during
anaesthesia, and was immediately frozen and stored in liquid
nitrogen. The myocardium was then rapidly removed and freeze- Six, previously designed, Assay-on-Demand TaqmanH primer
and probe sets were purchased (Applied Biosystems), MuRF1
(#Rn00590197_m1);
MAFbx/atrogin-1
(#Rn00591730_m1);
20S proteasome subunit a1 (PSMA1; #Rn00568675_m1); TNFa PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e6945 2 Endotoxin and the Myocardium (#Rn00562055_m1); IL-6 (#Rn00561420_m1) and the housekeep-
ing gene hydroxymethlybilane synthase (HMBS; #Rn00565886_m1). Each Taqman primer and probe set was validated by performing
real-time PCR with a series of 4-fold cDNA template dilutions to
obtain standard curves of cycle threshold number (Ct) against log
relative concentration (data not shown). All six genes were found to be
amplified with equal efficiency allowing the subsequent use of the
comparative Ct method (DDCt) for the relative quantification of gene
expression. All sample and non-template control reactions were
performed in the ABI Prism 7000 Sequence Detection System
(Applied Biosystems) in triplicate. The Ct values for each triplicate were
averaged and the DCt calculated by the subtraction of the
corresponding mean Ct value for the normalisation gene HMBS. Statistical analysis All data reported are means6S.E.M. For within-group analysis
of HR and BP data, a nonparametric equivalent of analysis of
variance allowing for multiple comparisons was utilised (Fried-
man’s test). Where temporal measures of mRNA and protein levels
have been made, the effects of time, treatment and time-treatment
interaction were determined by two-way ANOVA. For all other
analysis, or when differences reached statistical significance by
two-way ANOVA, separate one-way ANOVA were performed
and, where appropriate, the LSD post-hoc test used to locate any
significant differences between time points. Significance was
accepted at the 5% level. Ubiquitin-proteasome system, TNFa and IL-6 In the EDL there were markedly increased levels of MAFbx/
atrogin-1 and MuRF1 mRNA within 6 h after the onset of LPS
infusion (Fig. 3A & 3B respectively). Furthermore, interesting
differences in the time course of change in the levels of these two
ligases were observed. Thus, elevated levels of MAFbx/atrogin-1
mRNA at 6 h plateaued, whereas in contrast, MuRF1 mRNA
levels were substantially elevated from the 6 h to the 24 h time-
point. In addition, LPS administration resulted in the increased
levels of the a1 proteasome subunit mRNA in the EDL at all
examined time-points relative to control animals, but the change
was particularly pronounced 24 h after the start of the LPS- In stark contrast to the EDL, the myocardium did not display
altered mRNA levels for either MAFbx/atrogin-1 or MuRF1
following LPS administration at any of the three time-points
examined (Fig. 3A & 3B). Interestingly, however, increased mRNA
levels of the a1 proteasome subunit were detected in the
myocardium 6 h and 24 h from the start of the LPS infusion
(Fig. 3C), but the protein levels for multiple subunits of the 20S
proteasome were unaltered (Fig. 3F). The mRNA levels of the two cytokines, TNFa and IL-6, were
examined to determine if they were in part responsible for any
tissue specific changes in translation initiation or muscle protein
breakdown pathways that were observed following LPS adminis-
tration. Transient and significant elevations of TNFa mRNA were
detected by 2 h of LPS administration in both EDL and
myocardium,
peaking
at
11-fold
and
13-fold
vs
controls,
respectively (Figure 3D). Similarly, elevated levels of IL-6 mRNA
were detected by 2 h of LPS infusion in both tissues, although of a
much larger magnitude, peaking at 176-fold elevation of IL-6
mRNA in the EDL and .1000-fold in the myocardium vs
controls, following 2 h and 6 h LPS administration respectively
(Fig. 3E). Figure 2. Protein to DNA ratio in muscle and myocardium
following LPS infusion. A) ASP to DNA ratio following 2 h, 6 h and
24 h LPS infusion expressed as a percentage of treatment group
relative to control groups. Values represent mean6SEM. n = 5–8 per
group. B) Quantity of alkaline soluble proteins and DNA in a 20 ml
aliquot of muscle extract. Translation initiation signalling In the EDL, the total protein level of AKT was unaffected by a
24 h LPS infusion (Fig. 4A), although a trend (P = 0.092) towards
reduced phosphorylation of AKT residue Ser473 was detected
(Fig. 4B). 24 h LPS infusion had no effect on the protein level or
phosphorylation state of residues Thr421 and Ser424 of p70 S6K
(Fig. 4C & 4D), nor on the phosphorylation state of Thr37 and
Thr46 of 4E-BP1 (Fig. 4E), where both p70 S6K and 4E-BP1 are
located downstream of AKT. In contrast to the EDL, a significant increase in AKT protein
was detected after 24 h LPS administration in the myocardium
(Fig. 4A). However, LPS-induced endotoxaemia did not appear to
have any significant effect on the phosphorylation state of residue
Ser473 of AKT in the myocardium (Fig. 4B) nor on p70 S6K
protein levels (Fig. 4C). However, 24 h LPS-infusion did result in a
rise in the phosphorylation state of residues Thr421 and Ser424 of
p70 S6K (Fig. 4D) and Thr37 and Thr46 of 4E-BP1 (Fig. 4E),
indicative of a drive towards increased translation initiation in the
myocardium. Figure 2. Protein to DNA ratio in muscle and myocardium
following LPS infusion. A) ASP to DNA ratio following 2 h, 6 h and
24 h LPS infusion expressed as a percentage of treatment group
relative to control groups. Values represent mean6SEM. n = 5–8 per
group. B) Quantity of alkaline soluble proteins and DNA in a 20 ml
aliquot of muscle extract. Solid lines indicate alkaline soluble proteins;
dashed lines, DNA; (%) mean of values from EDL of saline treated
animals; (D) EDL/LPS; (&) myocardium/saline and (m) myocardium/LPS. By two-way ANOVA, significant differences were observed for main
effects of treatment (P = 0.02) and time (P = 0.03) and treatment-time
interaction (P = 0.02). * indicates significantly different from control
(P,0.05). Ubiquitin-proteasome system, TNFa and IL-6 Solid lines indicate alkaline soluble proteins;
dashed lines, DNA; (%) mean of values from EDL of saline treated
animals; (D) EDL/LPS; (&) myocardium/saline and (m) myocardium/LPS. By two-way ANOVA, significant differences were observed for main
effects of treatment (P = 0.02) and time (P = 0.03) and treatment-time
interaction (P = 0.02). * indicates significantly different from control
(P,0.05). doi:10.1371/journal.pone.0006945.g002 Protein and phosphorylation levels of AKT, p70 S6K and
4E-BP1 Proteins were extracted from approximately 30 mg of muscle by
homogenisation in 0.8 ml of pH 7.5 homogenisation buffer,
consisting of 50 mM Tris-HCL, 1 mM EDTA, 1 mM EGTA, 1%
(v/v) TritonH X-100 and 0.1% (v/v) b-mercaptoethanol. In
addition, to every 10 ml of homogenisation buffer was added 1
tablet of a protease inhibitor cocktail (Roche, Burgess Hill, UK)
and 100 ml of phosphatase inhibitor cocktail II (Sigma). Extracted
proteins were subsequently quantified using the method of Lowry
et al. [27] and diluted to a concentration of 2 mg/ml with 2x SDS
buffer (25% (w/v) glycerol, 16% (v/v) 0.625 M Tris pH 6.8, 4%
(w/v) SDS, 0.0025% (w/v) bromophenol blue and 10% (v/v)
b-mecaptoethanol). Protein samples (50 mg) were separated by
molecular weight on 5–20% SDS-PAGE gels in 1x Tris/Glycine/
SDS buffer (Biorad) and transferred overnight at 4uC to PVDF
membranes (Amersham Bioscience). Equal loading of samples was
confirmed by Ponceau S staining. Membranes were subsequently
blocked with 5% (w/v) bovine serum albumin in 1x tris-buffered
saline for 1 h, followed by incubation overnight at 4uC with a
primary polyclonal rabbit antibody (all sourced from Cell
Signaling Technology Inc., Danvers, USA) for either AKT (Cat
No. #9272), AKT Ser473 (#4058), p70 S6K (#9202), p70 S6K
Thr421/Ser424 (#9204) or 4E-BP1 Thr37/Thr46 (#9459). While, Figure 1. Heart rate and mean arterial pressure during
continuous LPS infusion. Values are mean6SEM; n = 6 animals. * significantly different from baseline (P,0.05). doi:10.1371/journal.pone.0006945.g001 PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e6945 September 2009 | Volume 4 | Issue 9 | e6945 PLoS ONE | www.plosone.org 3 Endotoxin and the Myocardium infusion (Fig. 3C). Since the mRNA expression of individual
subunits of the 20S proteasome can vary in response to a catabolic
stimulus [30], the protein levels of subunits a1–3 and 5–7 were
also determined. In agreement with findings for the a1 subunit,
maximal protein levels for subunits a1–3 and 5–7 were observed in
the EDL following 24 h LPS administration (Fig. 3F). While only
animals subjected to 24 h LPS treatment underwent implantation
of
arterial
catheters
into
the
distal
abdominal
aorta,
the
reproducibility of the catabolic response to LPS in skeletal muscle
to previous findings where arterial catheters were not used [3],
suggest that the results observed were not due to prior surgical
intervention. infusion (Fig. 3C). Skeletal muscle and myocardial protein content keletal muscle and myocardial protein content A significant reduction (23%; P,0.05) in the ASP:DNA ratio
relative to controls was observed in the EDL after 24 h of LPS
infusion (Fig. 2A). This appeared the result of a decline in levels of
ASP in muscle extracts as opposed to a change in DNA content
(Fig. 2B), indicative of a decline in muscle protein content. In
contrast to skeletal muscle, there were no significant changes in
ASP levels, DNA content (Fig. 2B), or the ASP:DNA ratio (Fig. 2A)
of the myocardium at any time point compared to controls. Protein and phosphorylation levels of AKT, p70 S6K and
4E-BP1 Since the mRNA expression of individual
subunits of the 20S proteasome can vary in response to a catabolic
stimulus [30], the protein levels of subunits a1–3 and 5–7 were
also determined. In agreement with findings for the a1 subunit,
maximal protein levels for subunits a1–3 and 5–7 were observed in
the EDL following 24 h LPS administration (Fig. 3F). While only
animals subjected to 24 h LPS treatment underwent implantation
of
arterial
catheters
into
the
distal
abdominal
aorta,
the
reproducibility of the catabolic response to LPS in skeletal muscle
to previous findings where arterial catheters were not used [3],
suggest that the results observed were not due to prior surgical
intervention. September 2009 | Volume 4 | Issue 9 | e6945 Discussion doi:10.1371/journal.pone.0006945.g003 propose that the use of the LPS model is more appropriate to the
clinical situation than CLP when considering the impact of
endotoxaemia on mechanisms regulating protein turnover in
rodent myocardium. However, given the sparse number of studies
that have examined this issue to date, it is not possible to preclude
temporal effects of endotoxaemia, induced by any one of the
various means, on myocardial protein content. reduce EDL protein content by 23%. Furthermore, we were able
to show this disparity between the two tissues in their response to
experimental
endotoxaemia,
is
underpinned
by
differential
responses in the processes governing muscle protein synthesis
and muscle protein breakdown. The results of the present study
clearly demonstrate that the events associated with UP-mediated
muscle protein breakdown, as have been shown to occur in fast-
twitch muscle following LPS administration [3], appear largely
unaltered in the myocardium of LPS treated rats. Furthermore,
the results highlight several interesting changes that occur in the
mechanisms that regulate protein translation initiation in the
myocardium, changes that would generally be suggestive of an
enhancement of translation initiation. These observations, accom-
panied by the maintenance of myocardial, but not EDL protein
content, suggest that the myocardium during early endotoxaemia
does not experience the same catabolic fate as skeletal muscle. Consistent with our previously reported findings [3], we
observed 24 h LPS administration to result in reduced fast-twitch
skeletal muscle protein content, elevated mRNA levels of MAFbx/
atrogin-1 and MuRF1, and an increase in protein levels for
multiple subunits of the 20S proteasome; observations that are in
accordance with the reported rise in UP-mediated protein
degradation during endotoxaemia in fast-twitch muscle [32]. Interestingly, while a trend towards a fall in phosphorylation of
AKT residue Ser473 was observed, it failed to reach statistical
significance
(P = 0.097),
and
moreover,
elements
examined
downstream of AKT (phosphorylation levels of p70 S6K and
4E-BP1) were unchanged by the LPS treatment protocol described
here. This is in contrast to our previously published findings where
declines in AKT Ser473 phosphorylation and total protein levels of
AKT were reported in EDL following 24 h LPS treatment [3]. Furthermore, while levels of p70 S6K and 4E-BP1 were not
examined as part of the previous study, phosphorylation of other
downstream targets of AKT kinase activity, namely Foxo1 and
Foxo3, were shown to be similarly reduced. Likewise, Lang and
colleagues [16] have shown the bolus i.v. Discussion Protein levels of 20S proteasome subunits a1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values
represent mean6SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all
measures (P#0.02), and for time and treatment-time interaction in all measures bar TNFa and IL-6 mRNA levels (P#0.02). In the myocardium,
significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit
protein levels (P#0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit
a1 and IL-6 mRNA levels in the myocardium (P#0.002). * indicates different from corresponding control (P,0.05); { different from 2 h LPS-treated
muscle (P,0.05); { different from 6 h LPS-treated muscle (P,0.05). d i 10 1371/j
l
0006945 003 r proteins associated with proteolysis in muscle and myocardium following LPS infusion. Bars denote fold Figure 3. mRNA levels for proteins associated with proteolysis in muscle and myocardium following change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit a1, D) TNFa and E) IL-6, compared to corresponding control
values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value.1 indicates greater than control mRNA levels and ,1 is lower then control
mRNA levels. Protein levels of 20S proteasome subunits a1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values
represent mean6SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all
measures (P#0.02), and for time and treatment-time interaction in all measures bar TNFa and IL-6 mRNA levels (P#0.02). In the myocardium,
significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit
protein levels (P#0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit
a1 and IL-6 mRNA levels in the myocardium (P#0.002). * indicates different from corresponding control (P,0.05); { different from 2 h LPS-treated
muscle (P,0.05); { different from 6 h LPS-treated muscle (P,0.05). PLoS ONE | www.plosone.org Discussion Endotoxaemia in the rat results in a reduction in fast-twitch
skeletal muscle protein content by reducing protein synthesis [2]
and increasing protein breakdown [1], but it is currently unclear if
a decline in myocardial protein content also occurs under these
conditions, and whether any change observed is regulated by the
same signalling and genomic pathways as skeletal muscle. Here we
have shown myocardial protein content to be maintained at
normal levels following an administration of LPS sufficient to September 2009 | Volume 4 | Issue 9 | e6945 September 2009 | Volume 4 | Issue 9 | e6945 PLoS ONE | www.plosone.org 4 Endotoxin and the Myocardium Figure 3. mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion. Bars denote fold
change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit a1, D) TNFa and E) IL-6, compared to corresponding control
values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value.1 indicates greater than control mRNA levels and ,1 is lower then control
mRNA levels. Protein levels of 20S proteasome subunits a1–3 and 5–7 were measured to confirm mRNA results for subunit alpha-1 (panel F). Values
represent mean6SEM. n = 6–8 per group. By two-way ANOVA, significant differences were observed in the EDL for the main effects of treatment in all
measures (P#0.02), and for time and treatment-time interaction in all measures bar TNFa and IL-6 mRNA levels (P#0.02). In the myocardium,
significant differences for the main effect of treatment were observed in all measures bar MAFbx/atrogin-1 mRNA levels and proteasome subunit
protein levels (P#0.03). Significant differences in main effects of time and treatment-time interaction were only observed for 20S proteasome subunit
a1 and IL-6 mRNA levels in the myocardium (P#0.002). * indicates different from corresponding control (P,0.05); { different from 2 h LPS-treated
muscle (P,0.05); { different from 6 h LPS-treated muscle (P,0.05). doi:10.1371/journal.pone.0006945.g003 Figure 3. mRNA levels for proteins associated with proteolysis in muscle and myocardium following LPS infusion. Bars denote fold
change in mRNA levels of A) MAFbx/atrogin-1, B) MuRF1, C) 20S proteasome subunit a1, D) TNFa and E) IL-6, compared to corresponding control
values in response to either a 2 h, 6 h or 24 h i.v. LPS infusion. A value.1 indicates greater than control mRNA levels and ,1 is lower then control
mRNA levels. Discussion administration of LPS
(1 mg kg21) to be sufficient to reduce muscle 4E-BP1 phosphor-
ylation after 24 h. Reasons for discrepancies between the results
described here with those of previously reported findings, are While
the
ASP
to
DNA
ratio
was
maintained
in
the
myocardium of LPS treated animals, previous published reports
detailing myocardial protein content during endotoxaemia, have
reported variable findings [15,17,31]. It is interesting to note that
where a decline in myocardial protein content has been reported,
it was following the induction of endotoxaemia via the use of the
cecal ligation and puncture technique in combination with the use
of non-sham-operated controls [15]. O’Leary and colleagues
demonstrated that performing a laporatomy, as required by the
CLP technique, results in significant reductions in myocardial
fractional protein synthesis rates and protein content, lasting for a
minimum of 72 h post completion of the surgical procedure [17]. Thus, the variations observed between studies could be the result
of methodological differences. Given the above findings, we would September 2009 | Volume 4 | Issue 9 | e6945 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org 5 Figure 4. Phosphorylation and protein levels of translation initiation intermediates in muscle and myocardium following LPS
infusion. Bars denote fold change in A) total AKT protein, B) phosphorylation levels of AKT residue Ser473, C) total p70 S6K protein, D)
phosphorylation levels of p70 S6K residues Thr421 and Ser424 and E) phosphorylation levels of 4E-BP1 residues Ser65 and Thr70, in response to 24 h LPS
administration relative to control animals. n = 8 per group. Values represent mean6SEM. * Indicates different from corresponding control (P,0.05). doi:10.1371/journal.pone.0006945.g004
Endotoxin and the Myocardium Endotoxin and the Myocardium Figure 4. Phosphorylation and protein levels of translation initiation intermediates in muscle and myocardium following LPS
infusion. Bars denote fold change in A) total AKT protein, B) phosphorylation levels of AKT residue Ser473, C) total p70 S6K protein, D
phosphorylation levels of p70 S6K residues Thr421 and Ser424 and E) phosphorylation levels of 4E-BP1 residues Ser65 and Thr70, in response to 24 h LP
administration relative to control animals. n = 8 per group. Values represent mean6SEM. * Indicates different from corresponding control (P,0.05
doi:10.1371/journal.pone.0006945.g004 Figure 4. Phosphorylation and protein levels of translation initiation intermediates in muscle and myocardium following LPS
infusion. Discussion Bars denote fold change in A) total AKT protein, B) phosphorylation levels of AKT residue Ser473, C) total p70 S6K protein, D)
phosphorylation levels of p70 S6K residues Thr421 and Ser424 and E) phosphorylation levels of 4E-BP1 residues Ser65 and Thr70, in response to 24 h LPS
administration relative to control animals. n = 8 per group. Values represent mean6SEM. * Indicates different from corresponding control (P,0.05). doi:10.1371/journal.pone.0006945.g004 skeletal muscle and myocardium during LPS-induced endotoxae-
mia is the increased rate of myocardial contraction that occurs, as
evidenced by the early, pronounced and sustained tachycardia
seen following the start of LPS administration. Two observations
described here are in support of this notion. Firstly, elevated
phosphorylation levels of residues Thr421 and Ser424 for p70 S6K
were observed in the myocardium of LPS-treated rats. p70 S6K
indirectly promotes the translation of target oligopyrimidine
(TOP)
mRNAs
which
are
almost
exclusively
confined
to
transcripts that encode for ribosomal proteins and translation
factors. Contraction, at least in skeletal muscle, promotes the
increased phosphorylation of p70 S6K [33] and is known to be
involved in cardiac hypertrophy [34]. Secondly, we observed no
change in MAFbx/atrogin-1 and MuRF1 mRNA levels in
myocardial tissue following LPS administration. Jones et al. [29]
had previously shown contraction to be sufficient to reverse unclear and difficult to discern. However, if real, reduced AKT
Ser473 phosphorylation would appear to have had no significant
impact on the two downstream translation initiation intermediates
p70 S6K and 4E-BP1. The findings as reported, would suggest
that derangements in UP-mediated muscle protein degradation
were predominantly responsible for the observed reductions in
EDL protein content, although direct measures of muscle protein
synthesis and muscle protein breakdown would have been useful in
providing further support of this view. skeletal muscle and myocardium during LPS-induced endotoxae-
mia is the increased rate of myocardial contraction that occurs, as
evidenced by the early, pronounced and sustained tachycardia
seen following the start of LPS administration. Two observations
described here are in support of this notion. Firstly, elevated
phosphorylation levels of residues Thr421 and Ser424 for p70 S6K
were observed in the myocardium of LPS-treated rats. p70 S6K
indirectly promotes the translation of target oligopyrimidine
(TOP)
mRNAs
which
are
almost
exclusively
confined
to
transcripts that encode for ribosomal proteins and translation
factors. PLoS ONE | www.plosone.org Discussion Contraction, at least in skeletal muscle, promotes the
increased phosphorylation of p70 S6K [33] and is known to be
involved in cardiac hypertrophy [34]. Secondly, we observed no
change in MAFbx/atrogin-1 and MuRF1 mRNA levels in
myocardial tissue following LPS administration. Jones et al. [29]
had previously shown contraction to be sufficient to reverse In stark contrast to the EDL, the protein content of the
myocardium was unaffected by 24 h LPS administration, although
an increase in phosphorylation of downstream elements of
translation initiation consistent with an enhancement of muscle
protein synthesis, in addition to the maintenance of MAFbx/
atrogin-1 and MuRF1 mRNA at normal levels, were observed. One possible explanation for the divergent response seen between PLoS ONE | www.plosone.org September 2009 | Volume 4 | Issue 9 | e6945 6 Endotoxin and the Myocardium limb-immobilisation-induced upregulation of the two ligases in the
vastus lateralis of healthy human subjects. Similar observations have
also been made in the gastrocnemius and soleus muscles of rats
subjected to spinal cord isolation, where brief bouts of high-load
isometric
contractions
were
able
to
prevent
the
increased
expression of MAFbx/atrogin-1 and MuRF1 mRNA [35]. It is
possible therefore that myocardial contraction could be responsi-
ble for the maintenance of MAFbx/atrogin-1 and MuRF1 mRNA
at basal levels. In further support of this notion, elevated levels of
MAFbx/atrogin-1 and MuRF1 mRNA have been observed in the
myocardium following chronic heart failure induced via the
ligation of the left coronary artery, a condition where reduced
contractile activity of the myocardium is observed [36] and, more
importantly, regular exercise training has been shown to be
sufficient to significantly reduce myocardial MAFbx/atrogin-1 and
MuRF1 mRNA levels following experimental myocardial infarc-
tion in rats [37]. elevation of IL-6 mRNA are not possible to discern; however,
the dual association of IL-6 with both contractile dysfunction [43]
and cardioprotection [44] is intriguing and worthy of further
investigation. In summary, the myocardium does not experience the same
reductions in protein content that are characteristically observed
in skeletal muscle following continuous LPS administration. It is
feasible that this is in part a consequence of the failure of the
myocardium to elicit events typically associated with UP-mediated
protein degradation, most notably MAFbx/atrogin-1 and MuRF1
mRNA elevation. References 13. Wray CJ, Mammen JMV, Hershko DD, Hasselgren PO (2003) Sepsis
upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle. Int J Biochem Cell Biol 35: 698–705. 1. Lecker SH, Solomon V, Mitch WE, Goldberg AL (1999) Muscle protein
breakdown and the critical role of the ubiquitin-proteasome pathway in normal
and disease states. J Nutr 129: 227S–237S. 1. Lecker SH, Solomon V, Mitch WE, Goldberg AL (1999) Muscle protein
breakdown and the critical role of the ubiquitin-proteasome pathway in normal
and disease states. J Nutr 129: 227S–237S. 2. Lang C, Frost R, Vary T (2007) Regulation of muscle protein synthesis during
sepsis and inflammation. Am J Physiol Endocrinol Metab 293: E453–459. 2. Lang C, Frost R, Vary T (2007) Regulation of muscle protein synthesis during
sepsis and inflammation. Am J Physiol Endocrinol Metab 293: E453–459. 14. Dehoux MJM, van Beneden RP, Fernandez-Celemin L, Lause PL, Thissen JPM
(2003) Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal
muscle after LPS injection. FEBS Lett 544: 214–217. 3. Crossland H, Constantin-Teodosiu D, Gardiner SM, Constantin D,
Greenhaff PL (2008) A potential role for Akt/FOXO signalling in both protein
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muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl
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of starvation, surgery, and sepsis on cardiac protein synthesis in rats: Effects of
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types of skeletal muscle atrophy involve a common program of changes in gene
expression. FASEB J 18: 39–51. 18. Acknowledgments The authors wish to thank Julie March and Philip Kemp for their excellent
technical skills. Discussion It is also possible that events typical of enhanced
protein translation initiation occur in the myocardium during early
endotoxaemia, but this remains speculative with the lack of a
robust reduction of AKT phosphorylation in fast-twitch muscle. The exact ‘trigger’ responsible for the tissue-specific response
remains unknown. Whether myocardial contraction is responsible
for mediating some of the observed differences during endotox-
aemia is unclear, but is consistent with the rapid and sustained
elevation in heart rate observed. Collectively, these findings
represent a significant addition to our understanding of the
response of skeletal and cardiac muscle to endotoxaemia and
highlight possible explanations for the disparity in observations
between these two tissues. The mechanisms responsible for initiating the tissue specific
changes in protein catabolism during sepsis are poorly understood. It has been widely suggested that the catabolic cytokines, TNFa
and IL-6, could, in part, be responsible for triggering the catabolic
events in skeletal muscle. Indeed, it has been demonstrated that
both can facilitate protein catabolism in skeletal muscle and
myocardium [20,38,39]. However, despite elevated local levels of
both TNFa and IL-6 in the skeletal muscle of septic animals, their
necessity
for
muscle
atrophy
to
occur
remains
equivocal
[12,40,41]. It is interesting to note that significantly elevated levels
of TNFa and IL-6 mRNA were observed here in both the
myocardium and skeletal muscle, occurring as early as 2 h after
the start of LPS administration, a finding similar to that of others
[14,40,42]. Thus, elevation of TNFa and IL-6 mRNA levels
precedes increases in both MAFbx/atrogin-1 and MuRF1. However, mechanisms by which the myocardium is protected
from perceived localised increases in TNFa and IL-6 remains
unclear. Furthermore,
reasons
for
the
significant
transient Author Contributions Conceived and designed the experiments: AJM SG DCT RL TB PG. Performed the experiments: AJM NA SG DCT. Analyzed the data: AJM
NA SG DCT RL TB PG. Contributed reagents/materials/analysis tools:
SG DCT RL TB PG. Wrote the paper: AJM SG PG. Conceived and designed the experiments: AJM SG DCT RL TB PG. Performed the experiments: AJM NA SG DCT. Analyzed the data: AJM
NA SG DCT RL TB PG. Contributed reagents/materials/analysis tools:
SG DCT RL TB PG. Wrote the paper: AJM SG PG. Endotoxin and the Myocardium lipopolysaccharide infusion in conscious rats. Am J Physiol Heart Circ Physiol
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https://openalex.org/W1976101448
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https://dro.deakin.edu.au/articles/journal_contribution/The_effect_of_gestational_age_on_angiogenic_gene_expression_in_the_rat_placenta/20939950/1/files/37221346.pdf
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The Effect of Gestational Age on Angiogenic Gene Expression in the Rat Placenta
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PloS one
| 2,013
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cc-by
| 9,599
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The effect of gestational age on angiogenic gene expression in the rat
placenta AUTHOR(S) AUTHOR(S) K Vaswani, M C Hum, H W Chan, J Ryan, Ryan Wood-Bradley, M Nitert, M Mitchell, James Armitage, G Rice
C
O K Vaswani, M C Hum, H W Chan, J Ryan, Ryan Wood-Bradley, M Nitert, M Mitchell, James Armitage, G Rice This is the published version of the power point
presentation: Vaswani, Kanchan, Hum, Melissa Wen‐Ching, Chan, Hsiu‐Wen, Ryan, Jennifer, Wood‐Bradley, Ryan
J., Nitert, Marloes Dekker, Mitchell, Murray D., Armitage, James A. and Rice, Gregory E. 2013, The
effect of gestational age on angiogenic gene expression in the rat placenta, PLOS one, vol. 8, no. 12,
Article number e83762, pp. 1‐11. 10536/DRO/DU:30063665 Downloaded from Deakin University’s Figshare repository
Deakin University CRICOS Provider Code: 00113B Abstract MDN received support from the Royal Brisbane and Women’s Hospital Foundation. JAA
was a Monash Fellow and Heart Foundation Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: k.vaswani@uq.edu.au looping of the vessels. Obtrusion of both capillary loops and new
sprouts results in the formation of terminal villi. Abstract The placenta plays a central role in determining the outcome of pregnancy. It undergoes changes during gestation as the
fetus develops and as demands for energy substrate transfer and gas exchange increase. The molecular mechanisms that
coordinate these changes have yet to be fully elucidated. The study performed a large scale screen of the transcriptome of
the rat placenta throughout mid-late gestation (E14.25–E20) with emphasis on characterizing gestational age associated
changes in the expression of genes invoved in angiogenic pathways. Sprague Dawley dams were sacrificed at E14.25,
E15.25, E17.25 and E20 (n = 6 per group) and RNA was isolated from one placenta per dam. Changes in placental gene
expression were identifed using Illumina Rat Ref-12 Expression BeadChip Microarrays. Differentially expressed genes (.2-
fold change, ,1% false discovery rate, FDR) were functionally categorised by gene ontology pathway analysis. A subset of
differentially expressed genes identified by microarrays were confirmed using Real-Time qPCR. The expression of thirty one
genes involved in the angiogenic pathway was shown to change over time, using microarray analysis (22 genes displayed
increased and 9 gene decreased expression). Five genes (4 up regulated: Cd36, Mmp14, Rhob and Angpt4 and 1 down
regulated: Foxm1) involved in angiogenesis and blood vessel morphogenesis were subjected to further validation. qPCR
confirmed late gestational increased expression of Cd36, Mmp14, Rhob and Angpt4 and a decrease in expression of Foxm1
before labour onset (P,0.0001). The observed acute, pre-labour changes in the expression of the 31 genes during gestation
warrant further investigation to elucidate their role in pregnancy. Citation: Vaswani K, Hum MW-C, Chan H-W, Ryan J, Wood-Bradley RJ, et al. (2013) The Effect of Gestational Age on Angiogenic Gene Expression in the Rat
Placenta. PLoS ONE 8(12): e83762. doi:10.1371/journal.pone.0083762 itor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America Editor: Vladimir V. Kalinichenko, Cincinnati Children’s Hospital Medical Center, United States of America Copyright: 2013 Vaswani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Vaswani et al. This is an open-access article distributed under the terms of the Creative Commons
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: GER was in receipt of an NHMRC Principal Research Fellowship. The Effect of Gestational Age on Angiogenic Gene
Expression in the Rat Placenta Kanchan Vaswani1*, Melissa Wen-Ching Hum1, Hsiu-Wen Chan1, Jennifer Ryan1, Ryan J. Wood-
Bradley2,3, Marloes Dekker Nitert1, Murray D. Mitchell1, James A. Armitage2,3, Gregory E. Rice1
1 University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital Campus, Herston, Queensland, Australia, 2 School of Medicine (Optometry),
Deakin University, Waurn Ponds, Victoria, Australia, 3 Department of Anatomy & Developmental Biology, Monash University, Clayton, Victoria, Australia http://hdl.handle.net/10536/DRO/DU:30063665 http://hdl.handle.net/10536/DRO/DU:30063665 Reproduced with the kind permission of the copyright owner Copyright : 2013, Public Library of Science Copyright : 2013, Public Library of Science December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org Introduction A)
The total number of differentially expressed genes that were up and
down regulated between the 3 gestational groups i.e. E14.25 vs. E15.25,
E15.25 vs. E17.25 and E17.25 vs. E20 are displayed as fold change (cut-
off .2 and FDR,1%). B) Overall gene expression fold changes from the
earliest time point under study E14.25 right up to E20. doi:10.1371/journal.pone.0083762.g001 nutrient demands of the growing fetus, particularly in late
gestation [6]. In human trophoblasts, System A, which includes
the amino acid transport genes such as Slc38A1, Slc38A2 and
Slc38A4 has been shown to increase as pregnancy progresses,
coinciding with increased fetal nutrient demands [7]. During late
gestation in rat, glucose transporter genes Slc2A3 and Slc2A1 are
upregulated and downregulated respectively, in placenta [6]. The
molecular and cellular mechanisms that regulate vascularisation
and angiogenesis within the placental villous tree during pregnan-
cy remain to be fully elucidated. Previous studies have established
the utility of rat and mouse models to eluciate the molecular and
cellular mechanisms involved in placentation and placental
development since it is more difficult to obtain human placenta
at various gestational stages [8–10]. Genome-wide gene expression
in rat placenta has been studied in late pregnancy [11] [12]. At
embryonic day 18.5 (E18.5), RNA sequencing showed differential
gene expression between the rat labyrinth zone, junctional zone
and metrial gland [12]. At E21, Buffat et al. reported changes in
genome-wide placental gene expression in rats exposed to an
isocaloric low protein diet [11]. Goyal et al. (2010), studied global
gene expression in placentae from two different rat species at 17.5
days of gestation and found 272 genes differentially expressed [13]. Large scale molecular analysis of the transcriptome of the normal
rat placenta across mid to late gestation, however, has not been
performed. In particular, gestational variation in the expresssion of
genes known to be involved in angiogenesis within the rat placenta
is yet to be documented. Figure 1. Differentially genes expression during gestation. A)
The total number of differentially expressed genes that were up and
down regulated between the 3 gestational groups i.e. E14.25 vs. E15.25,
E15.25 vs. E17.25 and E17.25 vs. E20 are displayed as fold change (cut-
off .2 and FDR,1%). B) Overall gene expression fold changes from the
earliest time point under study E14.25 right up to E20. Introduction A successful outcome to pregnancy depends on establishing an
effective materno-fetal exchange interface. Two processes critical
for materno-fetal exchange are adequate perfusion of the placenta
by maternal blood, and formation of the placental villous tree (the
exchange surface of the placenta) and its vascular network. Failure
to achieve either of these processes is associated with adverse
pregnancy outcome, including: miscarriage; intrauterine growth
restriction; preeclampsia and preterm delivery. Furthermore,
epidemiological studies identify a strong association between
compromised placental structure and function, impaired fetal
growth and the development of adult-onset diseases [1–3]. The placental vasculature continues to develop and adapt
during pregnancy in response to the increasing requirements of the
fetus. Reynolds et al., [4] reported that the large increase in
transplacental exchange, that supports the exponential increase in
fetal growth during the last half of gestation, depends primarily on
the dramatic growth of the placental vascular beds and the
resultant large increases in uterine and umbilical blood flow [5]. The microvasculature of the placenta develops by the process of
branching angiogensis that increases capillary numbers and
surface densities. In the human placenta, branching angiogenesis,
the formation of new vessels through sprouting, occurs from 5
weeks gestation through to 24 weeks gestation, while non-
branching angiogenesis, the formation of capillary loops through
elongation, predominates thereafter to term. Development of the vascular network within placental villi
during first trimester is a critical process that involves both
vasculogenesis and angiogenesis. In humans, vasculogenesis starts
during the third week post-conception. Hemangioblastic cell cords
differentiate in situ from placental mesenchymal stem cells in the
villous cores. The cords elongate through proliferation and cell
recruitment, and connect with the vasculature of the developing
fetus. A feto-placental circulation starts around 8 weeks of
gestation and perfusion of the placental villi by maternal blood
occurs 4–6 weeks later. Elongation of the capillaries results in Available data supports the hypothesis that fetal growth and
energy demand regulates placental growth and vascularization. For instance, the activity and expression of some nutrient
transporters, in the mouse placenta, is modulated by fetal nutrient
demands for growth [6]. Mutant mouse small placentae are
capable of increasing glucose and amino acid transfer to meet the December 2013 | Volume 8 | Issue 12 | e83762 1 PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 Angiogenic Gene Expression in the Rat Placenta Figure 1. Differentially genes expression during gestation. Materials and Methods Total RNA was extracted from 30 mg of pulverized frozen
placental tissue (n = 6 placentae per gestational age group, one
placenta per dam), using the AllPrep DNA/RNA Mini Kit
(Qiagen) as per manufacturers’ instructions. Genomic DNA was
removed by On-column Dnase1 treatment. Following extraction,
total RNA was quantified via NanoDrop ND-1000 spectropho-
tometer (Thermo Scientific, DE, USA). RNA quality was verified
using an Agilent 2100 Bioanalyzer (VIC, Australia) prior to the
analysis. RNA samples that fulfilled the following criteria were
selected for microarray analysis: (i) RIN .8.5; (ii) 260/280 ratio
.2; (iii) 260:230.1. RIN (RNA Integrity Number) values were
greater than 8.7. For qPCR, the RNA was reverse transcribed
using the QuantiTect Reverse transcription kit (Qiagen) using
1 mg of RNA per sample. Microarray Analysis on Illumina Rat Ref Arrays Microarray Analysis on Illumina Rat Ref Arrays
For the microarray analysis, 500 ng of total RNA was
converted to double stranded cDNA and this was used to
generate biotinylated cRNA probes using the Illumina Total-
Prep RNA Amplification Kit. Biotin-labelled cRNA were then
hybridized to Illumina RatRef-12 Expression BeadChip (San
Diego, CA, USA). Slides were scanned on a BeadStation 500
System using Beadscan software Version 3.5.31. No RNA
samples were pooled in this analysis, each of the placental
samples was analyzed independently. Samples were hybridized
into wells at random. Two chips of 12 wells were used for the Introduction doi:10.1371/journal.pone.0083762.g001 The aim of this study, therefore, was to characterize gestational
variation in rat placental gene expression using microarray
analysis (22,000 gene probes) and specifically identify changes in
the molecular pathways involved in angiogenesis. The hypothesis
to be tested was that genes involved in regulating angiogenesis are
differentially expressed during late gestation compared to late mid-
gestation. Placentae were collected at four gestational ages
(E14.25; E15.25; E17.25 and E20) and gene expression was
compared. 14.25, 15.25, 17.25 and 20 (n = 6 per gestational age). Whole
placentae were collected from the pregnant dams, weighed and
then snap frozen in liquid nitrogen. Tissues were stored at 280uC
until processed and analysed. Animals and Diets All animal experiments were performed at the Department of
Anatomy and Developmental Biology, Monash University (Mel-
bourne, Australia) with the approval of The School of Biomedical
Sciences
Animal
Ethics
Committee
of
Monash
University. Experiments were carried out in accordance with the National
Health and Medical Research Council of Australia ‘‘Australian Code
of Practice for the Care and Use of Animals for Scientific Purposes’’ (7th
edition, 2004). Sprague Dawley dams were allowed to adapt to the animal house
for one week, consuming standard chow diet and water ad libitum
prior to start of the study. The animals were maintained in a light
controlled environment (12 h light/dark cycle) throughout this
study and fed on a standard chow diet. Animals were time mated
in a 3 hour period, with male Sprague Dawley rats. This was
designated as Day 0 of pregnancy. The rationale of using a 3 h
window mating time is to reduce variability of gestational age
among the offspring and to maximize the accuracy in staging of
gestation. Pregnancy was confirmed at the time of sacrifice. After
mating, dams were housed individually. All animals had access to
food and water ad libitum throughout this study. Tissue Collection The pregnant dams were anaesthetized (Isoflurane Rhodia
Australia P/L, VIC, Australia) and sacrificed at embryonic day (E) PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 2 Angiogenic Gene Expression in the Rat Placenta Figure 2. Heat Map of relative expression of Angiogenic genes. Of the 31 differentially expressed genes, 22 were up regulated (A) and 9 w Figure 2. Heat Map of relative expression of Angiogenic genes. Of the 31 differentially expressed genes, 22 were up regulated (A) and 9 were
down regulated (B). The heat map diplays data for 6 individual placentae for each gestational age. The genes correspond to the gene list in Table 1,
where a change from green, black to red indicates and increase in signal. The colour legend above each map shows relative microarray signal ranges
between 0 to 1. Each coloured rectangular box within the heat map represents a separate rat placenta sample. The samples are grouped according to
gestational age i.e. E14.25, E15.25, E17.25, E 20. doi:10.1371/journal.pone.0083762.g002 Figure 2. Heat Map of relative expression of Angiogenic genes. Of the 31 differentially expressed genes, 22 were up regulated (A) and 9 were
down regulated (B). The heat map diplays data for 6 individual placentae for each gestational age. The genes correspond to the gene list in Table 1,
where a change from green, black to red indicates and increase in signal. The colour legend above each map shows relative microarray signal ranges
between 0 to 1. Each coloured rectangular box within the heat map represents a separate rat placenta sample. The samples are grouped according to
gestational age i.e. E14.25, E15.25, E17.25, E 20. doi:10.1371/journal.pone.0083762.g002 Shapiro-Wilk
tests,
respectively. Normally
distributed
and
homogenous data were assessed by ANOVA otherwise data
were analysed using non-parametic Kruskal-Wallis tests. experiment. Illumina Whole-Genome Gene Expression Bead-
Chips consist of oligonucleotides immobilized to beads held in
microwells on the surface of an array substrate. Labelled sample
cRNA were detected by hybridization to 50 mer probes on
BeadChip. Post washing and staining, BeadChips were scanned
on a BeadArray reader. Array experiment readout deposited on
ArrayExpress. ArrayExpress Accession number is E-MTAB-
1987. BioInformatic Anayses and Statistics Bioinformatic analysis was performed by using the Illumina
Beadstudio
and
Significance
Analysis
of
Microarry
(SAM,
Stanford University) software. Data were normalized by perform-
ing a probe-intensity transformation and normalization via the
Lumi package, Bioconductor. After normalization, differentially
expressed placental genes (i.e. .2 fold expression, false discovery
rate of ,1%) were identified using SAM and were further
analysed using Web-based Gene Set Analysis Toolkit (WebGestalt,
http://bioinfo.vanderbilt.edu/webgestalt/). Genes were assigned
to their respective functional classes based on the Gene Ontology
(GO) database. Differences in group means were assessed by post-
hoc comparisons (Bonferroni tests). Quantitative Real-Time PCR Validation Experiments Where * = p,0.05, p,0.0005 = **; p,0.0001 = *** and ns = p.0.05 using one-way ANOVA and post-hoc tests (Bonferroni test). doi:10.1371/journal.pone.0083762.g003 Mircoarray Analysis qPCR data were consistent with microarray data and estab-
lished that both Ptgs2 and Pla2g2a remain relatively constant
throughout E14.25, E15.25 and E17.25 and increase dramatically
(and also consistent with literature) towards the end of gestation at
E20 (Figure 3). Nos2 on the other hand, is downregulated towards
E17.25 and E20 (Figure 4 and Figure S2). To characterize gestational age dependent changes in gene
expression, microarray analysis was performed on placental cDNA
obtained from pregnant rats at gestational days E14.25, E15.25,
E17.25 and E20. A cutoff of .2 fold change in expression and
false discovery rate of less than 1% was used to ascribe the
differential expression of genes. Between gestational age E14.25
and E15.25, 40 gene transcripts were differentially expressed,
between E15.25 vs E17.25, 143 gene transcripts were differentially
expressed and between E17.25 vs E20, 678 were differentially
expressed using SAM Analyses (Figure 1). Specific differentially
expressed angiogenic genes were then selected from the SAM
readout and a heat map (Figure 2) displays expression changes
over time. Quantitative Real-Time PCR Validation Experiments Quantitative Real-Time PCR Validation Experiments
qPCR was used to validate the expression of three placental
genes (Ptgs2, Pla2g2a and Nos2) known to display gestational
age changes in expression. Each experiment was performed in
duplicate and normalized against the expression of b-Actin
(Actb)
using
the
ddCt
method
[14]. RNA
was
reverse
transcribed into complentary DNA strands, using the Quantitect
Reverse transcription kit (Qiagen, VIC, Australia) following
manufacturers instructions. Primers used for qPCR throughout
the study are detailed in Table S1 and were all designed using
PRIMER-BLAST/ncbi against Rattus Norvegicus mRNA. qPCR
analyses was performed using ABI 6000 using standard SYBR
green from Life Technologies (VIC, Australia). Normalized Ct
ratios were subjected to ANOVA. Spearman’s Rank Correla-
tion, p,0.05 was used for correlation plots. Prior to statistical
analysis of gene expression during gestation, homogeneity of
variance and normality were assessed using Bartlett’s and Normalized
expression
data
were
subjected
to
Principal
Component Analyses (PCA, XLSTAT). 2D observation plots
were generated for individual gestational age samples and
correlation circles for the 31 angiogenic genes analyzed. December 2013 | Volume 8 | Issue 12 | e83762 3 PLOS ONE | www.plosone.org Figure 3. Comparison of qPCR and microarray expression data. qPCR and Microarray data for Ptgs2 are shown in panels A and B respectively;
statistical significance (p values) is presented in panel G. C represents linear correlation between qPCR and microarray data for Ptgs2 (p,0.05). qPCR
and microarray results for Pla2g2a are shown in panels D and E respectively; statistical significance (p values) are presented in panel G. Panel F
indicates the linear correlation (Spearman’s rank correlation) between qPCR and microarray data (p,0.05). Both genes display late gestational
increase. Where * = p,0.05, p,0.0005 = **; p,0.0001 = *** and ns = p.0.05 using one-way ANOVA and post-hoc tests (Bonferroni test). doi:10.1371/journal.pone.0083762.g003
Angiogenic Gene Expression in the Rat Placenta Angiogenic Gene Expression in the Rat Placenta Figure 3. Comparison of qPCR and microarray expression data. qPCR and Microarray data for Ptgs2 are shown in panels A and B respectively;
statistical significance (p values) is presented in panel G. C represents linear correlation between qPCR and microarray data for Ptgs2 (p,0.05). qPCR
and microarray results for Pla2g2a are shown in panels D and E respectively; statistical significance (p values) are presented in panel G. Panel F
indicates the linear correlation (Spearman’s rank correlation) between qPCR and microarray data (p,0.05). Both genes display late gestational
increase. Morphogenesis and Angiogenesis Genes were classsified into pathways using GO (gene ontology)
based upon their Entrez IDs. Within the Blood Vessel Morphogenesis
and Angiogenesis pathway a total of 31 differentially expressed genes
were identified (GO:0048514 : blood vessel morphogenesis [412
gene products] and GO:0001525 : angiogenesis [333 gene
products]). 22 of these genes displayed increased expression and PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 4 Angiogenic Gene Expression in the Rat Placenta Figure 4. Comparison of qPCR and microarray data for Nos2. qPCR and microarray results of Nos2 are presented in panels A and B,
respectively. Statistical significance (p values) is presented in panel D. Panel C depicts linear correlation (Spearman’s rank correlation) between qPCR
and microarray data (p,0.05) Both qPCR and microarray data display a significant decrease in expression with gestational age. (* = p,0.05,
p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). doi:10.1371/journal.pone.0083762.g004 Figure 4. Comparison of qPCR and microarray data for Nos2. qPCR and microarray results of Nos2 are presented in panels A and B,
respectively. Statistical significance (p values) is presented in panel D. Panel C depicts linear correlation (Spearman’s rank correlation) between qPCR
and microarray data (p,0.05) Both qPCR and microarray data display a significant decrease in expression with gestational age. (* = p,0.05,
p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). doi:10.1371/journal.pone.0083762.g004 9 genes decreased expression across the gestational age groups. Between E14.25 and E15.25 none of the 31 genes were
differentially expressed, between E15.25 and E17.25, 15 genes
were differentially expressed and between E17.25 and E20, 26
genes were differentially expressed (Table 1, Figure 2, Figure S1
and S2). Figure S4 displays the mean centroid analyses of A)
upregulated and B) downregulated genes. Discussion The aim of this study was to identify mid-late gestation changes
in the expression of genes in the whole rat placenta using
microarray analysis. In particular, this study focused on changes in
genes involved in angiogenic pathways. During mid-late gestation,
fetal demand for energy substrates and gas exchange increases
dramatically in the absence of a concomitant increase in placental
mass. An increase in branching angiogenesis contributes to
placental accommodation during this later gestational period. The data obtained establish that the expression of approximately
7% of genes profiled (22,000) within the placenta change
significantly between E14.25 and E20 days of gestation. Of this
7%, 42% of genes increased expression and 58% decreased
expression. A subset of 31 genes known to be involved in the Blood
Vessel Morphogenesis pathways were identified, of which ,70%
displayed
increased
expression
with
gestational
age. RNA
sequencing of different rat placental regions at E18.5 showed that
the blood vessel development pathway was prominent in genes
that were differentially expressed in the labyrinth zone versus the
junctional zone and the metrial gland [12]. In the metrial gland,
the vascular development pathway was enriched [12]. These
results indicate that the vasculature in the rat placenta is
developing late into gestation. Quantitative RT-PCR of Angiogenic Genes The expression of five angiogenic genes (Mmp14, CD36, Angpt4,
Rhob and Foxm1) was confirmed by qPCR (Figure 5). For all genes
examined, a significant correlation between microarray and qPCR
data was established (Spearman’s Rank Correlation, p,0.05). December 2013 | Volume 8 | Issue 12 | e83762 Principal Component Analysis PCA of placental angiogenic gene expression separated data
into three distinct groups. E14.25 and E15.25 samples clustered
together, while E17.25 and E20 samples partitioned independently
(with F1 accounting for up to 70% of sample variation, Figure 6). These data are consistent with the hypothesis that the expression
profile of Blood Vessel Morphogenesis and Angiogenesis genes in the rat
placenta changes significantly after 70% of gestation is reached
and continues to change thoughout the remainder of gestation. Gestational age based PCA analyses are presented in Figure S3. Furthermore, PCA indicates that the biological variation within
the gestational age category for these genes is small (n = 6). This
conclusion was additionally supported by ANOVA where the
variance in microarray data was partitioned between, gene
(p,0.00001), gestational age (p,0.00001) and biological replicates
(p.0.9). The microarray expression data for eight genes were orthog-
onally validated by qPCR. These genes included three genes
previously known to display gestational age and labour-associated
changes and five genes identified in this study that displayed
altered expression during gestation. Previously, we and others,
have identified late gestation and pre-labour changes in the
expression of enzymes involved in the biosynthesis of eicosanoids
(i.e. prostaglandin synthase 2, Ptgs2 ([15–18] and phospholipase A2
(Pla2a) [19]) and in nitric oxide (i.e. inducible nitric oxide synthase,
Nos2 [20]). Consistent with these observations, the gene expression
of prostaglandin synthase 2 and secretory Pla2a increased (from PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 December 2013 | Volume 8 | Issue 12 | e83762 5 Angiogenic Gene Expression in the Rat Placenta Table 1. List of Angiogenic genes identified and pairwise comparison of gene expression between gestational age groups. Table 1. List of Angiogenic genes identified and pairwise comparison of gene expression between gestational age groups. Principal Component Analysis Gene Identifiers
E14.25 vs E15.25
E15.25 vs E17.25
E17.25 vs E20
E14.25 vs E20
Gene name
Entrez ID
Fold change
p value
Fold change
p value
Fold change
p value
Fold change
p value
Up- regulated
22 genes
Bmp4
25296
–
ns
–
ns
3.8
***
6.77
***
Vegfa
83785
–
ns
,2
*
–
ns
3.04
***
Mmp14
81707
–
ns
,2
***
–
ns
3.44
***
Acvrl1
25237
–
ns
2.8
***
–
ns
3.42
***
Edn1
24323
–
ns
–
ns
,2
***
3.61
***
IL18
29197
–
ns
–
ns
6.4
***
6.74
***
Gpx1
24404
–
ns
–
ns
3.7
***
3.69
***
Id1
25261
–
ns
–
ns
,2
***
4.27
***
Cd36
29184
–
ns
2.8
***
,2
***
4.87
***
Hs6st1
316325
–
ns
–
ns
,2
***
,2
***
Emcn
295490
–
ns
,2
**
,2
***
2.94
***
Rhob
64373
–
ns
–
ns
,2
***
2.31
***
Prl7d1
84377
–
ns
–
ns
,2
***
9.29
***
Angptl4
362850
–
ns
3.1
***
3.7
***
6.93
***
Sox18
311723
–
ns
,2
**
–
ns
,2
***
Klf5
84410
–
ns
,2
*
,2
**
,2
***
Pf4
360918
–
ns
–
ns
3.0
***
2.38
***
Cav1
25404
–
ns
6.7
***
,2
***
15.46
***
Sgpl1
286896
–
ns
–
ns
,2
***
2.64
***
Notch4
406162
–
ns
–
ns
,2
***
,2
***
Tek
89804
–
ns
,2
*
2.2
***
4.72
***
Tgm2
56083
–
ns
,2
**
,2
***
2.34
***
Down-regulated
9 genes
Itgb2
309684
–
ns
–
ns
.0.5
**
.0.5
***
ItgaV
296456
–
ns
–
ns
.0.5
*
0.41
***
Itga4
311144
–
ns
.0.5
*
0.4
***
ns
***
Foxm1
58921
–
ns
.0.5
**
0.4
***
0.20
***
Anpep
81641
–
ns
–
ns
0.2
***
0.2
***
Gbx2
114500
–
ns
–
ns
0.2
***
0.13
***
Atg5
365601
–
ns
.0.5
**
–
ns
.0.5
***
Cited2
114490
–
ns
–
ns
0.5
***
0.5
***
Nos2
24599
–
ns
.0.5
*
0.2
***
0.16
***
E14.25 vs E15.25 - no significant differences in gene expression. E15.25 vs E17.25–15 of 31 genes were differentially expressed. E17.25 vs E20–26 of 31 genes were
differentially expressed. E14.25 vs E20 shows gene expression changes for each gene from E14.25 vs E20. Principal Component Analysis Significance was ascribed where gene expression was changed
by ,2 (* = p,0.05, p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). The order of the genes listed in the table corresponds to the Heat Map on Figure 2. doi:10.1371/journal.pone.0083762.t001 E14.25 vs E15.25 - no significant differences in gene expression. E15.25 vs E17.25–15 of 31 genes were differentially expressed. E17.25 vs E20–26 of 31 genes were
differentially expressed. E14.25 vs E20 shows gene expression changes for each gene from E14.25 vs E20. Significance was ascribed where gene expression was changed
by ,2 (* = p,0.05, p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). The order of the genes listed in the table corresponds to the Heat Map on Figure 2. doi:10 1371/journal pone 0083762 t001 E14.25 vs E15.25 - no significant differences in gene expression. E15.25 vs E17.25–15 of 31 genes were differentially expressed. E17.25 vs E20–26 of 31 genes were
differentially expressed. E14.25 vs E20 shows gene expression changes for each gene from E14.25 vs E20. Significance was ascribed where gene expression was changed
by ,2 (* = p,0.05, p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). The order of the genes listed in the table corresponds to the Heat Map on Figure 2. doi:10.1371/journal.pone.0083762.t001 of Cd36, Mmp14, Angpt4, Rhob and Foxm1 was confirmed by qPCR
on same samples. In all cases, concordance between microarray
and qPCR data was confirmed. These five genes were chosen on
the basis of their function and the fold changes shown in Table 1. CD36 is a, cell membrane scavenger receptor involved in
angiogenesis, inflammation and lipid metabolism [21]. In micro-
vascular endothelial cells it functions as a receptor for Thrombos-
pondin-1 (TSP-1) [22]. CD36 has been implicated in inflamma-
tory processes via the activation of phospholipases and the
formation of prostaglandins [23] and the cellular uptake of fatty
acids via the activation of peroxisome proliferator–activated
receptor c [24]. In this study, Cd36 expression increased by 2.8 E17.25 to E20) and Nos2 decreased (from E15.25 to E20) during
gestation (as determined by both microarray analysis and real-time
PCR). This finding is consistent with current literature. Further-
more, the data for Ptgs2 are consistent with the literature where
expression is known to increase prior to the onset of labour
[10,12,13]). Phospholipases hydrolyze phospholipids into fatty
acids. December 2013 | Volume 8 | Issue 12 | e83762 Principal Component Analysis For Mmp14, qPCR are microarray data are presented in panels A and B, respectively; for Rhob
in panels C and D; for CD36 in panels E and F; for Angpt4 in panels G and H; for Foxm1 in panels I and J. Statistical significance (p values) is presented
in panel K. Where p,0.05 = *; p,0.0005 = **; p,0.0001 = *** as assessed by one way ANOVA and post-hoc tests (Bonferroni test). The x-axes in all
graphs represent the 4 gestational age groups. Y-axes for qPCR graphs denote relative fold change normalised to b-actin. The y-axes for the
microarray bar charts denote microarray hybridisation signals. doi:10.1371/journal.pone.0083762.g005 Figure 5. qPCR validation of 5 Angiogenic genes. For Mmp14, qPCR are microarray data are presented in panels A and B, respectively; for Rhob
in panels C and D; for CD36 in panels E and F; for Angpt4 in panels G and H; for Foxm1 in panels I and J. Statistical significance (p values) is presented
in panel K. Where p,0.05 = *; p,0.0005 = **; p,0.0001 = *** as assessed by one way ANOVA and post-hoc tests (Bonferroni test). The x-axes in all
graphs represent the 4 gestational age groups. Y-axes for qPCR graphs denote relative fold change normalised to b-actin. The y-axes for the
microarray bar charts denote microarray hybridisation signals. doi:10.1371/journal.pone.0083762.g005 Figure 5. qPCR validation of 5 Angiogenic genes. For Mmp14, qPCR are microarray data are presented in panels A and B, respectively; for Rhob
in panels C and D; for CD36 in panels E and F; for Angpt4 in panels G and H; for Foxm1 in panels I and J. Statistical significance (p values) is presented
in panel K. Where p,0.05 = *; p,0.0005 = **; p,0.0001 = *** as assessed by one way ANOVA and post-hoc tests (Bonferroni test). The x-axes in all
graphs represent the 4 gestational age groups. Y-axes for qPCR graphs denote relative fold change normalised to b-actin. The y-axes for the
microarray bar charts denote microarray hybridisation signals. doi:10.1371/journal.pone.0083762.g005 fold during E15.25 to E17.25 days of gestation and remains
significantly elevated till labour onset. Interestingly, increased expression of Cd36 mRNA and protein is observed in cord blood
from late pregnancy compared with early pregnancy samples. expression of Cd36 mRNA and protein is observed in cord blood
from late pregnancy compared with early pregnancy samples. Figure 5. qPCR validation of 5 Angiogenic genes. For Mmp14, qPCR are microarray data are presented in panels A and B, respectively; for Rhob
in panels C and D; for CD36 in panels E and F; for Angpt4 in panels G and H; for Foxm1 in panels I and J. Statistical significance (p values) is presented
in panel K. Where p,0.05 = *; p,0.0005 = **; p,0.0001 = *** as assessed by one way ANOVA and post-hoc tests (Bonferroni test). The x-axes in all
graphs represent the 4 gestational age groups. Y-axes for qPCR graphs denote relative fold change normalised to b-actin. The y-axes for the
microarray bar charts denote microarray hybridisation signals.
doi:10.1371/journal.pone.0083762.g005 Principal Component Analysis Phospholipase A2 is an important mediator of arachidonic
acid formation, which are the substrates of eicosanoids such as
prostaglandins. It has been reported that there is a relative
abundance of phospholipase protein in late pregnancy [19]. E17.25 to E20) and Nos2 decreased (from E15.25 to E20) during
gestation (as determined by both microarray analysis and real-time
PCR). This finding is consistent with current literature. Further-
more, the data for Ptgs2 are consistent with the literature where
expression is known to increase prior to the onset of labour
[10,12,13]). Phospholipases hydrolyze phospholipids into fatty
acids. Phospholipase A2 is an important mediator of arachidonic
acid formation, which are the substrates of eicosanoids such as
prostaglandins. It has been reported that there is a relative
abundance of phospholipase protein in late pregnancy [19]. Of the 31 Blood Vessel Morphogenesis pathway genes that displayed
differental expression during gestation in this study, the expression December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org 6 fold during E15.25 to E17.25 days of gestation and remains
significantly elevated till labour onset. Interestingly, increased
expression of Cd36 mRNA and protein is observed in cord blood
from late pregnancy compared with early pregnancy samples. Figure 5. qPCR validation of 5 Angiogenic genes. For Mmp14, qPCR are microarray data are presented in panels A and B, respectively; for Rhob
in panels C and D; for CD36 in panels E and F; for Angpt4 in panels G and H; for Foxm1 in panels I and J. Statistical significance (p values) is presented
in panel K. Where p,0.05 = *; p,0.0005 = **; p,0.0001 = *** as assessed by one way ANOVA and post-hoc tests (Bonferroni test). The x-axes in all
graphs represent the 4 gestational age groups. Y-axes for qPCR graphs denote relative fold change normalised to b-actin. The y-axes for the
microarray bar charts denote microarray hybridisation signals. doi:10.1371/journal.pone.0083762.g005
Angiogenic Gene Expression in the Rat Placenta Angiogenic Gene Expression in the Rat Placenta fold during E15.25 to E17.25 days of gestation and remains
significantly elevated till labour onset. Interestingly, increased
expression of Cd36 mRNA and protein is observed in cord blood
from late pregnancy compared with early pregnancy samples. Figure 5. qPCR validation of 5 Angiogenic genes. Principal Component Analysis December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 7 Angiogenic Gene Expression in the Rat Placenta Figure 6. Principal Component Analysis (PCA). Principal components F1 vs. F2 for individual gestational age groups (n = 6, E14.25, E15.25,
E17.25 and E20) for all 31 angiogenic genes are plotted to identify variation in gene expression. Day E14.25 and E15.25 samples cluster together,
indicating a similar genes expression profile between these 2 groups. Samples within the E17.25 and E20 groups, however, display independent
clustering, indicative of significantly altered gene expression profiles. Biological replicates within each gestational age group are tightly clustered. doi:10.1371/journal.pone.0083762.g006 Figure 6. Principal Component Analysis (PCA). Principal components F1 vs. F2 for individual gestational age groups (n = 6, E14.25, E15.25,
E17.25 and E20) for all 31 angiogenic genes are plotted to identify variation in gene expression. Day E14.25 and E15.25 samples cluster together,
indicating a similar genes expression profile between these 2 groups. Samples within the E17.25 and E20 groups, however, display independent
clustering, indicative of significantly altered gene expression profiles. Biological replicates within each gestational age group are tightly clustered. doi:10.1371/journal.pone.0083762.g006 Mmp14 encodes for MMP14 (EC 3.4.24.80, also known as
MT1-MMP) which is a key extracellular matrix-remodeling
enzyme
that
promotes
collagen
remodeling. MMP14
is
a
dominant cell-surface protease required for endothelial cell tube
morphogenesis, invasion and for the creation of vascular guidance
tube [23]. However, there is only limited information about the
specific role of MMP14 role in placental angiogenesis. In mouse
placentae, MMP14 is strongly expressed in the labyrinth region
[25]. This region of the placenta is critical for the formation of
syncytiotrophoblast and the subsequent formation of fetal vessels. In human placentae, immunoreactive MMP14 is expressed in
term and
preterm syncytiotrophoblast
cells,
where it may
contribute to the cleavage and release of endoglin [26] and in
the endothelium of feto-placental vessels. Endoglin has been
suggested to be a major factor in the development of preeclampsia
[27]. Placental expression of MMP14 is greater in pregnancies
complicated by gestational diabetes mellitus [28] and, in vitro it is
induced by insulin and IGF-II in vitro [28] and HIF-1a [24]. In this
study, Mmp14 mRNA expression increased from E15.25 to
E17.25 (the peak of trophoblast invasion takes place at this stage)
[29]. Principal Component Analysis Another gene that is involved in prostaglandin regulation is
Sgpl1 (SGPL1, EC 4.1.2.27) which catalizes the irreversible
degradation of the sphingosine-1-phosphate (S1P) [30]. S1P is a
bioactive lipid mediator that promotes cell proliferation, survival,
migration,
adherence,
inflammation
and
angiogenesis
[31]. SGPL1, thus, regulates the available pool of S1P and inhibits its
signalling activities. SGPL1 may regulate S1P-induced Ptgs2 expression in the rat myometrium [32]. In this study, Sgpl1
expression increased 2.6 fold during gestation from E14.25 to E20. Angiopoeitin like Protein 4 (EC 2.6.1.2) expression was low at
E14.25 and E15.25 and increases at E17.25 remaining constant till
E20. Over the gestational period assessed in this study, Angpt4 gene
expression increased ,7-fold. Angpt4, like the other angiopoietins 1
and 2 has been reported to play a role in angiogenesis [33]. Yamakawa et al., reported that ANGPT4 increased endothelial
cell migration and tube formation in vitro and reduced vascular
leakage [34]. Angiogenic effects of ANGPT4 have also been
observed in glioblastoma [35]. To date little is known about the
biological functions of ANGPT4 in the placenta. However,
ANGPT1 has been shown to potentiate VEGF activity and work
together to increase the luminal diameter of blood vessels in sheep
placenta [36]. In this study, the receptor for ANGPT1, ANGPT2
[36] and ANGPT4, TEK (Tek) showed an increase in placental
expression during from E15.25 to E17.25 and further from E17.25
to E20 (fold change 2.2) of gestation. TEK has been implicated in
the regulation of angiogenesis and cell proliferation, migration and
survival [36]. Tek expression was significantly, positively correlated
(p,0.05) with Angpt4 expression during gestation, (upregulated
prior to labour) indicating the possibility that they are working
together to promote late gestational angiogenesis. TIE 1 is the
other receptor for ANGPT4 and in our Microarray study,
interestingly, Tie1 expression does not change from E14.25 right
up to E20 (Data not shown). expression in the rat myometrium [32]. In this study, Sgpl1
expression increased 2.6 fold during gestation from E14.25 to E20. Angiopoeitin like Protein 4 (EC 2.6.1.2) expression was low at
E14.25 and E15.25 and increases at E17.25 remaining constant till
E20. Over the gestational period assessed in this study, Angpt4 gene
expression increased ,7-fold. Angpt4, like the other angiopoietins 1
and 2 has been reported to play a role in angiogenesis [33]. Yamakawa et al., reported that ANGPT4 increased endothelial
cell migration and tube formation in vitro and reduced vascular
leakage [34]. Angiogenic Gene Expression in the Rat Placenta Additionaly, during late gestation in rat, glucose transporter
genes Slc2A3 and Slc2A1 are upregulated and downregulated
respectively, in placenta [6]. This is consistent with our microarray
data where Slc2A3 expression increases from E15.25 to E17.25
and further increases at E20 and Slc2A1 is increased from E15.25
to E17.25 and then gets downregulated at E20 (data not shown). Amino acid transport genes such as Slc38A1, Slc38A2 and Slc38A4
have been shown to increase with gestation, coinciding with
increased fetal nutrient demands [7], which is also consistent with
our microarray data. We can relate the Angiogenic expression
changes in this study to the expression of several transporter genes. In conclusion, the data obtained in this study extends our
understanding of placental genes that may contribute to placental
vascular accommodation during mid-late gestation and late-
gestation, in particular, those involved in regulating placental
angiogenesis. Several of these gene products work together either
directly or indirectly. However, we are not able to ascertain that
the pre labour associated changes seen in these 32 angiogenic
genes, has any link with the actual labour process, even though
some of the changes are acute. Further studies need to be carried
out to prove a link between some or all of these genes with the
onset or trigger of labour. The study identified gestational age-
dependent changes in placental gene expression within the Blood
Vessel Morphogenesis pathways that have not previously been
characterized in rat placenta. Furthermore, it confirms and
extends data for genes previously reported to display gestational
and potential labor-associated changes. We also observed that,
most expression changes for these genes, occurred between E17.25
and E20, prior to labour onset. One caveat that must be
considered in interpreting the results obtained in this study is that
mRNA was extracted from whole placental tissue. Therefore, it is
not possible to attribute observed changes in gene expression to
specific cell types that comprise the rat placenta. Nevertheless, the
data obtained identify lead candidate genes for subsequent cell
specific analyses (e.g. based cell selection using laser capture
microscopy) and provide opportunity to further elucidate func-
tional pathways that may be of significance in mid-late placental
function in both normal and pathological pregnancies. ,
p
p
The transcriptional regulator Foxheadbox M1 (Foxm1) was
expressed during late midgestation (E14.25–E15.25, as assessed by
both microarray and qPCR) but decreased 5-fold by E20. Angiogenic Gene Expression in the Rat Placenta microarray and qPCR) establish that Rhob expression gradually
increases during gestation (from E14.25 to E20), by a fold change
of 2.3. Recently, RHOB (EC 3.6.1.47) was shown to regulate
endothelial cell migration, sprouting, and capillary morphogenesis
[37], although the mechanism by which RHOB regulates
angiogenesis is not well understood. Howe and Addison (2012)
concluded that RHOB plays a significant role in VEGF-induced
endothelial cell morphogenesis, in part, by negatively modulating
the activity of RHOA, [37]. Both RHOA (Ras homolog family
member A) and RHOB are essential downstream effectors of
VEGF signalling in the angiogenic process. siRNA-inhibition of
RhoB results in increased RHOA activation in response to VEGF
(vascular edothelial growth factor) stimulation [37]. Vegf was also
seen to upregulate the expression of RhoB [26]. In our study, we
see that both RhoB and Vegf are up regulated towards late gestation,
first Vegf expression increases at E15.25 and then Rhob expression
increases downstream at E17.25, indicating a positive correlation. Placental expression of Vegfa was increased 3-fold between E14.25
and E20 in the present study. VEGF is known to be an important
regulator of angiogenesis in placenta [38], [39] and its increased
expression during late gestation has been previously reported [5],
while its receptor Flk (i.e VegfR2) is downregulated from E14.25 vs
E20, prior to labour (Microarray data not shown).NOTCH-4 is a
modulator of angiogenesis in human placenta and regulates
placental cell fate [40–42]. Similar to RHOB expression, Notch
signalling acts downstream of VEGF. Notch signalling helps to
regulate endothelial cell morphogenesis via activation of MMPs
[43]. In this study, Notch-4 expression doubled between E15.25
and E20, where its expression is elevated prior to term/labour. have been previously identified in placentae [26,28] [49]. ITGA4
forms a heterodimer with integrin beta 1 to function as a receptor
for collagen. Increased expression of ITGA4 has been implicated
in inhibition of endothelial cell migation during angiogeneis [49]. In this study, Itga4 gene expression decreases during gestation
(from E15.25 right up to E20) and Itgb2 expression decreases
acutely prior to labour onset. Cited2 gene expression also decreases
during late gestation from E17.25 to E20 by two fold. In the mouse
placenta, lack of CITED2 is characterized by disorganization of
the placental fetal vasculature and a fewer trophoblast giant cells,
spongiotrophoblasts and glycogen cells [50]. Hence Cited2 is
important for normal placental vascularisation. Angiogenic Gene Expression in the Rat Placenta Little,
however, is known about FOXM1 function in angiogenesis in the
placenta. Interestingly, the onset of labour in humans is associated
with decreased placental expression of Foxheadbox O4, (both
FoxO4 mRNA and protein) where it may function as a negative
regulator of Ptgs2 expression and prostaglandin biosynthesis [44]. In cancer development and progression, FOXM1 has been
implicated in regulating the expression of the human endothelial
cell caveolae-marker CAV-1 and Foxm1 expression has also been
linked to growth of glioma cells in tumour angiogenesis [45,46]. Placental Cav-1 mRNA expression increased during gestation in
this study. Caveolin has been reported to facilitate VEGF/NO-
mediated angiogenesis [47]. Our data show, Cav-1 expression
increased 6.7 fold between E15.25 and E17.25 and further
increased till E20. CAV-1 is known to interact with FGFR1which
modulates FGF2 related placental angiogenesis [48]. Unlike in
cancer progression we show that an increase in Cav-1 and Vegf
expression is negatively correlated with Foxm1 expression. An
increase in FOXM1 could have a negative effect on CAV-1
expression or its expression is perhaps acutely switched off prior to
labour onset. Figure S5 shows CAV-1 protein localised intensively
around placental blood vessels indicating its important role in
Angiogenesis. Principal Component Analysis Angiogenic effects of ANGPT4 have also been
observed in glioblastoma [35]. To date little is known about the
biological functions of ANGPT4 in the placenta. However,
ANGPT1 has been shown to potentiate VEGF activity and work
together to increase the luminal diameter of blood vessels in sheep
placenta [36]. In this study, the receptor for ANGPT1, ANGPT2
[36] and ANGPT4, TEK (Tek) showed an increase in placental
expression during from E15.25 to E17.25 and further from E17.25
to E20 (fold change 2.2) of gestation. TEK has been implicated in
the regulation of angiogenesis and cell proliferation, migration and
survival [36]. Tek expression was significantly, positively correlated
(p,0.05) with Angpt4 expression during gestation, (upregulated
prior to labour) indicating the possibility that they are working
together to promote late gestational angiogenesis. TIE 1 is the
other receptor for ANGPT4 and in our Microarray study,
interestingly, Tie1 expression does not change from E14.25 right
up to E20 (Data not shown). Ras homolog family member B, Rhob has not been extensively
studied in the placenta. The data obtained in this study (both December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 8 Angiogenic Gene Expression in the Rat Placenta References Aplin J (2000) Maternal influences on placental development. Seminars in Cell &
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Mean Centroid of Angiogenesis. A) for upregu-
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and E20 vs E17.25 = ns and B)E20 vs E14.25 = *, E20 vs
E15.25 = ***
and
E20
vs
E17.25 = ns. (* = p,0.05,
p,0.0005 = **; p,0.0001 = *** and ns = p.0.05). (TIF) The assistance of Mr. Nick Matigan, Eskitis Institute, Griffith University,
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Gestational Variation in Angiogenic gene
expression of 22 genes that display a late gestational
increase. The x-axis indicates the gestational ages whereas the y-
axis indicates the signal obtained from the microarray hybridisa-
tion using the Illumina Bead reader. These 22 genes had SAM p
values ,0.0001. Angiogenic genes of significance that were upregulated (based
on microarray data alone) during gestation included: bone
morphogenetic protein 4 (Bmp4); transglutaminase (Tgm EC
2.3.2.13); sphingosine-1 phosphate lyase (Sgpl1, EC 4.1.2.27);
endomucin (Emcn); Interleukin-18 (IL-18) and glutathione perox-
idase 1 (Gpx1, EC 1.11.1.9). Figure S2
Gestational Variation in Angiogenic gene
expression of 9 genes that display a late gestational
decrease. The x-axis indicates the gestational ages in days
whereas the y-axis indicates the signal obtained from the Figure S2
Gestational Variation in Angiogenic gene
expression of 9 genes that display a late gestational
decrease. The x-axis indicates the gestational ages in days
whereas the y-axis indicates the signal obtained from the Microarray analysis also identifed a suite of Blood Vessel
Morphogenesis pathway genes that were downregulated during late
gestation, prior to labor onset. These included, Itga4, Itgb2, Anpep
and Cited2. The integrin subunits alpha 4 (Itga4) and beta 2 (Itgb2) December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org 9 Angiogenic Gene Expression in the Rat Placenta microarray hybridisation using the Illumina Bead reader. SAM p
values were ,0.0001. (TIF) vessels (A and B), BMP4 (C and D) in endothelial cells lining blood
vessels and Rabbit IgG Isotype Negative Control (E). Haemotox-
ylin staining of nuclei shown in blue. (TIF) Figure S3
Two Dimensional PCA Observational plots
for Gestational Age Comparison Groups: A) E14.25 vs. E15.25. PCA plot F1 vs. F2. B) E15.25 vs. E17.25. PCA plot F1 vs. F2. C) E17.25 vs E20. PCA plot F1 vs. F2. Data were analyzed
using Pearson Correlation Matrix (XLSTAT, p,0.05). Table S1
List of Primer oligonucleotide sequences used
for qPCR experiments. (TIF) Author Contributions Conceived and designed the experiments: GER KV MDN. Performed the
experiments: KV HWC MWCH. Analyzed the data: KV GER JR. Contributed reagents/materials/analysis tools: MDM JAA RJWB. Wrote
the paper: KV GER. Figure S5
Immunohistochemical Localization of CAV-1
and BMP4. Intense staining for CAV-1 around placental blood Figure S5
Immunohistochemical Localization of CAV-1
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Cited2 affects trophoblast formation and vascularization of the mouse placenta. Developmental biology 294: 67–82. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83762 PLOS ONE | www.plosone.org 11 11
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Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited. 1. Introduction Autism Spectrum Disorder (ASD) is diagnosed along a continuum of behavioural variants in
social communication and repetitive behaviours (American Psychiatric Association, 2013). Most individuals on the autism spectrum also experience differences in sensory perception. Some individuals on the spectrum are ‘high-functioning’ and able to cope in every day
environments, while others are severely affected, non-verbal, and may have co-occurring
diagnoses, such as intellectual disability, epilepsy, and/or obsessional, conduct, or mental
health disorders. These individuals require substantial support, caring and careful manage‐
ment, and evidence-based, effective interventions. ASD diagnosis can be detected from as early as 6-months to 1-year of age, although it is more
common that children are aged 2-3 years before diagnosis is affirmed. Frequently, higher
functioning individuals are not diagnosed until adolescence, or even adulthood. Present
figures indicate that approximately 1:50 children are affected worldwide (CDC, 2013), with
parent reported prevalence rates even higher in some countries, e.g., 1:38 (2. 6%) in South Korea
(Kim, Leventhal, Koh et al., 2011) and 1:29 (3. 5%) in the UK, based on data of 11-year old
children (n=13, 287) from the Millennium Cohort Study [19]. Similar prevalence rates have
been found across racial, ethnic and socioeconomic groups; it seems that boys are affected more
frequently than girls (estimated ration of 4:1), although this may be due to under diagnosis in
girls. Karola Dillenburger Additional information is available at the end of the chapter http://dx.doi.org/10. 5772/58983 http://dx.doi.org/10. 5772/58983 Evidence-Based Management and Intervention for
Autism Spectrum Disorders Karola Dillenburger Karola Dillenburger © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited. 2. Etiology The exact etiology of ASD remains unknown even though genetic, immunological, neurolog‐
ical, neurotoxins, electromagnetic radiation, and allergenic causes have been investigated. Autism Spectrum Disorder - Recent Advances 246 Early theories of maternal unresponsiveness have been discredited as mentalistic and sexist,
while recent neurological studies have shown physical differences in early brain growth and
functioning. Sibling and twin studies point to the possibility of genetic links. Ultimately, it is
likely that ASD is caused by a combination of genetic and environmental risk factors [11]. In
fact, it is to be expected that one day we will be able to differentiate symptomatology along
the spectrum much more precisely and different ‘causes’ will be linked with different mani‐
festations along the autism spectrum. Early theories of maternal unresponsiveness have been discredited as mentalistic and sexist,
while recent neurological studies have shown physical differences in early brain growth and
functioning. Sibling and twin studies point to the possibility of genetic links. Ultimately, it is
likely that ASD is caused by a combination of genetic and environmental risk factors [11]. In
fact, it is to be expected that one day we will be able to differentiate symptomatology along
the spectrum much more precisely and different ‘causes’ will be linked with different mani‐
festations along the autism spectrum. General Practitioners (GP) are the first port of call for most parents who are concerned about
their children’s behaviour. A referral is made to an assessment team, commonly lead by a
pediatrician working in collaboration with a team of allied health professionals. A full
diagnosisis based on behavioural observations and caregiver reports of their behavioural
observations [19]. Although presently, there are no medical tests, ASD remains a medical
diagnosis that requires a medical as well as educational response. The Vice President for State Government Affairs of Autism Speaks, the world’s largest autism
charity, Professor Lorri [87] made this point clearly when arguing for health cover: Autism is diagnosed by a doctor, not a school principal. Treatment is prescribed by
a doctor, not a teacher. Here are some things autism families deal with daily:
swallowing aggression, teeth grinding, feces eating, depression, tantrum, drooling,
elective mutism, food refusal, food theft, genital stimulation, hallucinations,
hyperactive behaviour, hyperventilation, inappropriate vocalizations, insomnia,
public disrobing, rectal digging, seizure behaviour, self-injurious behaviour,
tongue protrusion and vomiting. Does anyone think these should be treated in our
school classrooms? (p. 1) Of course not everyone agrees. 2. Etiology Proponents of the neurodiversity movement [48, 58, 66] argue
that autism represents a neurological difference that should be celebrated rather than treated
with interventions. [44] maintain that these arguments are valid with regards to rights,
recognition and acceptance, however, they rightly argue that ‘[o]nly a narrow conception of
neurodiversity, referring exclusively to high-functioning autists, is reasonable’ (p. 20). 3. Economic impact For ‘low-functioning’ individuals with ASD, the lifetime cost to society is estimated to bein
excess of £1. 4 million and these figures are similar internationally [7]. Of course, the cost for
quality of life for the individual and their family is much higher. For example, 86% of parents
of children with disabilities have to pay above average childcare costs and 72% of these families
have given up work or reduced their working hours, because of childcare problems [Buckland,
R., Glass, P., Baroness Eaton, et al., 2014] and only about 15% of adults with ASD are in gainful
employment [79]. The potential positive impact of effective interventions is enormous. It is not surprising, therefore, that the intervention market is booming [17, 32]. As governmental
investments into ASD services are increasing, fad treatments abound. In fact, the struggle for Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 247 a slice of the market has been called the ‘Autism Wars’ [31]. Primary care, allied health, social
care, and education professionals have a key role to play in protecting families and individuals
affected by ASD from fads and ineffective, controversial, or even dangerous treatments that
are peddled by self-proclaimed autism experts for commercial reasons [15]. 5. Evidence base The evidence for ABA-based interventions spans all valid and recognised research method‐
ologies, including Single-System Design (SSD), Randomised Controlled Trials (RCT), Meta-
analysis and Sequential Meta-analysis, Systematic Reviews, Social Validity studies,
Neuroscience studies, and Cost-benefit analysis. Single System Designs (SSD) include reversal designs, multiple baseline designs (across
behaviours, settings, or subjects), changing criterion designs, and alternating treatment
designs [46]. In SSD studies internal validity is achieved by each participant serving as his/her
own control, while external validity/generality is achieved through numerous replications of
carefully described SSD methodologies. Hundreds, if not thousands, of Single-System Design (SSD) studies have been published
evidencing the effectiveness of ABA for individuals with autism [22]. While most of these
studies are published in flagship journals, such as Journal of Applied Behavior Analysis, increas‐
ingly other mainstream journals publish SSD evidence for ABA-based interventions, for
example, the British Journal of Special Education [15]. A good example of an SSD is Garcia-Albea, Reeve, Brothers, and Reeve (2014), who used
a multiple-probe design across participants to teach 4 boys with autism to initiate and
participate in social interactions without vocal prompts from adults. The procedure
involved the use of a script and script-fading procedure. The boys quickly learned to talk
independently about a whole range of relevant things in their environment without the
help of adults. While this kind of research methodology lends itself particularly well to the
action-based researcher/scientist-practitioner model inherent in ABA, it can be usefully
employed in a range of different settings [49]. Randomised controlled trial (RCT), sometimes held up as the ‘gold standard’ for evidence of
effectiveness of interventions, originated from medical research. RCTs were developed to
compare outcomes for one group of people who receive a certain type of medication (treatment
group), to that of another group of people who are not receiving the same medication, i. e.,
who may have received a placebo or ‘treatment as usual’ (control group). The basic assumption
underpinning RCT is that, if both groups of people are well matched, any differences that are
observed after the intervention are due to the intervention [37]. While RCTs may have their utility in relatively clear-cut medical research, there are many
problems when they are used in social care or educational research, not least the ethical
dimension of withholding a potentially beneficial treatment from the control group. 4. Interventions Given that there are no medical indicators for ASD, it is not surprising that currently there are
no pharmacological treatments for the core symptoms of autism. There are, however, phar‐
macological treatments for some of the co-occurring symptoms, but due to lack of evidence of
effectiveness and potentially serious side effects, the National Institute for Clinical Excellence
(NICE, 2013) advises against the use of pharmacological interventions. There are some commercially available intervention packages. However, commonly they are
very expensive and make unsubstantiated claims and promises of recovery or ‘cure’ for autism. These claims are predatory on vulnerable parents, especially since there is generally very little
evidence of effectiveness. The very few studies that exist for some of these commercial
packages are usually not very rigorous and/or conducted by people who have a financial
interest (Houghton, Schuchard, Lewis, & Thompson, 2013). Some interventions have been developed and are frequently used or recommended by allied
health professionals despite the fact that, after a thorough review of all available research
evidence was carried out by the large team of multidisciplinary professionals for National
Autism Centre (NAC, 2009), these interventions have been categorised as unestablished. For
example, Sensory Integration Therapy is recommended widely by occupational therapists [9],
yet there is evidence that it has very little or no effects and can even be counterproductive or
detrimental [55]. Sensory Integration Therapy is classed as not recommended also by the
Australian Department of Families, Housing, Community Services and Indigenous Affairs
(FaHCSIA) [74]. Facilitated Communication is another unestablished treatment (NAC, 2009) that is still in use,
despite the fact that it is highly controversial and has been exposed as being based on deception
[57] and has the potential of causing harm [56]. Virtually all interventions that have been categorised as established are based on knowl‐
edge and applications of the scientific discipline of behaviour analysis [NAC, 2009; 89]. Even some of the most ardent doubters or opponents of applied behaviour analysis (ABA)
have come to the realisation that behavioural interventions are the key to enhancing quality
of life for individuals on the autism spectrum and their families across the lifespan [41,
[Howlin et al., 2014], 47, 60, 95]. It is important to know that the term 'behaviour' when used by behaviour analysts refers to
anything we do and therefore includes feeling and thinking [16]. Autism Spectrum Disorder - Recent Advances 248 5. Evidence base Of course,
there are safeguards, such as cross-over designs or the Hippocratic Oath to ‘do no harm’ [90]. However, some of the main drawbacks in autism research are that, for RCT results to be valid,
all members of the ‘treatment group’ have to receive the exact same treatment and this has to
be held stable for the agreed duration of the intervention. Of course, when interventions are
based on a functional analysis of behaviour, as is the case in ABA, they are tailored to the needs
of the individual, i. e., they are person/child-centered. Data-based decisions are made with Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 249 regards to intervention adjustments, that are implemented immediately, for ethical reasons,
in order to avoid harm and enhance treatment effects [14]. These kinds of progressive,
systematic, individualized, data-based intervention revisions and adjustments would inva‐
lidate RCT data (see Single-System Designs). Of course ABA (i. e., the application of the scientific discipline of behaviour analysis) itself
cannot be assessed via RCTs, yet some specific intervention packages, such as Early Intensive
Behaviour Interventions (EIBI) or the Early Start Denver Model, have been assessed in RCTs. A good example is Howard, Sparkman, Cohen, Green, and Stanislaw (2005), who evaluated
29 pre-school children who received intensive behaviour analytic intervention (treatment
group) and two matched control groups of 16 children each, receiving either intensive or non-
intensive ‘‘eclectic’’ interventions. While the scores for cognitive, language, and adaptive skills
were similar at intake, at follow-up the treatment group had statistically significant higher
mean standard scores in all areas. These data were confirmed at the 2 year follow-up [40]. Other RCTs or quasi-experimental control studies have compared Treatment as Usual with
ABA-based interventions, such as specific commercially available intervention packages [34),
high vs low intensity ABA-based interventions [30, 59], or waitlist controls [67]. Meta-analysis and sequential meta-analysis are increasingly used to give a summary of multiple
small n studies that provide individual participant data, with the expectation that combining
these data (commonly calculated in effect sizes) will allow for the identification of patterns
and thus increase statistical powerto show that treatment effects are not due to measurement
error, variation in sample, etc. Sequential meta-analyses are conducted where enough cumulative
knowledge is available through meta-analysis to draw convincing statistical conclusions about
effect size. 5. Evidence base Of course as in all research, there are a number of issues related to researcher bias
and declaration of interest, however, over recent years meta-analyses have become a welcomed
addition to the evidence-based practice literature. With regards to autism interventions, a recent overview of meta-analyses [77] found that early
intensive ABA-based treatment was significantly related to enhanced outcomes (effect sizes 0. 30 to > 1). Further meta analyses [22, 23, 24, 72] and a recent sequential meta-analysis [54] have
confirmed these findings [1]. Systematic reviews are based on detailed searches of data banks with clearly defined inclusion/
exclusion criteria. Usually teams of multidisciplinary experts summarise selected studies, such
as RCTs, single-system research design studies, and meta-analyses. Given the wide reach of
evidence covered in systematic reviews, they have gained a strong place in evidence-based
practice in ASD. The number of systematic reviews of ASD interventions has risen recently [77]. By-and-large
ABA-based interventions, in particular Early Intensive Behavioural Interventions (EIBI), are
endorsed by systematic reviews. A good example of a comprehensive systematic review was
carried out by the large scale multidisciplinary team of the National Autism Center (2009); 11
interventions were designated as established, of these all but one are explicitly based on ABA;
22 intervetions were categorised as emerging, most of these were also based on ABA. All other
systematic reviews came to similar conclusions [6, 36, 70, 75, 91]. Autism Spectrum Disorder - Recent Advances 250 The review by [43] is the notable exception, in that it does not fully concur with these conclu‐
sions. Howlin et al. concluded that ‘this review provides evidence for the effectiveness of EIBI
for some, but not all, preschool children with autism’ (p. 20). Given that this review is fre‐
quently cited in the UK as a basis against the roll-out of EIBI for all children with ASD who
need it [42], it is important to note here that Howlin et al. misinterpret a number of important
points. First, it is in the mathematical nature of all group average data (such as those calculated
for RCTs) that some individual data are above while others are below the average; such is the
nature of group averages (see also [77]; second, Howlinet al. ‘cherry pick’ results by ignoring
the fact that obviously some children must do extremely well, otherwise the group average
would not be what it is. Thus, Howlin et al. 5. Evidence base 5772/58983 Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 251 ‘indistinguishable’ from neuro-typical peers, but that they also lead to improved neurological
development, i. e., neurological plasticity allowing for compensatory development. Cost-benefit analyses are an important way to substantiate evidence of effective interventions. A recent study estimated the annual ‘cost of autism’ between £0. 8-1. 4+ million per lifetime
depending on the level of functioning; these costs were similar in UK and USA [7] and in other
parts of the world [61, 71]. There is evidence that effective ABA-based interventions can reduce this cost substantially in
the long-term, i. e., $1+million per year [45]. However, due to the fact that intensive interven‐
tions generally are rather costly in the short-term, there has been resistance to their imple‐
mentation. The key question is how effective high-quality programs can be delivered in a more
cost-effective sustainable model, without loosing out on effectiveness [1]. All of these studies supply ample evidence of the effectiveness and efficacy of ABA-based
interventions, in achieving individual potential in a full range of areas, including intellectual,
social, and verbal, functioning, ASD symptomatology, and challenging behaviour. On the basis of this evidence, ABA-based interventions are now widely endorsed in the USA,
Canada, Australia, and some European countries. On a federal level in the USA, for example,
Medicaid now covers ABA-based interventions and the Affordable Care Act covers behav‐
ioural health treatments [83], which include ABA-based interventions generally, and is not
restricted to ASD diagnosis. More recently, [94] recommended More recently, [94] recommended
that principles of applied behaviour analysis (ABA) and behaviour intervention
strategies be included as important elements in any intervention program for young
children with autism. (p. 33) that principles of applied behaviour analysis (ABA) and behaviour intervention
strategies be included as important elements in any intervention program for young
children with autism. (p. 33) 6. Endorsement In the USA, interventions for individuals with ASD that are based on ABA are endorsed as
medically, as well as educationally, necessary and covered by health insurance in the vast
majority of States [2]. In fact, they are now considered ‘treatment as usual’ [28]. As early as
1999 the [84] endorsed ABA-based interventions: Thirty [now 45] years of research demonstrated the efficacy of applied behavioural methods
in reducing inappropriate behaviour and in increasing communication, learning, and appro‐
priate social behaviour. (p. 164) More recently, [94] recommended 5. Evidence base contradict themselves in their conclusions. First
they call for large sample comparisons and group averages (i. e., RCTs) and then they do not
accurately interpret group data. In a subsequent paper, Howlin and colleagues (2014) report extremely poor long-term
outcomes in a 40-year follow-up study of children diagnosed with autism at the Institute of
Psychiatry/Maudsley Hospital, London between 1950 and 1979. Intriguingly, they explicitly
link these findings to the fact that none of these children had received early intensive behav‐
ioural interventions and claim that EIBI is available now. Praising the potential positive effects
of EIBI stands in contrast to their earlier conclusions [42, 43]. It will be interesting to see how
this new evidence will translate into advice given to government bodies. Given that group average scores are neither sensitive to individual differences nor offer
sufficient generality, most behaviour analytic researchers prefer to rely on replicated single-
system designs (SSD) instead of group averages [14, 18, 29]. Clearly, SSD research data cannot
be ignored and should find their rightful place in future reviews of autism intervention
guidelines, such as NICE Guideline 170 (2013). Social Validity studies assess the social significance, appropriateness, and importance of
treatment goals, procedures, and intervention effects [93]. Social validity measures are
increasingly becoming integral part of research into interventions in ASD [27, 53]. A number of studies have shown clear evidence of high social validity of ABA-based inter‐
ventions, especially those that include parent participation and training [18, 92]. Interestingly,
while there is evidence of increased parental stress in families affected by ASD [10, 17], there
is evidence of parental stress reduction when effective interventions for children are in place
[17]. This is also true for education staff [26]. Neuroscience studies, including MRI scans are useful tools to bolster evidence-based practice
in particular in the area of ASD, where the plasticity of the brain during early childhood
constitutes an important focus of intervention [11]. There is evidence of differences in brain
activity between individuals diagnosed with ASD and those who do not have an ASD
designation [Dawson, Klinger, Panagiotides, Lewy, & Castelloe, 1995, 35]. There is further evidence that early behaviour analytic intervention can lead to measurable
change in brain activity [12]. For example, [28] found that ABA-based interventions not only
lead to behavioural improvements, with some optimal outcome individuals becoming Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. [8] recognized that: in areas such as social engagement, language, coping, and reduction of difficult
behaviours… Applied behavioural analysis is usually needed to assist a child to
gain skills and reduce negative or undesirable behaviours. (p. 10) Autism Spectrum Disorder - Recent Advances 252 The Federal U. S. Office of Personnel Management responsible for all federal government
employees concluded that ABA-based interventions should be covered not only for educa‐
tional but also for medical reasons: The Federal U. S. Office of Personnel Management responsible for all federal government
employees concluded that ABA-based interventions should be covered not only for educa‐
tional but also for medical reasons: based on ample scientific and empirical evidence, ABA therapy qualifies as a
medical treatment, rather than purely educational. [5], p. 1) In Canada, ABA-based interventions are supported, for example by the Ontario Department
of Education Policy/Program Memorandum [73] that support[s] incorporation of ABA
methods into school boards’ practices. . . The use of ABA instructional approaches may also
be effective for students with other special education needs. (p. 1) The Maine Administrators of Services for Children with Disabilities confirmed their support
in the Report of the Autism Task Force [6] It is important to note that ABA is frequently perceived to be synonymous with
discrete trial teaching. However, ABA is comprised of a broad scope of empirically
derived behavioural principles used in interventions. (p. 25) Despite this general endorsement of evidence-based behaviour analytic interventions across
most of the English speaking world, the highly controversial approach taken by governments
across the UK and Ireland is to support an ‘eclectic’ approach. There are no clear guidelines
as to what an ‘eclectic’ approach entails and not a single study is published anywhere to show
the effectiveness of an eclectic approach being equal or superior to ABA-based interventions
[14]. In fact, [21] and [39] findings show clearly that ABA-based interventions are superior to
an eclectic approach. Individually tailoring behavioural interventions to match child charac‐
teristics is key to effectiveness [82]. Yet in the UK, the National Institute for Clinical Excellence’s (NICE, 2013) response to
stakeholders, who asked for ABA-based interventions to be included in the NICE guidelines
for the management of children with ASD, was the following: In the review of evidence, the Guideline Development Group found no evidence to support
ABA, and therefore could not make a recommendation about ABA. (pp. [8] recognized that: 5& 8) They also asserted that: NICE clinical guidelines are based on the best quality evidence and are developed
according to rigorous and robust methodologies. The developers were unable to
identify high quality evidence of effectiveness of the ABA approach in managing
children and young people with autism. (pp. 5 & 8) NICE clinical guidelines are based on the best quality evidence and are developed
according to rigorous and robust methodologies. The developers were unable to
identify high quality evidence of effectiveness of the ABA approach in managing
children and young people with autism. (pp. 5 & 8) This view is informed mainly by relatively few, but well rehearsed anti-ABA arguments that
continue to circulate misinformation and misleading anti-ABA propaganda. As [33] points out: The most concerning issue affecting the quality of practices and policies in the
helping professions is the play of propaganda, which misleads us regarding what
is a problem, how (or if) it can be detected, its causes, and how (or if) it can be
remedied. Propaganda is defined as encouraging beliefs and actions with the least Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 253 thought possible. Censorship is integral to propaganda including hiding well-
argued alternatives and lack of evidence for claims. Evidence-based practice was
developed in part because of misleading claims in the professional literature. If
propaganda is an integral part of our society, we cannot escape its influence. But
we can become aware of it, encouraged by ethical obligations to avoid harming in
the name of helping. (p. 302) 7. A ‘new idea’ Anti-ABA propaganda generally comes from people not trained in the science (e.g., [41, 47]. Censorship comes in the form of excluding behaviour analysts from review bodies [14] or
ignoring data presented to review panels [68]. As a consequence of this exclusion, ABA remains
a ‘new idea’ in the UK, despite its extensively documented history and evidence base accessible
in the English language. The German philosopher Alfred Schoppenhauer (1788-1860) recognised that: All new ideas pass through three stages. First, they are ridiculed. Second, they are
violently opposed Third they are accepted as being self-evident All new ideas pass through three stages. First, they are ridiculed. Second, they are All new ideas pass through three stages. First, they are ridiculed. Second, they are
violently opposed. Third, they are accepted as being self-evident. violently opposed. Third, they are accepted as being self-evident. This is true for the evolution of the arguments by ABA opponents [15]. First, they ridicule ABA
as‘ one approach for autism’, while promoting the rather ill-defined eclectic approach. Of
course, one could argue that the eclectic approach is one approach as it precludes any other
approach, such as the dual approach taken in Germany, where psychotherapists are trained
in either behaviour therapy and psychotherapy, and the service user has the choice which
service they prefer to use [17]. In reality then, eclecticism is ‘one approach’ to autism inter‐
vention. When opponents of ABA state that they do not want one approach for all, they cannot
at the same time say that they promote the one approach called ‘the eclectic approach’. re of course further problems with eclecticism. There are of course further problems with eclecticism. • Staff training in all possible autism treatments is impossible. Training and skills of eclectic
practitioners necessarily remain limited to a certain number of preferred interventions. The
decision about what to include/exclude in an eclectic treatment package therefore is not
based on the child’s needs but on the practitioner skills. • Staff training in all possible autism treatments is impossible. Training and skills of eclectic
practitioners necessarily remain limited to a certain number of preferred interventions. The
decision about what to include/exclude in an eclectic treatment package therefore is not
based on the child’s needs but on the practitioner skills. • There is no coherent theoretical knowledge base and the potential for conflicting interven‐
tions means that synergy effects cannot be controlled. 7. A ‘new idea’ • There is no coherent theoretical knowledge base and the potential for conflicting interven‐
tions means that synergy effects cannot be controlled. • There is no evidence of effectiveness. On the other hand as mentioned earlier, ABA is not ‘one approach to autism’ [16], it is the
application of the scientific discipline of behaviour analysis. On the other hand as mentioned earlier, ABA is not ‘one approach to autism’ [16], it is the
application of the scientific discipline of behaviour analysis. ABA aims to discover and understand the underlying principles of behaviour with
the function of a particular behaviour considered in the design of behaviour change
interventions. Interventions are designed for the individual, recognizing that the
function of behaviour varies based on complex combinations of variables. [6], p. 25) Autism Spectrum Disorder - Recent Advances 254 There is nothing wrong with using one approach, if this ‘one approach’ is science [15]. Countless procedures have been developed from the science of behaviour analysis, many
specifically for ASD, e.g., Discrete Trail Teaching (DTT); Pivotal Response Training (PRT);
Natural Environment Training (NET); Verbal Behaviour Approach (VB); while other proce‐
dures have been developed for more general applications, e.g., Functional Analysis and
Functional Assessment; Preference Assessments; shaping, forward chaining, backward
chaining; differential reinforcement of low or zero rate and/or incompatible or alternative
behaviours; Time-out from Positive Reinforcement (TOR); etc. . Some of these procedures have
been combined into comprehensive packages for autism, such as Early Intensive Behavioural
Interventions (EIBI) or Early Start Denver Model (ESDM), while others are used more gener‐
ally, e.g., Programmed Instruction, Generative Instruction; Peer Tutoring; Habit Reversal
Training; etc. Given that the science of behaviour analysis underpins all of these programmes/
procedures and continuous data-based decision making is part and parcel of ABA, new
procedures and progammes are developed continuously to meet the individual or group needs
of service users. ABA has been further ridiculed and accused of intending to change the person, while others
pride themselves for accepting the person for who they are [69, 80]. In fact, the targets of ABA-
based interventions are socially relevant behaviours, linked to cultural and personal norms
and preferences [4]. The curricula are agreed with individuals with ASD and/or their caregiv‐
ers. 7. A ‘new idea’ They are generally based on wide-ranging target behaviours, including life skills, such as
dressing, toileting, attending; social skills, such as playing or imitation; academic skills,
including attending, reading, drawing, writing, and maths, and work/employment based
skills, including interviewing or team work. Basically, the aim of ABA is to enhance all skills necessary to lead a fulfilled life for individuals
who would otherwise be limited in the quality of life they experience. These are the same aims
that most parents have for all of their children, irrespective of a diagnosis. As such, ABA does
not intend to ‘change the person’, but to enhance skills and help individuals to break down
barriers to learning and achieve their full potential. After all, enhancing skills development
increases choice. Once a new idea can no longer be ridiculed, the second point Schoppenhauer made comes to
play: the new idea is opposed. In the case of ABA, this refers to statements such as there is no
evidence to support ABA and therefore no recommendation can be made (NICE, 2013). We
have outlined the wealth of evidence in favour of ABA-based interventions earlier in this
chapter. Given that behaviour analysts commonly are not included in review bodies, at least
in Europe, this mountain of evidence generally is excluded from reviews [68]. When the evidence can no longer be denied, the opposition turns to the behaviour analytic
scientists themselves, stating that research conducted by behaviour analysts is biased and
therefore not to be taken seriously. The idea, that it is objectionable that scientist conduct
scientific research in their own subject area is rather intriguing. Given that it is against ethical
guidelines of all social and health care as well as education professionals to work outside their
own area of expertise [90], clearly, multidisciplinary practice and interdisciplinary research
teams in ASD, should routinely include behaviour analysts, not least because others are not
qualified to make authoritative statements about behaviour analysis [16]. Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983 255 Once ridicule and opposition are not longer tenable, the third point of Schoppenhauer’s
concept of the evolution of a new idea comes to play, when finally, new ideas are considered
self-evident. Intriguingly, this is now starting to happen with regards to ABA. There is
evidence of a claim that all teachers and psychologists use ABA techniques. 7. A ‘new idea’ However, being
able to conduct one or two behavioural techniques [16] clearly does not equate to training in
applied behaviour analysis to international standards [3]. For example, clinical psychology
training typically includes (under Psychological Therapies) ‘competency in two evidence-
based therapeutic approaches including CBT and one other (e.g. psychodynamic, systemic,
social constructionist) ’ [76]. Other professionals commonly receive no training in behaviour
analysis and either none or very little training in ASD [19]. 8. Staff training A Board Certified Behaviour Analyst (BCBA®) has received fully approved training in the
science of behaviour analysis either at Masters or doctoral level, including at least 270 hours
of course work and 1500 hours of supervised practice in ABA [3]. While NICE (2013) did not make any recommendations regarding ABA or staff qualifications,
they recommend a ‘social-communication intervention’ that includes play-based strategies
with parents, carers and teachers to increase joint attention, engagement and reciprocal
communication in the child or young person. Table 1 offers a direct comparison of the basics of ABA-based interventions that were stipulated
by the [8] and the NICE (2013) recommendations. offers a direct comparison of the basics of ABA-based interventions that were stipulated
8] and the NICE (2013) recommendations. Table 1 offers a direct comparison of the basics of ABA-based interventions that were stipulated
by the [8] and the NICE (2013) recommendations. Basics of ABA-based interventions
A Psychosocial intervention (NICE,
2013)
Curriculum decision
The curriculum is organized around typical
developmental expectations;
Individualized approach is used to determine
developmental level of programme;
Functional analysis identifies the
communicative role of behavior;
Language- and communication-intensive;
Socialization and play are actively stimulated;
A specific social-communication
intervention for the core features of
autism in children and young people;
Be adjusted to the child or young
person's developmental level;
To increase joint attention,
engagement and reciprocal
communication;
Methods
An individualized approach is used to select or
develop developmentally appropriate methods;
Procedures are based on applied behaviour
analysis;
Includes structured as well as natural
environment training. Includes play-based strategies;
Include techniques of therapist
modelling and video-interaction
feedback;
Include techniques to expand the
child or young person's
communication, interactive play and
social routines; Autism Spectrum Disorder - Recent Advances
256 256 Basics of ABA-based interventions Basics of ABA-based interventions
A Psychosocial intervention (NICE,
2013)
Monitoring
A formalized assessment of skills (cognitive,
language, socialization, adaptive behavior, fine
and gross motor, and play) is conducted at
regular intervals;
Data are recorded to monitor progress and to
troubleshoot;
Assessment results are used as a guide for
planning what skills to teach next;
Integration of research and practice is used;
Generalisation &
Maintenance
Generalization and maintenance of skills are
built into the program;
Outcome targets
Mainstreaming opportunities with typically
developing peers are built into the program;
Transitional support is provided when the child
leaves one program and moves to the next. 8. Staff training The skills needed in the next situation are
taught and support needed is considered;
Aim to increase the parents', carers',
teachers' or peers' understanding of,
and sensitivity and responsiveness to,
the child or young person's patterns of
communication and interaction;
Parent involvement
Parent training and family support are used;
Education about options for intervention is
provided;
Training is culturally acceptable to individual
families;
With parents, carers and teachers;
Staffing
Collaboration of all team members is used;
Related services are included (i.e., speech,
occupational therapy, adapted physical therapy,
and/or augmentative communication);
Ongoing teacher/therapist training is included
to consider what new and experienced
personnel need to know. The intervention should be delivered
by a trained professional. For pre‑school children consider
parent, carer or teacher mediation. For school‑aged children consider
peer mediation. Table 1. Comparision of ABA-based interventions and NICE (2013) recommendation 9. Conclusion In a recent review for the Canadian Medical Journal, [1] summed up the evidence for ABA-
based intervention in ASD when they stated: In a recent review for the Canadian Medical Journal, [1] summed up the evidence for ABA-
based intervention in ASD when they stated: Current best practices for preschool-aged children with ASD include a focus on
improving language, cognitive and adaptive skills using applied behaviour analysis
(ABA) techniques. Applied behaviour analysis refers to the application of empiri‐ Evidence-Based Management and Intervention for Autism Spectrum Disorders
http://dx.doi.org/10. 5772/58983
25 257 cally derived learning principles (i. e., the antecedent–behaviour–consequence
contingency) to produce meaningful changes in behaviour. Such strategies are
carefully engineered and implemented through a variety of approaches (e.g.,
discrete trial teaching to more naturalistic learning contexts) to teach skills and
reduce problem behaviour. Applied behaviour analysis interventions can be
provided in a variety of settings (e.g., home, specialized treatment centres, special‐
ized or public schools) by a range of front-line therapists, ideally supervised by a
psychologist or board-certified behaviour analyst who specializes in ASD. (p. 515) It is, therefore, not surprising that increasingly reports link ABA-based intervention with
optimal outcomes. Individuals previously diagnosed with ASD are now living independent
productive happy lives or no longer meeting diagnostic criteria. Mukaddes, Tutkunkardas,
Sari, Aydin, and Kozanoglu (2014) suggested that ‘[i]t could be concluded that a group of
children with an autism diagnosis could lose the diagnosis of autism upon early intervention’
(p. 1). [65] for example, report a strong statistical significance of early intensive behaviour
analytic interventions for children previously reported to have optimal outcomes [28], while
[85] report on reductions of restricted and repetitive behaviours and [85] focus on improve‐
ments in academic skills following early intensive behavioural interventions. Evidently, ABA translates into evidence-based interventions that allow individuals with ASD
to overcome barriers by ensuring choice, human rights, equality and true active participation. ABA helps achieve potential by cherishing the person for who they really are and, by accepting
difference, it values the difference we can make in people’s lives. Author details Karola Dillenburger* Address all correspondence to: k. dillenburger@qub. ac. uk Centre for Behaviour Analysis, School of Education, Queens University Belfast, UK Parts of this chapter were previously published in National Association of Primary Care Review
(Spring/Summer 2012) p. 134-135. Parts of this chapter were previously published in National Association of Primary Care Review
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Effects of sevoflurane and clonidine on acid base status and long-term emotional and cognitive outcomes in spontaneously breathing rat pups
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RESEARCH ARTICLE Methods Copyright: © 2017 Almenrader et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. At postnatal day (PND) 7, male Sprague Dawley rat pups were randomized into four groups
and exposed to sevoflurane for one hour, to a single dose of intraperitoneal clonidine or to a
combination of both and compared to a control group. Blood gas analysis was performed at
the end of sevoflurane anaesthesia and after 60 minutes from clonidine or saline injection. Emotional and cognitive outcomes were evaluated in different group of animals at infancy
(PND12), adolescence (PND 30–40) and adulthood (PND 70–90). Editor: Antonella Gasbarri, University of L’Aquila,
ITALY Editor: Antonella Gasbarri, University of L’Aquila,
ITALY Received: November 27, 2016
Accepted: March 1, 2017
Published: March 20, 2017 Accepted: March 1, 2017
Published: March 20, 2017
Copyright: © 2017 Almenrader et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper. Funding: This work was supported by institutional
funds from the Sapienza University of Rome. The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. Effects of sevoflurane and clonidine on acid
base status and long-term emotional and
cognitive outcomes in spontaneously
breathing rat pups Nicole Almenrader1,2☯, Paola Colucci1☯, Valentina De Castro1†, Daniela Valeri1,
Maura Palmery1, Viviana Trezza3, Patrizia Campolongo1* 1 Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy, 2 Department of
Anaesthesia and Intensive Care, Policlinico Umberto I, Rome, Italy, 3 Department of Science, Section of
Biomedical Sciences and Technologies, University “Roma Tre”, Rome, Italy a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ☯These authors contributed equally to this work. † Deceased. * patrizia.campolongo@uniroma1.it OPEN ACCESS Citation: Almenrader N, Colucci P, De Castro V,
Valeri D, Palmery M, Trezza V, et al. (2017) Effects
of sevoflurane and clonidine on acid base status
and long-term emotional and cognitive outcomes
in spontaneously breathing rat pups. PLoS ONE 12
(3): e0173969. https://doi.org/10.1371/journal. pone.0173969 Background Numerous experiments in rodents suggest a causative link between exposure to general
anaesthetics during brain growth spurt and poor long-lasting neurological outcomes. Many
of these studies have been questioned with regard of their translational value, mainly
because of extremely long anaesthesia exposure. Therefore, the aim of the present study
was to assess the impact of a short sevoflurane anaesthesia, alone or combined with cloni-
dine treatment, on respiratory function in spontaneously breathing rat pups and overall
effects on long-lasting emotional and cognitive functions. Introduction Multiple studies have raised concerns regarding direct neurotoxic effects of general anaes-
thetics on the developing brain with possible long lasting impact on neurocognitive function
[1]. Alterations, such as attention deficit/hyperactivity disorders (ADHD) [2], learning deficits
[3], behavioural disturbances [4] and language deficits [5] have been reported in retrospective
cohort follow up studies in children. Time of exposure as well as the cumulative anaesthetic
dose have both been identified as determinant factors of neurotoxicity. There is clear evidence
from animal studies that the time of synaptogenesis is the most vulnerable period for anaesthetic
neurotoxicity. Different brain regions are vulnerable at different time windows. In rats, exten-
sive synaptogenesis takes place in the thalamus, hippocampus and neocortex at postnatal day
(PND) 7 [6]. Therefore, most studies in this field have been performed by exposing rodents to
general anaesthetics at this specific time frame. A recent study in young rodents suggested that
physiological disturbances during anaesthesia might contribute to volatile anaesthetic induced
neurotoxicity [7]. A higher overall mortality as well as a significant increase in hippocampal cell
death and worse performance in the Morris water maze test were reported in spontaneously
breathing, anaesthetized rats compared to those who were mechanically ventilated or control
group animals. Stratmann and coworkers observed a similar pattern of neuronal cell death in rat pups
exposed to either 4h of isoflurane or 4h of hypercarbia suggesting that part of isoflurane
induced cell death might be due to isoflurane induced hypercarbia [8]. However these findings
did not translate consistently into neurocognitive outcome as only those animals treated with
4 h of isoflurane anaesthesia showed long-term neurocognitive deficits. The impact of hypercapnic acidosis on neurocognitive outcome has also been questioned in
neonates undergoing thoracoscopic surgery for repair of congenital diaphragmatic hernia and
oesophageal fistula [9,10]. Interestingly, the European paediatric anaesthesia community is currently leading an active
debate on how to define ‘safe conduct of anaesthesia’ in neonates and young children, pointing
out the primary importance of controlling and maintaining stability of physiological parame-
ters [11]. So the current question is, if it is the anaesthetic agent itself or rather the anaesthesia
induced derangement of body homeostasis having detrimental effects on the developing brain
[12,13]. In the search of preventive and protective strategies, alpha-2-agonists gained an important
role being attributed with neuroprotective properties attenuating the apoptotic and neurode-
generative effects of NMDA-antagonists and GABAA-agonists [14–16]. Results Rat pups exposed to either sevoflurane or to a combination of sevoflurane and clonidine
developed severe hypercapnic acidosis, but maintained normal arterial oxygenation. Emo-
tional and cognitive outcomes were not found altered in any of the behavioural task used
either at infancy, adolescence or adulthood. Competing interests: The authors have declared
that no competing interests exist. 1 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Conclusions Sixty minutes of sevoflurane anaesthesia in newborn rats, either alone or combined with clo-
nidine, caused severe hypercapnic acidosis in spontaneously breathing rat pups, but was
devoid of long-term behavioural dysfunctions in the present setting. PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Randomization At PND7 rats were randomized by a computer generated list (Graph Pad Software, Inc., Cali-
fornia, USA) into four groups receiving: sevoflurane (group S), clonidine (group C), sevoflur-
ane/clonidine (group SC), saline (control group). Anaesthesia protocol Animals anaesthetized with sevoflurane (Sevorane, Abbott, Italy) were placed in a heated
anaesthesia chamber connected to a vaporizer system and to a gas anaesthesia machines (Fluo-
vac System, Harvard Apparatus, Holliston, US) breathing sevoflurane 2.5% in oxygen and air
(1:1) with a fresh gas flow of 2 l/min. Animals treated with clonidine (Catapresan 150 mcg/ml,
Bo¨hringer Ingelheim, Italy) received an intraperitoneal injection of 400 μg/kg diluted in sterile
saline to a standard volume of 200 l immediately before being placed in the anaesthetic cham-
ber. Control animals and those exposed to sevoflurane received an intraperitoneal administra-
tion of 200 l of sterile saline. The duration of stay in the gas chamber was set to a total time of
60 minutes for comparison with sevoflurane exposure. The chamber was heated and the tem-
perature maintained constant by an infrared light and a heating pad. The concentration and
duration of sevoflurane administration were established after preliminary results and previous
published studies on rat pups. We decided to omit anaesthesia induction with a high concen-
tration of sevoflurane in order to minimize cardiorespiratory depressant effects. Anaesthesia
induction was defined as time from the beginning of sevoflurane until loss of righting reflex. The clonidine dose was chosen according to Kesavan and coworkers [21]. Arterial pulse oxim-
etry and heart rate (MouseOx Plus1, Starr Life Sciences Corp., Oakmont, US) were recorded
continuously in one animal every set of four animals undergoing anaesthesia. All other ani-
mals were clinically observed for any sign of distress during the entire procedure. Materials and methods
Animals All animal experiments were approved by the Italian Ministry of Health (Rome, Italy) and per-
formed according to the guidelines of the Italian Ministry of Health (D.L. 96/1992) and the
European Community Directive 2010/63/EU of September 22nd 2010. Multiparous pregnant female Sprague-Dawley rats (Charles River1, Calco, Italy) were
housed in an air-conditioned room (temperature 21±1˚C) in a standard 12-h light-dark cycle
(lights on at 06:00 h), with access to food and water ad libitum. Newborn litters, found up to 5 pm, were considered to be born on that day (PND0). On
PND1 all litters were reduced to a size of 6 males and 2 females per litter as previously
described [20]. On PND21, pups were weaned and housed in groups of three in 42 × 27 × 14 cm Plexiglas
cages in air-conditioned rooms. One male pup per litter from different litters per treatment
group was used in each experiment. Each male rat was tested only once. Females were not
tested. Introduction The use of clonidine
in paediatric anaesthesia is expanding as a result of its’ sedative and analgesic properties [17–
19]. On this background we tested the hypothesis that clonidine and 1h of 2.5% sevoflurane,
either alone or in combination, would differently affect the respiratory system in spontane-
ously breathing animals. In order to evaluate if early exposure to anaesthesia—induced hyper-
carbia could lead to long-lasting behavioural alterations, emotional and cognitive functions
were assessed as secondary outcomes not only at infancy (PND12), but also at adolescence
(PND 30–40) and adulthood (PND 70–90). 2 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Behavioural tests at infancy Ultrasonic vocalization (USV). The ultrasonic vocalization (USV) analysis is a useful tool
to assess pup social communication skills in the first few days of life [22]. The USV test has pre-
viously been studied to assess autism-like behaviour in rodents after neonatal exposure to
sevoflurane [23]. When separated from the mother and the nest, newborn rats emit USVs with
frequencies between 30 and 90 kHz. These USVs are essential for the mother/pup interaction
and are critical for pups survival [22]. At PND 12, pups were removed from the nest and placed
in a soundproof arena (30 × 30 × 30 cm). The recording session lasted 3 min [20]. USV calls
were recorded by the Avisoft recorder (Avisoft Bioacoustics, Berlin, Germany). Homing test. One of the earliest expressions of spatial behaviour is the pups’ ability to return
towards their nest if separated from mother and littermates. Hippocampal formation plays a piv-
otal role in spatial cognition and navigation [24]. The homing test is a useful task for the evalua-
tion of these rudimentary cognitive abilities in infant rats [25]. At PND 12, each pup was placed
in a Plexiglas arena (36×22.5×10 cm) with the bottom covered by 2/3 clean litter and by 1/3 of lit-
ter from the mother’s cage, with the nest odour, representing the nest area. The pup was placed
close to the wall on the clean litter side and video-recorded for 4 min. The latency to reach the
nest area and the time spent in the nest area were recorded to evaluate homing performance. Blood gas analysis Blood samples for blood gas analysis were taken from the left ventricle after 60 minutes of
drug or saline administration or after 60 minutes of placement in the gas chamber. Arterial
blood was analysed immediately after blood collection with a blood gas analyser (GEM Pre-
mier 4000, Instrumentation Laboratory SpA, Milan, Italy). After blood sampling animals were
sacrificed by cervical dislocation. 3 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Behavioural testing The series of behavioural tests was chosen on the basis of the relatively well established ana-
tomical areas involved in these tasks. All behavioural tests were performed by the same opera-
tor, who was blinded to group assignment. Adolescent and adult rats were handled three times
for one minute each day, starting 72 hours before testing. Emotional and cognitive assessments
were performed at infancy (PND12), adolescence (PND30-40) and at adulthood (PND70-90). PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Behavioural tests at adolescence and adulthood Inhibitory avoidance. The inhibitory avoidance test is a commonly used task to investi-
gate learning and long-term memory processes in rodents [26]. This test has been used previ-
ously to measure sevoflurane-induced impairment of memory consolidation in rodents [27]. The procedure was carried out as previously described by Morena [28]. For training, adoles-
cent and adult rats were placed into the starting compartment and were allowed to explore the
apparatus. After the rats stepped into the dark compartment, the sliding door was closed, and
a single footshock (0.4 mA) was delivered for 1 s. Animals were removed from the shock com-
partment 15 s after termination of the footshock. Retention was tested 48 h later. On the reten-
tion test, rats were placed into the starting compartment, and the latency to re-enter the shock
compartment (maximum latency of 600 s) was recorded. Longer latencies were interpreted as
indicators of better retention. Elevated plus maze. The elevated plus maze (EPM) is a validated assay to study hippo-
campus dependent behaviour performance in rodents [29] and has been applied previously to
assess anxiety related behaviour in rodents after inhalational [30] and intravenous anaesthetics
[31]. The EPM apparatus comprised two open arms (50 × 10 × 40 cm) and two closed arms
(50 × 10 × 40 cm) that extended from a common central platform (10 × 10 cm). Confinement
to the closed arms was associated with the observation of significantly more anxiety-related
behaviours than confinement to the open arms [32]. The EPM test was performed following
the procedure described by Manduca and colleagues [33]. Adolescent and adult rats were
individually placed on the central platform facing a closed arm and a 5-min test period was
recorded. Behavioural analysis was carried out using the Observer1 XT 13.0 software (Nol-
dus, Wageningen, The Netherlands). The following parameters were analysed: PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 4 / 12 Results There were no differences in demographic data between groups (Table 1). Mean time and
SEM of anaesthesia induction was 4.8 ± 0.5 minutes in the sevoflurane group and 4.1 ± 0.3
minutes in the sevoflurane/clonidine group (p = 0.3). There was no anaesthesia related
mortality. Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Table 1. Demographic data. CONTROL
CLONIDINE
SEVOFLURANE
SEVOFLURANE/ CLONIDINE
BW (g)
17.90 ± 0.78
16.89 ± 0.72
16.30 ± 0.79
17.85 ± 0.80
T˚C pre- treatment
36.70 ± 0.08
36.63 ± 0.18
36.68 ± 0.11
36.68 ± 0.08
T˚C post- treatment
36.71 ± 0.08
36.64 ± 0.17
36.93 ± 0.05
36.89 ± 0.05
Data are presented as mean ± standard error of mean (SEM). There was no difference of body weight and temperature among groups (n = 10 per group;
p > 0 05) BW = body weight T˚C = axillary body temperature rd error of mean (SEM). There was no difference of body weight and temperature among groups (n = 10 per group;
xillary body temperature. Data are presented as mean ± standard error of mean (SEM). There was no difference of body weight and temperature among groups (n = 10 per group;
p > 0.05). BW = body weight. T˚C = axillary body temperature. Data are presented as mean ± standard error of mean (SEM). There was no difference of body weight and temperature among groups (n = 10 per group;
p > 0.05). BW = body weight. T˚C = axillary body temperature. https://doi.org/10.1371/journal.pone.0173969.t001 a. % Time spent on the open arms (% TO); b. % Open entries (% OE); c. Number of exploratory head dippings (HDIPS); d. Number of stretched-attend postures (SAP). Statistical analysis. Data are expressed as mean ± standard error of mean (SEM). To
assess the effects of different treatment, data were analysed using one-way ANOVA (Graph
Pad Prism version 6.0) followed by Tukey’s multiple comparison post-hoc test where appro-
priate. For all comparisons, p values of less than 0.05 where considered statistically significant. Values are reported as mean ± standard error of mean (SEM). * p<0.05. BE = base excess, pH, pCO2 and lactate levels were significantly lower in rat pups treated with sevoflurane or sevoflurane and clonidine. There
were no differences among groups for bicarbonate, pO2 and glucose levels (p > 0.05). https://doi.org/10.1371/journal.pone.0173969.t002 * p<0.05. BE = base excess, pH, pCO2 and lactate levels were significantly lower in rat pups treated with sevoflura * p<0.05. BE = base excess, pH, pCO2 and lactate levels were significantly lower in rat pups treated with sevoflura
were no differences among groups for bicarbonate, pO2 and glucose levels (p > 0.05). Values are reported as mean ± standard error of mean (SEM). Values are reported as mean ± standard error of mean (SEM).
* p<0.05. BE = base excess, pH, pCO2 and lactate levels were significantly lower in rat pups treated with sevoflurane or sevoflurane and clonidine. There
were no differences among groups for bicarbonate pO and glucose levels (p > 0 05) mean ± standard error of mean (SEM). Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Table 3. Ultrasound vocalization (USV) and homing test at infancy. CONTROL
CLONIDINE
SEVOFLURANE
SEVOFLURANE/ CLONIDINE
USV
Frequency (n)
691.08 ± 100.37
619.20 ± 104.39
448.80 ± 123.71
624.10 ± 107.17
HOMING TEST
Latency (s)
83.34 ± 23.25
91.34 ± 28.53
82.50 ± 27.27
136.95 ± 32.51
Nesting time (s)
145.42 ± 21.39
152.25 ± 21.37
190.49 ± 23.78
115.90 ± 27.48 Table 3. Ultrasound vocalization (USV) and homing test at infancy. Data are expressed as mean ± standard error of mean. There were no differences of behavioural tests among groups (n = 10 per group; p > 0.05). s = seconds; n = numbers of vocalizations https://doi.org/10.1371/journal.pone.0173969.t003 Lactate. Lactate levels were significantly lower in animals treated with sevoflurane alone
or in combination with clonidine compared to control and clonidine treated rats. Lactate levels
of rat pups treated with clonidine alone were significantly lower compared to the control
group (F (3,34) = 61.35; P < 0.0001). HCO3. One-way ANOVA did not reveal any significant difference in bicarbonate levels
among groups (F (3,34) = 0.8338; P = 0.48) HCO3. One-way ANOVA did not reveal any significant difference in bicarbonate levels
among groups (F (3,34) = 0.8338; P = 0.48) (
)
pO2. No cases of hypoxia were observed. There was no statistically significant difference
in pO2 between groups (F (3,34) = 1.115; P = 0.36) Base excess. One-way ANOVA did not show any significant difference among groups
(F (3,34) = 2.004; P = 0.13) Glucose levels. No significant differences were observed among groups (F (3,34) = 2.205;
P = 0.10) Behavioural tests USV and homing test. No significant effects of treatment on USVs were observed
(F (3, 38) = 0.91, P = 0.45, Table 3). As concerning the homing test, one-way ANOVA did not
reveal any significant effect of treatment on the latency time (F (3, 38) = 0.85, P = 0.48) and on
the nesting time (F (3, 38) = 0.44, P = 0.73) in the homing test (Table 3). USV and homing test. No significant effects of treatment on USVs were observed
(F (3, 38) = 0.91, P = 0.45, Table 3). As concerning the homing test, one-way ANOVA did not
reveal any significant effect of treatment on the latency time (F (3, 38) = 0.85, P = 0.48) and on
the nesting time (F (3, 38) = 0.44, P = 0.73) in the homing test (Table 3). Inhibitory avoidance. No significant differences were found in the inhibitory avoidance
test among groups either at adolescence or adulthood respectively (training: F (3, 28) = 0.55,
P = 0.65; test: F (3, 28) = 1.01, P = 0.40; training: F (3, 38) = 0.44, P = 0.72; test: F (3, 38) = 0.61,
P = 0.61, Table 4). Inhibitory avoidance. No significant differences were found in the inhibitory avoidance
test among groups either at adolescence or adulthood respectively (training: F (3, 28) = 0.55,
P = 0.65; test: F (3, 28) = 1.01, P = 0.40; training: F (3, 38) = 0.44, P = 0.72; test: F (3, 38) = 0.61,
P = 0.61, Table 4). Elevated plus maze. One-way ANOVA showed that there was no significant effect of
treatment at adolescence on %TO (F(3, 38) = 0.55, P = 0.65; Fig 1A), on %OE (F(3, 38) = 0.62,
P = 0.60; Fig 1B), on HDIPS frequency (F(3, 38) = 0.74, P = 0.54) and on SAP Frequency
(F(3, 38) = 0.55, P = 0.65) and at adulthood on: %TO (F(3,34) = 0.89, P = 0.46; Fig 1A), %OE
(F(3,34) = 0.27, P = 0.85; Fig 1B), in HDIPS frequency (F(3,34) = 1.51, P = 0.23), and in SAP fre-
quency (F(3,34) = 1.25, P = 0.31). Table 4. Inhibitory avoidance test (IA) at adolescence and adulthood. Table 4. Inhibitory avoidance test (IA) at adolescence and adulthood. Blood gas analysis Mean and SEM values for all parameters are reported in Table 2. Mean and SEM values for all parameters are reported in Table 2. pH and pCO2. Rat pups treated with either sevoflurane or a combination of sevoflurane
and clonidine developed severe respiratory acidosis, while there was no difference in pH and
pCO2 between control animals and those receiving clonidine (F (3,34) = 46.82, P < 0.0001;
F (3,34) = 41.16, P < 0.0001 for pH and pCO2, respectively). Table 2. Blood gas analysis. CONTROL
CLONIDINE
SEVOFLURANE
SEVOFLURANE/CLONDINE
pH
7.39 ± 0.01
7.39 ± 0.01
7.25 ± 0.04*
6.99 ± 0.04*
pCO2 mmHg
47.50 ± 1.53
50.70 ± 2.10
81.40 ± 7.81*
133.50 ± 9.70*
lactate mmol/L
3.55 ± 0.25
2.68 ± 0.14*
1.10 ± 0.13*
0.66 ± 0.11*
HCO3 mmol/L
26.39 ± 0.57
27.84 ± 0.58
27.79 ± 0.65
26.45 ± 1.66
BE mmol/L
3.89 ± 0.61
5.57 ± 0.83
7.25 ± 1.2
6.13 ± 1.37
glucose mg/dL
113 ± 4.27
141 ± 7.77
127 ± 7.82
112 ± 16.36
pO2 mmHg
93.40 ± 2.71
92.70 ± 2.68
88.30 ± 1.48
90.13 ± 1.7 Table 2. Blood gas analysis. Values are reported as mean ± standard error of mean (SEM). PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 5 / 12 Behavioural tests y
(
)
CONTROL
CLONIDINE
SEVOFLURANE
SEVOFLURANE/ CLONIDINE
ADOLESCENCE
Training Latency Time (s)
9.07 ± 1.75
8.13 ± 1.31
8.29 ± 2.36
6.06 ± 1.25
Test Latency Time (s)
140.42 ± 71.30
352.95 ± 97.78
285.52 ± 89.43
294.36 ± 98.42
ADULTHOOD
Training Latency Time (s)
10.03 ± 1.41
10.83 ± 2.10
12.66 ± 2.62
9.83 ± 1.41
Test Latency Time (s)
352.44 ± 74.76
295.91 ± 59.13
356.48 ± 69.81
239.12 ± 73.12 Latency times are measured in seconds (s). Results are expressed as mean ± standard error of mean (SEM). There was no difference among groups
(n = 10 per group; p > 0 05) PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 6 / 12 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Fig 1. (A): Elevated Plus Maze (EPM) at adolescence and adulthood. Percentage of entries in the open
arms (OE). Results are expressed as mean ± standard error of mean (SEM). There was no difference among
groups (n
10 per group; p > 0 05) (B) Elevated Plus Maze (EPM) at adolescence and adulthood Fig 1. (A): Elevated Plus Maze (EPM) at adolescence and adulthood. Percentage of entries in the open
arms (OE). Results are expressed as mean ± standard error of mean (SEM). There was no difference among
groups (n = 10 per group; p > 0.05). (B): Elevated Plus Maze (EPM) at adolescence and adulthood
Percentage of time spent in the open arms (TO). Results are expressed as mean ± standard error of mean
(SEM). There was no difference among groups (n = 10 per group; p > 0.05). https://doi.org/10.1371/journal.pone.0173969.g001 Fig 1. (A): Elevated Plus Maze (EPM) at adolescence and adulthood. Percentage of entries in the open
arms (OE). Results are expressed as mean ± standard error of mean (SEM). There was no difference among
groups (n = 10 per group; p > 0.05). (B): Elevated Plus Maze (EPM) at adolescence and adulthood
Percentage of time spent in the open arms (TO). Results are expressed as mean ± standard error of mean
(SEM). There was no difference among groups (n = 10 per group; p > 0.05). https://doi.org/10.1371/journal.pone.0173969.g001 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups the current study. HCA has been suggested as a determinant factor in anaesthesia related neu-
rotoxicity and long-term cognitive outcome [7,8]. Stratmann and coworkers showed that neonatal exposure to either two and four hours of
isoflurane or four hours of carbon dioxide caused a similar pattern and distribution of cell
death in the cortex, hippocampus and thalamus [8]. However long term neurocognitive defi-
cits were only found in rodents after four hours of isoflurane exposure suggesting a dose-
dependent threshold. In contrast Wu and co-workers [7] compared spontaneously breathing to mechanically
ventilated 14-day old rats that were exposed to four hours of either sevoflurane or isoflurane. Self—ventilating animals showed a higher percentage of overall mortality (30%), neuronal cell
death and alterations of spatial learning compared to mechanically ventilated and control ani-
mals. HCA has important biochemical and physiological effects [35]. The effect of HCA on brain function in neonates is of uttermost interest among neonatolo-
gist when applying protective ventilation strategies with permissive hypercapnia. Extremes of
hypercapnia and especially rapid changes of PaCO2 have been linked to adverse neurological
outcome in premature neonates [36]. Furthermore, HCA was shown to inhibit neuronal
metabolism and to increase expression of pro-apoptotic mediators in the cerebral cortex of
newborn piglets [37]. Notwithstanding the above concerns, several animal studies have sug-
gested neuroprotective effects of hypercapnia on impaired neuronal function in the injured
neonatal rat brain [38–40]. Vannucci and coworkers showed that hypercapnia protects the immature rat brain from
hypoxic—ischaemic damage. Improved cerebrovascular perfusion and oxygen delivery as well
as enhanced cerebral glucose utilization were observed in animals exposed to hypoxia and
hypercapnia. Besides a marked attenuation in cerebral tissue lactacidosis, a reduction of the
excitatory amino acid neurotransmitter glutamate in the CSF was found [38,39]. There has been recently a debate on safety of thoracoscopic surgery for congenital dia-
phragmatic hernia (CDH) and oesophageal atresia in the newborn [9,10]. A prospective pilot
randomized controlled trial at Great Ormond Street Hospital provided evidence that thoraco-
scopy in neonates is associated with significant hypercapnia and acidosis. The levels of hyper-
capnia and acidosis were of such concern that thoracoscopic repair of CDH in this age group
was suspended. Six out of 10 neonates experienced paCO2 levels above 105 mmHg for a dura-
tion ranging from 30 minutes up to three and a half hours. PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Discussion To date there is an open debate on whether impaired long-term neurocognitive outcome after
anaesthesia in newborn rodents and humans might be a result of a direct drug related neuro-
toxic effect or rather a consequence of physiological disturbances occurring during anaesthesia
[7,11,34]. The present findings show that even a relatively brief anaesthesia with sevoflurane causes
profound hypercapnic acidosis (HCA) in spontaneously breathing rat pups. This effect is
aggravated by the contemporary administration of clonidine. However, no correlation could
be found between anaesthesia–induced hypercapnic acidosis at PND 7 and emotional or cog-
nitive alterations at infancy, adolescence and adulthood in any of the behavioural tasks used in 7 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups In the present study neonatal rodents were exposed to sevoflurane and as a consequence of
anaesthesia induced respiratory depression also to hypercapnic acidosis. However, oxygen-
ation and tissue perfusion were guaranteed as shown by normal serum paO2 and lactate levels. The battery of neurocognitive tests utilized focused on the detection of more subtle changes
related to social communication, autism-like behaviour and anxiety. More recent studies in neonatal rodents assessing autism-like behaviour, social behaviour and
fear conditioning after sevoflurane exposure [23,30] reported conflicting results. Noteworthy,
most of these experiments used extremely long anaesthesia protocols with up to six hours of expo-
sure to inhalational anaesthetics. When translating time from newborn rat pups to human beings,
six hours in a rat pups’ life would have an equivalent of more than two days in human life [44]. Not surprisingly very high mortality rates up to 30%7 are reported in these experiments, especially
in the final hours of the anaesthesia. A direct correlation between the cumulative dose of general
anaesthetics and the risk of neurocognitive impairment has been postulated [1,2,45]. The role of alpha-2-agonists in the context of neuroprotection needs still to be defined. Few
studies in rodents have been performed suggesting protective properties [14–16]. Pre- and postconditioning with dexmedetomidine and clonidine has been reported to
attenuate cerebral ischaemia–reperfusion injury in rats [46–48]. Conversely, in relation to the possible neuroprotective effects of clonidine [15,16], our find-
ings suggest that clonidine alone has no impact either on respiratory function nor on neuro-
cognitive outcome in rat pups. In combination with sevoflurane a rather synergistic
depressant effect on the ventilatory response to carbon dioxide was observed. This is in accor-
dance with findings of Kesavan and coworkers [21] showing a 35% reduction in respiratory
rate after 90 minutes of intraperitoneal injection of clonidine 400 mcg/kg in neonatal rats. No
conclusions regarding possible neuroprotective effects can be drawn from the present study as
the battery of behavioural tests did not reveal any deficit in any group treated. More detailed
studies in this field are warranted. An unexpected finding of our study was the inverse relationship between serum lactate and
carbon dioxide. The regulation of serum lactate in rat pups is not fully understood and it has
been suggested that lactate may play a pivotal role as substrate of cerebral metabolism during
the neonatal period [49]. It has been shown that repeated tactile stimulation of neonatal rats
increases serum lactate levels by 207%. However, on PD7 this effect was reduced to 11%. No
increase in serum lactate levels was observed in anaesthetized rat pups [50]. This observation is
in accordance with our findings showing lower serum lactate levels in anaesthetized rat pups
compared to controls. Limits of the study The present study design does not allow a clear separation between anaesthesia and hypercar-
bia related effects as a hypercarbic (CO2) control group is lacking. However, results give insight
in an ‘overall’ combined effect of general anaesthesia and hypercapnic acidosis on the develop-
ing brain opening the hypothesis that general anaesthesia might be protective under these
conditions. There were no changes of arterial
oxygenation. Results from neurodevelopmental follow-up of these kids are still awaited and
might elucidate long-term effects of hypercapnia and acidosis on the developing brain [10]. There are no reports in the literature as yet indicating a deleterious cerebral effect of acidosis
and hypercapnia in the presence of normoxia. A case report of an 8—year old, asthmatic boy suffering more than 14 hours of severe
hypercapnic acidosis due to respiratory failure was followed by complete recovery [41]. A sec-
ond case of severe hypercapnia under sevoflurane anaesthesia was reported in a three—week
old baby undergoing incarcerated hernia repair. No neurological sequelae were observed [42]. A reduction in cerebral metabolic requirements due to deep sedation with sevoflurane as well
as adequate tissue perfusion and oxygenation were proposed as protective mechanisms for tol-
erating severe hypercapnia. Volatile anaesthetic preconditioning (APC) of the brain has been demonstrated both, in
vitro and in vivo, yet the underlying mechanism is not fully understood. Suppression of energy
requirements seems not sufficient to explain the neuroprotective effects of sevoflurane. Further
mechanisms such as inhibition of glutamate release, direct scavenging of free radicals, anti-oxi-
dative and anti-inflammatory effects as well as prevention of apoptotic cell death seem to play
an important role for increasing cerebral ischaemic tolerance [43]. 8 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Conclusions Results of the present study suggest that a brief exposure to sevoflurane and severe hypercarbia
might not affect long-term cognitive and emotional functions during the period of brain
growth spurt. However, the absence of behavioural and emotional deficits does not preclude
other forms of behavioural impairments, which might have been detected by more specific
tasks others than those used in the present study. 9 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
March 20, 2017 Sevoflurane and clonidine long-term effects in spontaneously breathing rat pups Author Contributions
Conceptualization: NA PCo PCa. Data curation: NA PCo VDC DV. Formal analysis: NA PCo PCa. Funding acquisition: PCa. Investigation: NA PCo VDC DV. Methodology: NA PCo VT MP PCa. Project administration: PCa. Resources: PCa. Visualization: NA PCo VT PCa. Visualization: NA PCo VT PCa. Writing – original draft: NA PCo PCa. Project administration: PCa. Resources: PCa. Supervision: PCa. Supervision: PCa. Validation: PCa. Author Contributions Investigation: NA PCo VDC DV. References Curr Opin Anesthesiol 2015; 28: 302–7 10 / 12 PLOS ONE | https://doi.org/10.1371/journal.pone.0173969
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https://openalex.org/W2592432936
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http://www.app.pan.pl/article/item/article/item/app002872016.html?pdf=39
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English
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Morphological and systematic re-assessment of the late Oligocene “Halitherium” bellunense reveals a new crown group genus of Sirenia
|
Acta Palaeontologica Polonica
| 2,017
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cc-by
| 343
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Morphological and systematic re-assessment of the late
Oligocene “Halitherium” bellunense reveals a new crown group genus of
Sirenia Manja Voss, Silvia Sorbi, and Daryl P. Domning
Acta Palaeontologica Polonica 62 (1), 2017: 163-172 doi:https://doi.org/10.4202/app.00287.2016 “Halitherium” bellunense is exclusively known from a single individual from upper
Oligocene glauconitic sandstone near Belluno, northern Italy. According to a review of its
morphological basis, which consists of associated cranial elements, some vertebrae and ribs,
this specimen is identified as a juvenile, because the first upper incisor (I1) and supposedly
second upper molar (M2) are not fully erupted. However, its juvenile status allowed only
cautious conclusions on its taxonomy and systematic affinity. The presence of a nasal
process of the premaxilla with a broadened and bulbous posterior end, and a lens-shaped I1,
corroborate an evolutionarily-derived status of this species that places it well within the
sirenian crown group Dugonginae. Considering these new data and in order to avoid
continued misuse of the inappropriate generic name of Halitherium, a new generic name, Italosiren gen. nov., and emended species diagnosis are supplied for this taxon. Key words: Mammalia, Tethytheria, Sirenia, Dugonginae, evolution, Oligocene, Italy. Manja Voss [manja.voss@mfn-berlin.de], Museum für Naturkunde, Leibniz
Institute for Evolution and Biodiversity Science, Invalidenstraße 43, 10115
Berlin, Germany. Silvia Sorbi [silviasorbi@gmail.com], Museo di Storia
Naturale, Università di Pisa, Via Roma 79, 56011 Calci, Pisa, Italy. Daryl P. Domning [ddomning@Howard.edu], Laboratory of Evolutionary Biology, Department of Anatomy,
Howard University, Washington, District of Columbia 20059,USA. This is an open-access article distributed under the terms of the Creative Commons
Attribution License (for details please see creativecommons.org), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and source are credited. This is an open-access article distributed under the terms of the Creative Commons Attribution License (for details please see creativecommons.org), which permits unrest
distribution, and reproduction in any medium, provided the original author and source a ion License (for details please see creativecommons.org), which permits unrestricted us
tion, and reproduction in any medium, provided the original author and source are credit Full text (358.1 kB)
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https://openalex.org/W2989469309
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https://hal.inria.fr/hal-03652052/file/491829_1_En_26_Chapter.pdf
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English
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Using Game-Based Environments to Measure Cognitive Decision Making
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Lecture notes in computer science
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cc-by
| 3,095
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To cite this version: Laura A. Waters, Karen L. Blackmore. Using Game-Based Environments to Measure Cognitive
Decision Making. 1st Joint International Conference on Entertainment Computing and Serious
Games (ICEC-JCSG), Nov 2019, Arequipa, Peru. pp.324-330, 10.1007/978-3-030-34644-7_26. hal-
03652052 Game-Based Environments to Measure Cognitive
Decision Making Laura A. Waters, Karen L. Blackmore Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03652052
https://inria.hal.science/hal-03652052v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License The University of Newcastle, New South Wales 2308, Australia
laura.a.waters@uon.edu.au Abstract. Within the area of serious games research, there is significant
potential for researchers and other stakeholders to use serious games to
gain more fundamental understanding of the underlying cognitive pro-
cesses of individual users or participants. In this research, we present the
results of an experiment to benchmark a visual search task presented
in a 3d game-like environment with a standard, controlled, lab based
implementation. Our results show similar trends in performance mea-
sures across experimental conditions in the two environments, however,
participants were faster and more accurate overall in the 3d game-like en-
vironment. There is significant potential for researchers and other stake-
holders to utilise serious games platforms as a means of measuring human
cognition within environments that are visually more closely related to
’real-life’ than those used in cognitive psychology. Keywords: 3D environment · cognition · visual search task · decision
making. 2.1
Measuring Human Cognition Cognitive psychology uses robust methodological approaches to test an indi-
viduals underlying processing abilities and capacity when performing particular
cognitive tasks. There are a number of robust tools and tasks that are used to
understand cognitive performance that can provide this broader understanding
of capability; for example, surveys, psychometrics, observation, randomised con-
trolled trials (RCTs). One key difficulty in being able to generalise task perfor-
mance to real world behaviours lies in the highly constrained nature of lab-based
environments and artificial stimuli used [5], such as simple shapes. These con-
cerns relate to the overall context of the results, and the potential impact of
this context on performance [8]. On the other hand, studies of entertainment
games and SGs tend to use quasi-experimental designs and surveys and largely
tend to measure post-intervention activity engagement [12]. While studies using
quasi-experimental designs and surveys have added to our understanding of the
outcomes and impacts of playing games, RCTs provide more rigorous evidence
about the impacts of games. Although, laboratory-based tests provide a robust
foundation for our understanding of decision making processes, more research is
required for conversion of highly constrained lab based tasks and measures into
other contexts such as more realistic virtual games-based environments. The rise
of SGs and immersive simulation environments provides a platform for a more
realistic and variable environment which could be used for cognitive testing and
training purposes. Simplistically, performance data can be used to profile players
or measure competency [1], and there is promise for game environments to be
used to capture more cognitive level data to assess cognitive processing archi-
tecture and capacity. 1
Introduction Serious games (SGs), or games that are used for a purpose other than purely en-
tertainment, are being used more frequently within industries for assessment and
training of specific skills and abilities. While some performance measures may be
captured, there is great benefit in understanding the types of cognitive processes
that underlie abilities. One important research area of cognitive psychology is
decision-making; that is, how we process information to make a judgment. This is
often done through lab-based experiments that ask participants to make simple
cognitive judgments about simple perceptual stimuli. Many SG models acknowledge that at the crux of any learning activity, there
is a cognitive process occurring [7, 15]. However, the incorporation of robust mea-
sures of cognitive performance in game based environments is not currently done,
even though the captured data might shine a light on understanding the pro-
cesses underlying their cognitive abilities [3, 7, 11]. Within the field of computer
science, there has been extensive research into 2D environments and evidence
has shown that visual search abilities improve with gaming [2], however these
findings were often incidental outcomes and visual search abilities have not been L. A. Waters & K. L. Blackmore L. A. Waters & K. L. Blackmore 2 explicitly considered in an immersive game-based environment in a way the re-
lates to the more formal, constrained approach used in cognitive psychology [6]. In this work we present the results of a pilot study to compare visual search
task performance, as measured through reaction time (RT), between a stan-
dard 2D constrained lab based task, and a 3D game-like visual environment. The focus here is to assess whether the deviations in RTs that occur when the
number of visual stimuli change maps between the 2D lab based task and the
3D counterpart. We begin by providing a brief background to the measurement
of human cognition, followed by an outline of our research focus, the experi-
mental methodology and the results of our pilot study. Lastly, we provide some
concluding discussion, and outline potential avenues for future work. explicitly considered in an immersive game-based environment in a way the re-
lates to the more formal, constrained approach used in cognitive psychology [6]. 2.2
Research Focus At the higher level, the purpose of this research is to develop the foundations of a
theoretical framework that uses the strengths of two currently separate research Cognitive Measures in 3D Environments 3 areas to address some of their respective limitations. On one hand, cognitive
psychology uses robust methodological approaches to design, measure perfor-
mance, empirically analyse, and make inferences about the underlying cognitive
processes and abilities that people use to complete a task or make decisions. We understand that human cognition plays a role in nearly every aspect of ev-
ery day life, however, due to the highly constrained nature of lab-based tasks,
it can be difficult to generalise results to how an individual might approach
tasks in their everyday environment. On the other hand, the gaming industry
has conducted extensive research into human-computer interaction, engagement
and player typing/preferences. By combining aspects of SGs with cognitive lab-
based approaches, we aim to develop a framework which provides guidelines to
both researchers and game designers on a) how to increase engagement and real-
ism when developing experiments in cognitive psychology, and b) how to embed
robust cognitive methodology in a SG. This would allow for finer grain data
and a greater depth of understanding about how individuals are processing and
engaging within the environment. This can help us to adapt or optimally design
games for learning to accommodate cognitive styles. This is particularly relevant
for SGs, where these games are typically deployed across a cohort of individuals
with different player profiles and styles. One overall end-goal of this research will consider how virtual environments
impact users; that is, do people behave and make decisions in a more realistic
and virtual environment in the same way they would in standard artificial lab-
based environments. However, in order to retain the robustness and validity of
any cognitive measures used in a game-based environment, we first need to lay
the groundwork for ensuring reliable measures of in-game performance by bench-
marking performance on simple tasks using the highly constrained approach of
cognitive psychology. Although we are restricting participant interaction with
the 3D game-like environment, the goal of this pilot study is to ensure that
the more visually immersive environment is still capturing valid and reliable
data that can be used as a performance baseline for future studies in which we
allow greater interactive possibilities for participants. 2.2
Research Focus As a means of benchmark-
ing performance on a cognitive task between a standard 2D lab-based cognitive
psychology test and a 3D game-like environment, we elect to design and pilot a
simple visual search task and replicate this in a restricted game-like environment. Python programming language was used to create the 2D lab-based task whereas
we used Virtual Battlespace 3 (VBS3) [4], a visually realistic game environment
with scenario creation tools, to create the 3D game-like environment. We hypothesise that participants will have a preference for the mechanics
used in the immersive environment of VBS3, and that this will be reflected
in this pilot experiment through faster RTs and greater accuracy even though
the cognitive processes involved should remain the same. If we find evidence to
support the hypothesis that game-based environments are a valid way to deliver
cognitive tasks and assess underlying cognitive processes, then we are developing
the capacity to re-deploy this back into the design of SGs and potentially allow
for greater flexibility in the realism of environments used for cognitive testing. L. A. Waters & K. L. Blackmore 4 3
Methodology In this pilot study, we designed a simple visual search task [14] within a 3D virtual
environment (Virtual Battlespace 3) which replicated the experimental design
of a typical 2D laboratory-based task requiring participants to make a simple
decision. Further details on the environments and visual stimuli are provided in
the materials section below. The research was conducted under the University of
Newcastle’s Human Research Ethics Committee approval number H-2018-0227. 3.1
Participants Participants were undergraduate students from the University of Newcastle. Of
the 41 students who completed the experiment, there were 28 males (M = 22.23
years, SD = 3.71) and 13 females (M = 25.38 years, SD = 9.91) ranging from
18 to 52 years of age. Participants were enrolled in either a second year soft-
ware engineering course (28 males and 2 females respectively) or a first year
introductory psychology course (11 females) and received course credit for their
participation. 3.2
Materials and Design The experiment was conducted in one of the University of Newcastle HCI labs
using an Alienware 17 R3 laptop. PsychoPy2 (v1.90.3) was used to develop and
run the 2D laboratory-based task, while the 3D game-based task was run using
VBS 3.7.0. Design In each of the environments, participants were presented with either 5,
10, 15 or 20 items (stimuli) in a single 4 second trial and required to make a
simple decision on whether a target item was present or not. The desired target
depended on the presented colour arrangement of an item. Stimuli used in stan-
dard cognitive experiments are often highly constrained shapes or letters which
are varied visually through the use of colours, location, or rotation [14]. Replica-
tion of our task in a 3D game-like environment meant that to achieve a higher
level of realism, abstract visual stimuli could not be used in both conditions. As such, this experiment used human avatars in VBS3 to address the criteria
of perceptual salience between targets and distractor items, relying on colour
arrangement of clothing to provide distinction. In each environment, the target
(2D: a rectangular, two toned shape, and 3D: a human avatar, see Figure 1 and
2 for example trials) would be tan on top and red on the bottom, while both
distractor types would be tan on the bottom half, and either red or yellow on
top. For each task, a practice block with feedback (correct or incorrect response)
was completed prior to undertaking 10 experimental blocks, each containing 40
trials (no feedback). We counterbalanced task and response key orders to account
for any potential ordering effects. We also asked participants to complete two (2)
questionnaires; one pre-test demographic survey containing additional questions Cognitive Measures in 3D Environments Cognitive Measures in 3D Environments 5 regarding gaming preferences and behaviours, and one post-test where we asked
participants about the difficulty of the two tasks and whether they were aware
of using a particular search strategy to reach their decision. Fig. 1. An example 15-item set presented in the 2D environment. The target item is
absent from this trial. Fig. 1. An example 15-item set presented in the 2D environment. The target item is
absent from this trial. Fig. 2. An example 20-item set from the VBS3 (3D) environment. The target avatar
is present on this trial. Fig. 2. An example 20-item set from the VBS3 (3D) environment. 3.2
Materials and Design The target avatar
is present on this trial. 4.1
Preliminary Analysis Surface level analysis of this pilot experiment revealed a significantly faster mean
RT (M = 0.74s) in the 3D environment compared to the 2D environment (M
= 1.02s), t = 74.515, p < .001, as well as higher accuracy overall (M = 99%
and M = 97% for 3D and 2D respectively), t = -9.191, p < .001. At first glance
it could be proposed that in each task participants respond ’yes’ as soon as
the target item has been identified (self-terminating cognitive architecture) on
target present trials, or exhaustively search all items in target absent trials before
responding ’no’. [13]. This is reflected in both 2D and 3D environments by the
slower average RTs on trials where no target item was present (see below figure). L. A. Waters & K. L. Blackmore 6 Fig. 3. The absolute RTs averaged across participants can be shown in the two graphs
above (Left graph: RTs for the 2D environment, Right graph: RTs for the 3D VBS3
environment). Participants were significantly faster when completing the task in VBS3
compared to the 2D python environment, however in both tasks were participants
tended to respond slower to trials without a target present. Fig. 3. The absolute RTs averaged across participants can be shown in the two graphs
above (Left graph: RTs for the 2D environment, Right graph: RTs for the 3D VBS3
environment). Participants were significantly faster when completing the task in VBS3
compared to the 2D python environment, however in both tasks were participants
tended to respond slower to trials without a target present. REFERENCES 7 5
Discussion Two main arguments in favour of using game-like or scenario based platforms
for teaching and training purposes are that firstly, they are more engaging and
therefore more effective as a training tool [9], and secondly, they can be more
closely related to the real world meaning an individuals performance is more
likely to reflect actual behaviour [10]. Unfortunately, the reported benefits and
effectiveness of these training approaches are often mostly subjective rather than
objectively measured, and assumptions are often made about generalisability to
real world behaviours. The findings of this simple pilot experiment indicate that embedding cog-
nitive tasks within virtual training environments with high levels of ’realism’
hold promise as a comparative environment for the capture of robust measures
of underlying cognitive processes. The group level trends of slower RTs on tri-
als where target item was absent or when there was a greater number of items
to be processed (as seen in figure 3), indicates that participants were engag-
ing similar underlying cognitive decision-making processes despite the different
environments. While the design of the task itself was highly constrained, it is
worth noting that the faster response times and improved accuracy in the VBS3
environment may be impacted by additional factors such as a discrepancy in
the perceived perceptual planes of the two environments. This factor warrants
further investigation and as such, future experimentation will focus on adjusting
the locations of the avatars as well as the viewing angle of the ’player avatar’ so
the spatial locations are more closely aligned with the 2D plane presented in the
comparative environment. By further researching performance measures within
these 2D and 3D comparative environments, we aim to develop a foundation for
designing and implementing tasks within SGs that can provide rich, robust and
valid measures of a users cognitive processing abilities. This would also provide
researchers, educators, or game designers with greater insight into the cognitive
abilities, interactions and learning styles of specific SG users. REFERENCES [15]
A. Yusoffet al. “A Conceptual Framework for Serious Games”. In: 2009
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video game experience on the time course of inhibition of return and the
efficiency of visual search”. In: Acta psychologica 119.2 (2005), pp. 217–
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Glenda Gunter, Robert F Kenny, and Erik Henry Vick. “A case for a formal
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